Neuroscience Matters Issue 02

Neuroscience Matters

Vol. 2 | No. 2 | Pages 1 - 3901 May 2014

at the University of Toronto© 2014

Revolutionary for Depression

Imaging the Whole-Brain

Gut Microbiotaon Disorders

The Circuitry Underlying the Revolutionary Use of

Ketamine to Treat Depression

The Effect of Gut Microbiota on Anxiety and Mood Disorders in

Mice

The Creation False Memories Through Optogenetic

Stimulation of the Hippocampus2 Page 22 Page 26

Jenise Chen is in a Chemistry and Human Biology: Health and Disease double major and is thus stuck with drawing molecules and formulas throughout her undergraduate career. In order to show off her creative and artistic talents, she has involved herself in many clubs and groups, including the Human Biology Students’ Union as the Webmaster and the Interneuron magazine as one of the Layout Designer and Editors.

Jenise is proud to have been given this amazing opportunity once again to take on designing and editing the journal created by Dr. Ju. She had a wonderful time creating the journal and hopes all the readers can enjoy both content and design aesthetics!

Happy reading, everyone!

Jenise Chendesign & layout editor:

Dear Readers,

I have the great fortune of working with some of the brightest young minds in our Neurosciences program at the University of Toronto. Prior to their graduation I ask my students to pick a topic or published research that they believe will have a significant impact within the next 10 years in the field of neuroscience. The 2013 class has chosen a very diverse but fascinating group of papers which are highlighted in the current issue of Neuroscience Matters. While only time will tell how accurate the students’ topics have been, I hope that you will enjoy their insights into some of the most interesting aspects of our discipline.

Signed,Bill

Dr. William Jueditor-in-chief:

Letter from the Editor:

Neuroscience Matters | Issue 02 | 20141

NeuroscienceMatters

HMB420F Class of 2013contributors:

Azubuogu AnuduRuoshi Cui

Stephany FranciscoLuisa Garzon

Michelle HayanoAilya JessaDehi Joung

Waldo LefeverEd (Zhouliang) Li

Kamalpreet MannVictoria MarsheBenjamin Ong

Chang Woo ParkIngrid Quevedo

Charlotte Chiarella-RedfernAnastasiya Slyepchenko

Kaai YeeJia Yan Zhang

Thanks to the following people for contributing their article to this journal!

Neuroscience Matters | Issue 02 | 2014 2

Neuroscience MattersDopamine D2R receptors in adolescence and its significant role in the development of synaptic connectionsJia Yan Zhang1 & Ingrid Quevedo1

1Human Biology Department, The University of Toronto. Toronto, Ontario CA.

Dopamine D2 receptors (D2R) are known to be involved in the regulation of synaptic connections and are important for different brain functions such as learning and working memory. Irregular D2R activity has also been observed in psychiatric disorders, particularly schizophrenia. Researchers employed various methods to examine the effects of D2R activity on dendritic spines and the resulting behaviour, such as pharmacological treatments, over-expressing or knocking down D2R in vivo and behavioural test such as the Y maze. This study found that increased D2R activation lead to a reduction in spine density in an age dependent manner. This effect increased D2R activity had on spine development was only observed in postnatal weeks 3-6, which corresponds to adolescence in mice. Increased D2R activity during this period caused by a mutation in the dysbindin (Dtnbp 1) gene led to a reduction of spines and impairments in the entorhinal-hippocampal circuit and working memory. Particularly, it

was found that these abnormalities could be alleviated by the application of D2R blockers during adolescence. Although the connection between D2R and schizophrenia had already been established this study indicated to a novel relationship between the two to better understand how it is that these receptors lead observed deficits. It was concluded that due to the age-dependent regulation of D2R in spine development that interventions during adolescence could prevent the irregular D2R activity thereby also prevent future cognitive impairments. The importance of these findings is that human adolescence at risk of developing schizophrenia could benefit from medication targeting D2R to prevent spine and cognitive abnormalities. Further research would have to be conducted before this could be established.

Key words: Dopamine D2 receptors (D2R), schizophrenia, dendritic spines, age-dependent


BACKGROUNDDopamine D2 receptors (D2R) are one of two subfamilies of dopamine receptors which includes D2, D3, and D4 receptors. These receptors have been implicated in a number of debilitating mental disorders including schizophrenia. For example, as early as 1970s researchers have associated the efficacy of antipsychotic drugs used to treat schizophrenia with their affinity of binding to dopamine receptors including D2R1. Additionally, there has been evidence of increased D2R occupancy by endogenous dopamine in schizophrenic patients compared to healthy individuals2. Moreover, genetic variants that have been associated with schizophrenia such as dysbindin (Dtnp1) are known to regulate cell surface expression of D2R3. Taken together, these evidences all indicate the importance of D2R in the development of schizophrenia.

Dopamine receptors are G-protein coupled receptors, and activation of these receptors can lead to a series of downstream signaling events. D2Rs are coupled with inhibitory G-proteins and upon activation of these receptors by agonist binding, G-protein α-subunit is released to inhibit a number of downstream signaling pathways including the cAMP-PKA pathway4. Activation of D2R

can also inhibit NMDA current transmission in prefrontal cortical and hippocampal neurons. Therefore, activated D2R is capable of altering synaptic transmission, which may lead structural changes of synapses and reform neuronal circuitry5.

One of the most predominant symptoms of schizophrenia is cognitive impairment6. It has been repeatedly reported from neuroimaging and postmortem studies that there is reduced spine density particularly in prefrontal cortex, reduced brain volume, and reduced grey matter volume in schizophrenic brain7-8. However, the pathophysiological mechanisms underlying such deficits remain as a mystery. Given the strong association between D2R and schizophrenia, and D2R’s ability of altering synaptic transmission, this research aims to experimentally investigate whether D2R regulate synaptic density and development.

RESULTSD2R regulates the formation of dendritic spinesTo determine whether D2R regulated spine development researchers injected male C57BL/6mice with vehicle, D2R agonistsquinpirole and bromocriptine, or D2R antagonist eticlopride. hippocampal slices were prepared twenty-four hours

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later for diolistic labeling. It was found that mice that had been injected with the D2R agonists had reduced spine density within the CA1 neurons and that multiple injections of the antagonist lead to an increase in spine density. Thus the activation of D2R inhibited spine development while its prolonged inactivation promoted it.

Researchers then wanted to further confirm the results of this experiment thus D2R was over-expressed or knocked down in vivo. The CA1 region was injected with a lentivirus expressing a fluorescent protein with either siRNA specific to Drd2 or over-expressing D2R. Brain slices were prepared 7 days later for examination. It was found that CA1 neurons treated with the D2R virus had a reduction of spine density whereas those with the Drd2 siRNA virus had an increase. These results were consistent with those of the pharmacological experiments.

D2R regulates maturation of spinesDendritic spines vary in shape and size – there are mushroom and thin spines which constitute the majority of spines found within mature neurons, and there are stubby spines which are mainly found within immature neurons. The effect of D2R activation on the different type of spines was analyzed within hippocampal neurons. Researchers analyzed the effect that D2R agonistswould have on the different spine types and on filopodia. Neurons treated with quinpirole and bromocriptine resulted in a reduction in the density of mushroom and thin spines whereas filopodium density increased, however stubby spine density was not affected (Fig. 1).

Figure 1. Quantification of culture hippocampal cells. D2R agonists reduced number of mushroom/thin spines and increased filopodium. Stubby spines remained unaffected.

Neurons were then treated with Drd2siRNA and a fluorescent protein to verify whether D2R mediated the effects of quinpirole and bromocriptine. Three days later, these same neurons were then treated with quinpirole for a total of 24 hours. Drd2 siRNA eliminated the effect quinpirole had on the density and length of mushroom and thin spines. The observed increase in filopodia

by the D2R agonists, which are normally found within immature neurons, indicated that D2R activation inhibited the ability of the spines to mature. To examine this, the same neurons were treated with quinpirole and were imaged before and one hour following treatment. It was observed that quinpirole increased the conversion of mushroom and thin spines to filopodia but reduced the conversion of filopodia to mushroom and thin spines. Overall these results suggested that activation of D2R reduced number of spines during development by preventing the maturation of the spines.

Increased D2R activation resulted in decreased spine density in sandy (dysbindin) miceThe dendritic spines of sandy mice were then examined to determine whether the increased D2R activation was involved in spine abnormalities observed in these mice. Sandy mice do not express the dysbindin protein and have increased D2R expression. Cultured neurons and slices from the sandy mice were used to examine spines. There was a decrease in the density of mushroom and thin spines and an increase in filopodia within the hippocampal neurons. Also when compared to wild-type mice there was significantly less spine density overall within the sandy mice.

Figure 2. Hippocampal neurons of wild-type and sandy mice. Drd2 siRNA increased the spine density of sandy mice implicating D2R in the observed abnormalities.

It still had to be determined whether the D2R activity within these mice contributed to the observed abnormalities. To determine whether the increase in D2R activity resulted in the spine abnormalities, sandy mice were injected with a virus expressing Drd2siRNA. These cells showed increased spine density (Fig. 2) and thus researchers concluded that the decrease in spine density resulted from the increased D2R activity within sandy mice.



Age-dependency of D2R regulation on spine densityTo observe if D2R regulation of spine development is age-dependent, 2-12 weeks old male mice were injected with vehicle or quinpirole, the D2R agonist, and hippocampal slices were prepared 24 hours after injection for diolistic labeling. It was found that in vehicle-injected mice, there was an increase in spine density from 2-8 weeks old mice, but there was a slight decrease in spine density in 12 week old mice. In quinpirole-injected mice, spine density of 2, 8, and 12 weeks old mice did not differ significantly from the same age vehicle-injected mice. However, there was significant decrease in spine density in 3-6 weeks old quinpirole-injected mice compared with vehicle-injected ones. These results suggest that there could be age-dependency of D2R regulation on spine density (Fig. 3 a, b).

To further validate this age-dependent relationship, spine density in hippocampal slices of the mutant sandy mice was compared to wild type mice at different ages. Again it was found that spine density in CA1 region of sandy mice at 2, 8, and 12 weeks old was comparable to the same age wild type mice. However, spine density of sandy mice from 3-6 weeks old was significantly lower than same age wild type mice (Fig. 3 c, d). Therefore, the age-dependency of D2R regulation on spine density is also evident in genetic mutant mice models.

Figure 3. The age-dependency of D2R regulation on spine development and rescue of spine deficiency by antipsychotic drug treatments. Hippocampal slices of male mice at different ages prepared for diolistic labeling 24 hours after injection of vehicle or D2R agonist quinpirole (a,b); sandy and wild-type mice at different ages (c,d); sandy and wild-type mice at 3 weeks of age injected with vehicle, potent D2R blocker loxapine, and weak D2R blocker clozapine (e,f). a, c, e showing Dil-labeled dendrites wtihin CA1 region of hippocampus. b, d, f showing spine density.

Since all antipsychotic drugs are D2R blockers, it was examined whether antipsychotic drugs could rescue the spine deficiency observed in sandy mice. Typical antipsychotic drug loxapine (which blocks D2R with high affinity) and atypical antipsychotic drug clozapine (which does not have high affinity for D2R) were injected into sandy and wild-type mice between at 3 weeks of age. Hippocampal slices were prepared 24 hours after injection for diolistic labeling. It was found that loxapine injection did not affect the spine density in wild-type mice, but was able to significantly increase spine density in sandy mice to a normal range. Clozaphine injection, on the other hand, did not affect spine density in sandy and wild-type mice (Fig. 3 e, f). These results suggest that typical antipsychotic drugs having high affinity for D2R can restore spine density during adolescence, but not atypical antipsychotic drugs having low affinity for D2R. Overall, the results indicate that there is age-dependency of D2R regulation, and adolescence is the critical period for D2R regulation of spine density. Hyperactivity of D2R during this critical period can result in significant spine deficiency, and such deficiency can be rescued by D2R blockers.

D2R-hyperactivity during adolescence can impair neural connectivityAlthough adult sandy mice have spine density comparable to wild-type mice, the D2R-hyperactivity induced loss of spines during adolescence could permanently impair neuronal circuitry, considering adolescence is the critical period in which brain networks mature. To investigate this possibility, a mono-synaptic connection from layer III of the entorhinal cortex to CA1 neurons was examined in adult sandy and wild-type mice. A retrograde tracer cholera toxin subunit B (CTB) was injected into CA1 region, and expression of CTB in entorhinal cortex was examined in horizontal brain sections prepared 24 hours after injection. In wild-type mice, expression of CTB was found both in medial entorhinal cortex (MEC) as well as the lateral entorhinal cortex (LEC). However, in sandy mice there was decreased number of CTB-labeled neurons in MEC, but increased number of labeled neurons in LEC compared to wild-type mice (Fig. 4).

To investigate whether changes in number of labeled neurons in sandy mice resulted from D2R hyperactivity during adolescence, the D2R antagonist eticlopride was both injected and fed once daily to 8 weeks old sandy mice. In both cases, eticlopride treatment restored number of labeled neurons in MEC and LEC to normal. Importantly, when 13 weeks old adult sandy mice were treated with eticlopride, the abnormal MEC-to-LEC ratio of labeled neurons was still observed after treatment (Fig. 4). Taken together, these results suggest that D2R hyperactivity during adolescence can result in permanent neuronal circuitry impairment, and treatment during adolescence, but not during adulthood, can restore the normal connectivity.



Figure 4. D2Rhyperactivity during adolescence impairs entorhinal-hippocampal synaptic connection. Wild-type mice were treated with quinpirole during adolescence, and sandy mice were treated with eticlopride during adolescence or adulthood. Retrograde tracer CTB were injected into CA1 hippocampal neurons 3-4 weeks after treatment. (a) The ratio of CTB-labeled MEC-to-LEC neurons. (b) the number of CTB-labeled neurons in MEC and LEC normalized to the total number of labeled neurons in entorhinal cortex.

D2R-hyperactivity during adolescence can impair working memoryBecause the entorhinal cortex-hippocampal connection is critical for spatial working memory, therefore abnormality in this circuitry could result in deficits of spatial working memory. To test this possibility, two behavioural tests were carried out in 8 weeks old adolescent wild-type and sandy mice under different treatments. The two behavioural tests were Y maze test used to measure spontaneous spatial working memory, and the open field test used to assess general motor activity in rodents. In the Y maze test it was found that in sandy mice, there was a lower alternation score reflecting decreased spatial working memory when compared to wild-type mice (Fig. 5a). Importantly, wild-type mice treated with quinpirole showed decrease in alternation score, suggesting D2R hyperactivity during adolescence indeed impairs spatial working memory. Conversely, sandy mice treated with eticlopride showed increase in alternation score, suggesting blocking D2R hyperactivity during adolescence can prevent such deficits(Fig.

5a). Interestingly, eticlopride treatment in adult sandy mice did not improve alternation score, suggesting again that adolescence is the critical period for treatment.

In the open field test it was found that in sandy mice, there was a longer total distance traveled reflecting motor hyperactivity when compared to wild-type mice (Fig. 5b).

Figure 5. D2R hyperactivity during adolescence impairs spatial working memory. Wild-type mice were treated with quinpirole during adolescence, and sandy mice were treated with eticlopride during adolescence or adulthood. Two behavioural tests were performed. (a) Y-maze test alternation score. (b) Total distance traveled in open field test.

However, adolescent sandy mice treated with eticlopride did not show decrease in total distance traveled, and adolescent wild-type mice treated with quinpirole did not show increase in motor activity as well.

Taken together, these results indicate that D2R-hyperactivity during adolescence can impair some cognitive function such as spatial working memory but not other behaviours such as locomotion. Importantly, treatment during adolescence is critical in restoring these cognitive deficits.



testing have been proposed to identify patients at the prodromal stage, the earliest disease stage in which patients may present nonspecific mood problems and transient psychotic symptoms12. As the results of this paper suggest, extensive future researches in humans can take place to investigate treatment targeting D2R during prodromal stage in hopes of preventing spine loss and cognitive deficits.

Additionally, all of the mice used in this research were male mice. Given the observed gender difference in age of onset in schizophrenia patients, future researches can investigate whether D2R activation would lead to gender-dependent synaptic regulation in mice. The findings can further help designing or optimizing preventative treatments in schizophrenia.

REFERENCES1. Howes, Oliver D., and ShitijKapur. “The dopamine hypothesis of schizophrenia: version III—the final common pathway.” Schizophrenia bulletin 35.3 (2009): 549-562.2. Abi-Dargham, Anissa, et al. “Increased baseline occupancy of D2 receptors by dopamine in schizophrenia.” Proceedings of the National Academy of Sciences 97.14 (2000): 8104-8109.3. Ji, Yuanyuan, et al. “Role of dysbindin in dopamine receptor trafficking and cortical GABA function.” Proceedings of the National Academy of Sciences 106.46 (2009): 19593-19598.4. Bonci, Antonello, and F. Woodward Hopf. “The dopamine D2 receptor: new surprises from an old friend.” Neuron 47.3 (2005): 335-338.5. Jia, Jie-Min, et al. “Age-dependent regulation of synaptic connections by dopamine D2 receptors.” Nature neuroscience 16.11 (2013): 1627-1636.6. Green, Michael F. “Stimulating the development of drug treatments to improve cognition in schizophrenia.” Annu. Rev. Clin. Psychol. 3 (2007): 159-180.7. McGlashan, Thomas H., and Ralph E. Hoffman. “Schizophrenia as a disorder of developmentally reduced synaptic connectivity.” Archives of General Psychiatry 57.7 (2000): 637.8. Glantz, Leisa A., and David A. Lewis. “Decreased dendritic spine density on prefrontal cortical pyramidal neurons in schizophrenia.” Archives of general psychiatry 57.1 (2000): 65.9. Sham, P. C., C. J. MacLean, and K. S. Kendler. “A typological model of schizophrenia based on age at onset, sex and familial morbidity.” ActaPsychiatricaScandinavica 89.2 (1994): 135-141.10. Dell’Osso, Bernardo, and A. Carlo Altamura. “Duration of untreated psychosis and duration of untreated illness: new vistas.” CNS Spectr 15.4 (2010): 238-246.11. Malla, Ashok K., et al. “Duration of untreated psychosis is associated with orbital–frontal grey matter volume reductions in first episode psychosis.” Schizophrenia research 125.1 (2011): 13-20.12. Miller, Tandy J., et al. “Symptom assessment in schizophrenic prodromal states.” Psychiatric Quarterly 70.4 (1999): 273-287.

All figure source: Jia, Jie-Min, et al. “Age-dependent regulation of synaptic connections by dopamine D2 receptors.” Nature neuroscience 16.11 (2013): 1627-1636.

CONCLUSIONThese results have indicated an age-dependent function of D2R activity for the regulation of spine development. There appeared to be a critical period when spines are sensitive to regulation by D2R and that increased D2R activation during this time resulted in an impairment in the formation of the entorhinal-hippocampal circuit and in the working memory of adult mice. The obtained results confirmed the theory that increased D2R activation during adolescence leads to impairments in the development of dendritic spines, neural circuits and working memory. During postnatal weeks 3-6, which would be the equivalent of adolescence in mice, was the only period in which this D2R regulation of spines was observed in both the wild-type and sandy mice. This regulation effect of D2R had long-lasting effects that continued on into adulthood.

SIGNIFICANCE OF THE WORKIn this paper the researchers used multiple cellular molecular methods to experimentally confirm that D2R regulates the development of spines in an age-dependent manner, and that D2R hyperactivity during adolescence can lead to spine deficiency and neuronal network disconnection. Additionally, the researchers demonstrated using behavioural tests that the observed molecular abnormalities can be translated into actual cognitive deficits, and these deficits can be rescued if treated in time. All of the results generated in this paper help to establish a novel link between D2R and schizophrenia, and provide a possible mechanism underlying the cognitive impairment symptom of schizophrenia.

Additionally, the age-dependency of D2R regulation demonstrated in this paper provide further explanation for the epidemiology of schizophrenia, as well as possible improvement opportunity in treatment. The onset of schizophrenia typically lies within early adulthood, with males developing symptoms slightly earlier (between ages 16 to 25) than females (between ages 25 to 30)9. It is relatively rare for schizophrenia to develop below the age of 10 or above the age of 409. Therefore, the finding that D2R hyperactivity during adolescence can lead to cognitive impairment supports the observed manifestation of schizophrenia in early adulthood. Taken together, this paper contributes to a better understanding of D2R downstream molecular effects and the pathogenesis of schizophrenia.

FUTURE DIRECTIONSThe finding that treatment with antagonist of D2R including antipsychotic drugs blocking D2R during adolescence can prevent and rescue spine deficiency and working memory provides new opportunities for treatment. It has been known for the past decades that identifying and treating schizophrenia patients as early as possible can lead to improved psychopathological outcome and reduced negative impact of brain pathology10-11. Multiple descriptive psychopathology assessments as well as genetic



Received December 6th, 2013; revised December 6th, 2013; accepted December 6th, 2013.

This work was supported by The Department of Human Biology of University of Toronto. The authors thank Dr. Ju, and the students in Seminar in Neurobiology of Behaviour for technical assistance, execution, and feedback on this lab exercise.

Address correspondence to: Jia Yan Zhang Email: [email protected] (Background, Results second part, Significance, and Future Directions)Ingrid Quevedo Email: [email protected] (Abstract, Results first part, and Conclusions)

Copyright © 2013 Ingrid Quevedo, Jia Yan Zhang Human Biology Program

Creating a false contextual memory in the presence of fear conditioning by optogenetically activating a hippocampal memory engramDehi Joung1 & Luisa Garzon1


Changes in synaptic formation relying on chemical and physical properties of neurons lead to neuronal representations in brain, which are considered to be involved in experience dependent memory formation. Memory can be formed distortedly. In order to explicitly understand memory formation, advanced methods for perturbation and observation of neuronal activity are required1. One way to efficiently perturb activity of neuron is optogenetics, which can either activate or silence the neuronal activity by using light-gated ion channel designed to be responsive to specific frequency of light. However, false memory formation was not well elucidated due to absence of relevant animal model. Ramirez and colleagues used this artificial optogenetic stimulation during contextual fear conditioning in context B to create false fear memories in C-fos-tTA transgenic mice in different context (context A)2. First, neuronal representations (contextual memory trace bearing cells) in the dentate gyrus or CA 1 of the hippocampus specifically combined with context A (genuine context) were identified and labeled with channelrhodopsin2 (ChR2) in the absence of doxycycline(DOX) which inhibits ChR2-mCherry

expression2. Then, during fear conditioning, mice were exposed to context B with simultaneous mild foot shock and artificial optical stimulation to labeled memory engram cell from context A2. Significant increase in freezing behavior of DG experimental group were demonstrated in context A2. Also, in a conditioned place avoidance (CPA) paradigm experiment, locomotion trace of the transgenic mice showed that chamber preference can be manipulated by creating false memory associated with DG optical stimulation leading to increase in tendency to avoid the chamber in which the mice never experienced mild electric foot shock2. Their experimental work demonstrated that the original experience for particular context could be substituted to contextual false fear memory by using artificial optical activation2.

Key words: c-fos, m-cherry, doxycycline (Dox), contextual fear conditioning, memory engram, Channelrhodopsin-2 (ChR2), optogenetics, false memory, dentate gyrus(DG), CA1, Hippocampus, conditioned place avoidance paradigm (CPA), locomotion trace



I. BACKGROUND Memories are created by traces that are left in our brains from what we experience, and they can be brought back to the surface during recall. Scientists have conceptualized these traces as memory engrams and they represent the lasting physical and chemical changes that are induced in brain cells and their connections upon the acquisition of memory. Furthermore, previous research has shown that such engrams activate specific groups of hippocampal granule cells in particular, and that this is sufficient enough to produce and recall memories3.

Memory however can be very unreliable and can mislead us terribly under certain conditions. For instance, about 70% of victims who were found guilty primarily based on eyewitness testimony were later acquitted based on DNA evidence4. Unfortunately, because of the lack of an animal model the mechanisms underlying false memories are a phenomenon that hasn’t been studied considerably up until now.

Having identified a population of memory engram cells in the dentate gyrus (DG) and the CA1 region of the hippocampus,

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environment (context A) so that cells can become labeled with ChR2-mCherry. Then, the mice were put back on Dox and the next day they were placed on context B. while on context B, simultaneous foot shocks were delivered along with blue light to re-activate those cells that were previously labeled on context A. Finally, freezing levels were measured in context A as well as in a completely new environment (context C). The degree of overlap between the cells that encoded each particular context was also calculated.

MAJOR RESULTS The animals froze in context A at levels significantly higher than the background levels,while freezing in context C was not statistically different from background levels, showing that the recall of the false memory was specific to context A only. Moreover, context A and C eachrecruited statistically independent populations of cells in the DG, and a second re-exposure to context A recruited substantially overlapping cell populations in the DG.

The false memory interferes with genuine memory in a competitive manner.Memory engram bearing cell for context A that was labeled with ChR2-mCherry by regulating level of Dox were exposed to context B for the fear conditioning (Figures 2, 3)2. When mice were re-exposed to context B(refer to B prime) with no light stimulation, there was significant difference in freezing behavior between the experimental group received light during fear conditioning and control group that did not received optic stimulation during the conditioning indicating that there is no interaction between false and original memories (Figure 2)2. However, in re-experiencing context B condition with light stimulation, freezing level of the control group decreased, whereas the experimental group froze significantly suggesting that labeled memory engram cell from context A triggered by light stimulation leads to interference between false and genuine memories (Figure 2)2. False fear memory can be artificially recalled in different context (context D) by reactivating labeled memory engram bearing cell in DG with light since significant increase (25%) in freezing level of experimental group was observed in context D in the presence of optical stimulation compared to background freezing level of control animals (Figure 3)2. This result suggests that the downstream event of elicited false fear memory is similar to genuine fear memory in terms of the recalling process 2.

Active fear behavior is induced by false fear memoryThe memory engram cells of specific chamber of conditioned place avoidance paradigm were labeled with either ChR2-mCherry as experimental group or mCherry as control group2. The experimental mice receiving light during fear conditioning in different context showed strong preference for unlabeled chamber(chamber preference 1.5, n=8subjects; *P=0.013; **P=0.008, unpaired student’s test) due to reactivation of ChR2-

the study carried out by Ramirez and colleagues aimed to incept or implant a false memory in a mouse brain with the use of optogenetics. This technique uses opsin genes (in this case Channelrhodopsin) to optically control action potentials patterns in very precise neuronal populations by injecting a virus in the target region and then activating the cell bodies with blue light1. The researchers saw that introducing the mice to a new environment could generate a contextual memory if they were allowed to explore the environment for a specific amount of time and then Channelrhodopsin gene expression could be induced only in those brain cells that carry the engram of that given memory. Finally, fear memory could be triggered by delivering foot shocks to test whether the mice now falsely associate these two things. Therefore, they hypothesized that by artificially activating a previously formed contextual memory engram while simultaneously delivering foot shocks can result in the creation of false fear memory for the context in which foot shocks were never delivered2.

II. PROPOSED RESEARCHMATERIALS AND METHODS Mice were bilaterally implanted with optical fibers in the DG and injected with an adeno-associated virus (AAV) virus to encode for regulatory DNA elements from the c-Fosgene and a red florescent protein known as mCherry. The gene that encodes for Channelrhodopsin-2 (ChR2) was also introduced along with the virus into the DG and CA1 regions.

When mice were raised in the presence of the antibiotic Doxycline (Dox) as part of their diet, the expression of ChR2-mCherry was null. However, allowing the mice to explore a novel environment after removing Dox from their diet induced the expression of these two genes. Therefore, Dox was used to regulate the expression of ChR2-mCherry.

Fig 1. Basic experimental procedure showing the steps in which mice where introduced to contexts A, B and c. Green shading represents presence of Dox in the diet, prime indicates the second exposure to a given context and the lighting symbol is the foot shocks that were administered in context B.Taken from Ramirez et al., 2013.

The basic experimental schemes (Figure 1) consisted on removing Dox from the animals’ diet and allowing them to explore a novel



mCherry memory engram cells in DG (Figure 4)2. This result indicates that false fear memory can force mice to avoid the labeled chamber where mice did not receive any foot shock by inducing natural fear response2.

Fig. 2. Freezing level of experimental (Blue line; light was delivered with foot shock during fear conditioning at context B) and control mice groups ( Red line; No light was delivered with foot shock during fear conditioning at context B) following application of re-exposing context B in addition to basic behavior protocol was measured responding to optical stimuli. Freezing level of control group decreased (approx20%), whereas freezing behavior of experimental group increased (approx20%) in the presence of light compared to light-off epochs in the context B prime. Taken from Ramirez et al., 2013.

Fig. 3. Freezing level of experimental (Blue line; light was delivered with foot shock during fear conditioning at context B) and control mice groups (Red line; No light was delivered with foot shock during fear conditioning at context B) following application of exposing distinct novel context D after re-exposing context B in addition to basic behavior protocol was measured responding to optical stimuli. Freezing level of control group remained at background freezing level, whereas freezing behavior of experimental group increased (approx20%) in the presence of light compared to light-off epochs in the context D. Taken from Ramirez et al., 2013.

Fig. 4. Chamber preference of the ChR2-mCherry experimental group (blue bar) and the mCherry control group (grey bar) was measured based on conditioned place avoidance paradigm. The experimental group tended to show strong preference for unlabeled chamber compared (approx1.4) to control group (approx0.8). Red dash line represents no preference. Taken from Ramirez et al., 2013.

DISCUSSIONRecalling process of memories can be easily and frequently distorted by fusing misinterpreted external information with previously formed memories2. Numerous activity of hippocampal neurons were identified in recalling process of human regardless of original and falsely formed memories5although restricted imaging techniques hinder understanding for hippocampal neuronal circuits involved in creating false memories in human2. In comparison to previous study, which used same experimental scheme but was unable to show increase in freezing level resulting from reactivating ontogenetically created false fear memories because of the wide range of target area, Ramirez and colleagues successfully showed increased freezing behaviour in mice by focusing small neuronal representation in DG (more restrict spatial and temporal manner) associated with light induced false memories during fear conditioning in different context2. Increase in freezing level and active fear response of experimental group froze were observed in context where the mild electric foot shock was never delivered (Figures 3, 4)2. The false and original memories were found to be dynamically interfered with each other at different stage including different context (context D) and same context without fear conditioning (context B prime) depending on presence of light that triggers reactivation of memory engram cell from context A (fig 2, 3)2. These results distribute establishing animal model to investigate false and genuine memories at the different level of memory trace to understand memory formation in human6. Based on these new finding of ontogenetically generated contextual false fear memory in transgenic mice2, internal recalling process of previously stored memory is speculated to be distorted to form some false memories through incorporating external information in human. In conclusion, this proposed animal model of creating false memory by interfering internally formed memory engram with contextual fear conditioning can confer capacity on



underlying CA1 memory trace is needed to be elucidated to understand memory formation. Also, the proposed animal model in this paper still remains to be tested in extended manner because previous studies were failed to address and reveal the false memory formation despite applying similar basic experimental scheme2. Moreover, it is also important to solve ethical issue by reappraise the memory trial value since the research showed that memory recalling process can be reconstructed by ontogenetically manipulating dynamic interaction between the false and original memories at different stages7.

REFERENCES1. Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M. &Deisseroth, K. Optogenetics in neural systems. Neuron71, 9–34 (2011).2. Ramirez, S. et al. Creating a False Memory in the Hippocampus. Science341, 387–391 (2013).3. Liu X., Ramirez S., Pang P.T., Puryear C.B., Govindarajan A., Deisseroth K., &Tonegawa S.. Optogeneticstimulation of a hippocampal engram activates fear memory recall. Nature484 (7394): 381–385 (2012).4. Innocence Project - Fact Sheets. Retrieved from http://www.innocenceproject.org/Content/DNA_Exonerations_Nationwide.php5. Cabeza, R., Rao, S. M., Wagner, A. D., Mayer, A. R. &Schacter, D. L. Can medial temporal lobe regions distinguish true from false? An event-related functional MRI study of veridical and illusory recognition memory. PNAS98, 4805–4810 (2001).6. McTighe, S. M., Cowell, R. A., Winters, B. D., Bussey, T. J. &Saksida, L. M. Paradoxical False Memory for Objects After Brain Damage. Science330, 1408–1410 (2010).7. Lacy, J. W. & Stark, C. E. L. The neuroscience of memory: implications for the courtroom. Nat Rev Neurosci14, 649–658 (2013).

Copyright © 2013 Dehi Joung & Luisa Garzon, Human Biology Program

Received December 06, 2013

This work was supported by the Human Biology Neuroscience Program at the University of Toronto. The authors thank Dr. Bill Ju, and the students in then HMB420 – Seminar in Neurobiology of Behavior class for their feedback and support and discussion on the presentation.

us to understand relevant neuronal circuit of memory formation for human cognitive neuroscience.

SIGNIFICANCE OF THE WORKBesides what is happening in the outside world, the human mind is capable of being imaginative to an immeasurable degree. On a daily basis we are processing information that comes from our perception, and we also maintain a constant amount of information that includes our thoughts and this all creates activity. Therefore, just like the mouse model used in this study, it is very likely that human beings can create false memories when exposed to a negative stimulus while unconsciously or consciously activating an unrelated memory, and this would then lead individuals to falsely associate the two. Basically, the person would erroneously associated what is going on in their mind rather than what is actually happening to them. Additionally, the stimulus may not necessarily have to be a negative one like the fear memory that was used in the study, but there is the possibility that a positive event with high valence can get falsely associated as well.

In relation to the flawed eyewitness testimonies it is also important to note that once false memories are formed they feel almost as real as any other memories. In fact, this study shows that the false memories created in the mice and the genuine memories has very similar and almost identical underlying brain mechanisms in the dentate gyrus. Therefore, it must become difficult for the individual to be able to distinguish between the two types of memory and this is a possible explanation as to why humans may believe that something really happened when it actually did not. Also, the work presented in this paper hints that there are some conditions that influence how strong memories either false or genuine can be formed and now having a model to work on can open new possibilities for further research to be carried out.

FUTURE DIRECTIONSSince false memory formed by reactivation of the memory engram bearing cell in CA1 during fear conditioning in distinct context did not increase freezing level of mice despite huge overlapped neuronal representation in CA1 involved in different contexts compared to false memory in DG2, the relevant circuit





The Circuitry Underlying the Revolutionary Use of Ketamine to Treat DepressionAilya Jessa1,2, Stephany Francisco1,2 & Michelle Hayano1,2

1Human Biology Department, University of Toronto, Toronto, ON. 2Psychology Department, University of Toronto, Toronto, ON.

Depression is one of the most common psychiatric illnesses affecting nearly 350 million people worldwide1. Traditionally, moderate to severe levels of depression is treated with common antidepressant (i.e. serotonin reuptake inhibitors) however, these drugs are known to be slow-acting and in some cases unsuccessful. While considerable evidence has supported the hypothesis regarding serotonergic influence on MDD, research on the antidepressant effects of ketamine suggests an alternative pathway, that is the glutamatergic system, to be involved in MDD as well. Ketamine, widely used as an anesthetic, has been found to alleviate depressive symptoms in major depressive disorder (MDD) within approximately 24 hours after administration2,3. Due to its addictive nature, ketamine is not commonly used because of the stigma surrounding its use, which has also prevented its testing

in clinical trials. Scheidegger and colleagues examine the neural networks that ketamine acts on in order to elucidate the neural changes underlying ketamine action3. Their study supports the use of ketamine to treat depression and addresses the potential it has to revolutionize the treatment of psychiatric disorders if successful in clinical trials. Understanding the changes in brain networks following ketamine application will help us understand the drug action and bring us one step closer to using this powerful drug to change the lives of many individuals suffering from depression.

Key words: Depression; Ketamine; Major Depressive Disorder (MDD); Functional Mode Network (FMN); Default Mode Network (DMN)

I. BACKGROUNDDepression is one of the most common mental illnesses, but the neural abnormalities underlying it still remain unclear. It has been estimated that antidepressant drugs only work in 50% of people1, and. Ketamine which is a hallucinogen commonly used as an anesthetic in medical practices has been show to lead to dramatic improvements in depressive symptoms within 24 hours when given at sub-anasthetic doses. The rapid improvements seen after Ketamine application have been known for many years but it has not been clear how it effects the brain. Scheidegger and colleagues examine the changes in connectivity in neural networks to determine what ketamine is doing and how it is leading to such dramatic improvements in mood in treatment-resistant patients3. This study is important for two reasons, firstly it helps elucidate the brain abnormalities that may underlie depression and it also paves the way for clinical trials for ketamine as a treatment for depression. If ketamine is successful in clinical trials, it could have major implications for treatment of depression especially for patients with suicidal tendencies because it can intervene before depression leads to serious symptoms like suicide.

A number of neuronal circuits have previously been identified to play a role in depression. The limbic-cortico-striato-pallido-thalamic circuit has been identified to play a role in mood regulation and is thought to have abnormal connectivity in depressed patients. Past research has also shown that in the

depressed brain there are low levels of glutamatergic signalling. This is relevant to the current study because ketamine is thought to stimulate glutamatergic signalling in certain areas of the brain. The Dorsal Nexus (DN) is an area of the brain that is involved with with integration of brain functions particularly with regards to memory, cognitive control and many affective states. The DN has been shown to have over connectivity in the brains of individuals with depression and may be involved in action of ketamine2.

Ketamine is an NMDA receptor antagonist3. A study by Zarate et al. conducted in 20113 found that antagonizing NMDA receptors leads to an increase in activity of AMPA receptors which may be involved in the changes in glutamatergic activity and improvement in depressive symptoms. Most antidepressants target the serotonergic systems, so the success of ketamine suggests that the glutaminergic system may also be involved. Unlike interventions that act on the serotonergic system, ketamine leads to an almost immediate reduction in symptoms4. This is great for interventions. However, like other anti-depression interventions, ketamine is not effective in all patients. A study done in 2012 found that carriers of the SNP Val66Met do not respond well to ketamine5.

II. MATERIALS AND METHODSIn the reviewed study, 19 healthy participants were tested. Initially a baseline rsfMRI scan was taken. Then S-isomer ketamine or placebo (saline) was administered via IV. The s-form of ketamine

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suggest that both ketamine and placebo did not affect subjective states of mood. However these tests were in healthy participants, who did not have depression.

rsfMRI on the four seed regions There were a number of changes seen in the DMN. When ketamine was administered, there was a focal decrease in connectivity between the left and right posterior cingulate sulcus (PCC) and bilateral dorsomedial prefrontal cortex (DMPFC). Changes were also seen in connectivity between the posterior anterior cingulate cortex (PACC) and medial prefrontal cortex (MPFC). In the affective network, ketamine subjects had a reduction in functional connectivity between the subgenual anterior cingulate cortex (sgACC) and right DMPFC, however these changes were seen in the placebo condition. Ketamine did not affect connectivity in dorsolateral prefrontal cortex (DLPFC), and no change in connectivity between amygdala and prefrontal cortical area. The results did not suggest any changes in focal connectivity in the cognitive control network (CCN) or the amygdala in the ketamine treatment condition.

In terms of changes in the overall dorsal nexus connectivity, in subjects in the ketamine condition, there was significant decrease in functional connectivity exclusively between DN and PCC. It was suggested by the researchers that there was an overall reduction in hyperconnectivity of DN. It was suggested that 24 hours was required to see effects because ketamine is leading to adaptive changes in neuroglialglutamatergic all over the brain and changing information processing in specific neural circuits. Overall the results suggest that ketamine reconfigures resting state functional connectivity.

Figure 2. Functional connectivity between the PCC seed regions and the DMPFC have decreased during ketamine (red) administration, while placebo (blue) condition show no significant reduction. The bar graph illustrates z values demonstrating changes in functional connectivity 24 hours after administration.

used is 3-4 times more potent at certain receptions, so the dosage was reduced by 50%. After 24hrs, a follow up fMRI scan was taken. Past studies have shown that the antidepressant effect is most prominent after one day6. In order to avoid carry-over effects, they waited 10-14 days before doing a second baseline measurement. The group that was initially given ketamine were then given IV for the second round, and the placebo group was given ketamine, on the second round. The brain areas analyzed were 4 “seed regions”, (1) the cognitive control network (CCN) found in the left and right dorsolateral prefrontal cortex (2) the default mode network (DMN) located in the left and right posterior cingulate cortex (3) the affective network (AN) found in the subgenual anterior cingulate cortex and (4) the amygdala. Three of the seed regions, CCN, DMN and AN, are known to be connected to the dorsal nexus.

Further, psychometric tests were administered in order to test for mood and depressive symptoms.

Figure 1. The following diagram illustrates the four seed regions that were analyzed. Three of the seed regions (CCN, DMN, and AN) are each connected to the dorsal nexus (DN).

RESULTSPsychometric testsSubjects that were given ketamine had a significant increase in psychotomimetic symptoms compared to subjects given placebo. However, no correlation was found linking these psychotomimetic symptoms to a change in functional connectivity in affective network (AN) and default mode network (DMN) regions relative to the dorsal nexus (DN). Using the SHAPS scale, the results



Figure 3. Significant reduction in functional connectivity between the PCC seed regions and the PACC/MPFC for ketamine (red) condition, but no change in reduction for the placebo (blue) condition, after 24 hours.

DISCUSSIONThe mechanism of action of ketamine is thought to be through reduction of hyperconnectivity in the brain particularly in the dorsal nexus and default mode network. However this study was conducted in healthy participants, so it is unclear if the same neural changes would be seen in the brain of depressed patients. In order to elucidate the neural changes of ketamine in the depressed brain a similar study is needed using participants who have been diagnosed with depression.

It is well established that ketamine has addictive properties which is why many clinicians may be aversive to its use. In order to understand the true efficacy of ketamine the pros and cons must be factored in or a method to avoid the addictive properties must be developed. The long-term effects of ketamine are also unclear. Although it may have effects as soon as within 24 hours it is unclear how long these symptoms last. If ketamine is reducing over-connectivity in the brain so quickly, it is also important to consider whether ketamine has the potential to reduce these connections to an extremely underconnected level. Also, it is important to consider if patients become resistant to the drug over time and if depressive symptoms return regardless of dosage. Although past studies have highlighted the benefits of ketamine it is necessary to understand the long-term effects of use. Further research should be done to determine which individuals respond to ketamine. Recent studies have indicated that depressed individuals with a Val66Met single-nucleotide polymorphism do not show any response to ketamine administration5. This particular SNP is located on the gene which regulates expression of brain-derived neurotrophic factor (BDNF)5.

SIGNIFICANCE OF THE WORKGiven the high prevalence of depression, these findings are significant, particularly if ketamine is successful, as it can impact the lives of many. The speed at which it can cause behavioral changes is astounding and can be useful especially for patients who may be at high risk of suicide. Understanding the changes in neural networks also gives us insight into the cortical and subcortical areas that are overactive and underactive during depression.

Ketamine operates through a different mechanism of action compared to traditional antidepressants, and generates the potential of creating a new class of pharmaceutical drugs targeting the glutamatergic system in depression. Currently, there are several alternative drugs that replicate effects of ketamine. For instance, Lanicemine7, effectively reduced depressive symptoms in patients without the typical side effects associated with ketamine use9. The most promising ketamine variant has been GLYX-138. It is a NMDAR partial agonist that rapidly alleviates depressive symptoms for up to two weeks. However, unlike ketamine GLYX-13 is found to have minimal side effects and did not impair consciousness8.

FUTURE DIRECTIONSRecent clinical trials have shown promising results for the use of ketamine as an antidepressant. For instance, Murrough et al. found that Ketamine was effective in reducing symptoms in treatment-resistant subjects after 24 hours9. This suggests that modulating NMDA receptors is an effective mechanism for reducing depressive symptoms especially in severe cases.

Despite the fact-acting benefits of ketamine, the addictive properties of ketamine creates the potential for abuse. Finding a way to make Ketamine less addictive will allow its use as an antidepressant. Instead of administering ketamine via injection, manufacturing a tamper-resistant tablet could prevent abuse. The following addiction prevention strategies have been utilized with Oxycontin and if these strategies can be applied to ketamine it would help control the addictive aspects of the drug.

And they all lived happily after – by becoming Neuroscientists

REFERENCES1.http://www.who.int/mediacentre/factsheets/fs369/en/index.html2. Sheline, Y.I., Price, J. L., Yan, Z., &Mintun, M. A. Resting-state functional MRI in depression unmasks increased connectivity between networks via the dorsal nexus. PNAS, 107(24), 11020-11025, (2010).3. Zarate, C. et al. A randomized trial of N-methy-D-aspartate in treatment-resistant major depression.Arch Gen Psychiatry, 63(8): 856-864 (2006).



Received December, 6, 2013; Reviewed immediately; Accepted there after

This work was supported by The Human Biology Program (HMB), The Neuroscience Faculty (NF). The authors thank Dr. Ju, and the students in HMB420 for their general awesomeness.

Address correspondence to: Ailya Jessa, Stephany Francisco, Michelle HayanoEmail: [email protected]@[email protected]

Copyright © 2013 Jessa, Francisco, Hayano, Human Biology Program

4. Scheidegger, M. et al. Ketamine Decreases Resting State Functional Network Connectivity in Healthy Subjects: Implications for Antidepressant Drug Action. PloS ONE, 7(9) (2012).5. Laje, G., Lally, N., Mathews, D., Brutsche, N., Chemerinski, A., Akula, N., Kelmendi B; Simen A; McMahon FJ; Sanacora G; Zarate C Jr: Brain-derived neurotrophic factor Val66Met polymorphism and antidepressant efficacy of ketamine in depressed patients. Biol Psychiatry. (2012)6. Sinner, B., & Graf, B. M. (2008) Ketamine. Hand ExpPharmacol: 313-333. doi:10.1007/978-3-540-74806-9_157. Sanacora G, Smith MA, Pathak S, Su H, Boeijinga P H, McCarthy D Jand Quirk MC. Lanicemine: a low-trapping NMDA channel blocker produces sustained antidepressant efficacy with minimal psychotomimetic adverse effectsMolecular Psychiatry 2013 8. Moskal J.R. Burgdorf J. Kroes, R.A. Brudzynski S. M. Panksepp J. GLYX-13, a NMDA Receptor Glycine-Site Functional Partial Agonist, Induces Antidepressant-Like Effects Without Ketamine-Like Side Effects. Neuropsychopharmacology, 38, 729–742.(2013)9. Murrough JW; Iosifescu DV; Chang LC; Al Jurdi RK; Green CM; Perez AM; Iqbal S; Pillemer S; Foulkes A; Shah A; Charney DS; Mathew SJ: Antidepressant efficacy of ketamine in treatment-resistant major depression: a two-site randomized controlled trial. Am J Psychiatry 2013



Free and nanoencapsulated curcumin suppress β-amyloid-induced cognitive impairments in rats: Involvement of BDNF and Akt/GSK-3β signaling pathway

Waldo Lefever1, Ed (Zhuoliang) Li1 and Benjamin Ong1


Curcumin has been implicated to be a therapeutic agent against Alzheimer’s disease1,2,3. The prevalence of Alzheimer’s disease is significantly lower in populations that consume curcumin regularly as curry spice in India. Alzhemer’s disease is histologically characterized by extracellular amyloid beta protein plaques, intracellular neurofibrillary tangles of phosphorylated tau protein and astrocyte reactivity2. Curcumin is natural polyphenol associated with anti-oxidative, ant-inflammatory and anti-amyloidogenic properties3. Thapa et al. demonstrates that detrimental interactions between amyloid beta protein and cell membranes of neuroblastoma cultures is attenuated by curcumin administration in a dose-dependent manner1. Similar cell culture studies illustrate the effects of curcumin on other molecular trends associated with Alzheimer’s disease. While the therapeutic effects of curcumin have been established in Alzheimer’s disease in vitro models, it is unknown whether its effects are replicable with in vivo models. This review focuses on describing the behavioural

and molecular effects of curcumin administration in Alzheimer’s disease mouse models. Clinical studies focusing on the effects of curcumin administration in Alzheimer’s disease patients illustrate incongruent results4-5. Curcumin’s therapeutic effects may be compromised by its low water solubility and half-life leading to ineffective access to the brain3. Furthermore, curcumin must diffuse through the blood-brain-barrier, a challenge for various potential drug treatments. It is expected that the benefits of curcumin could be amplified if the drug’s molecular disadvantages are resolved. This review explores the hypothesis by using nanotechnology as a medium to administrate curcumin into the brain. A curcumin loaded lipid-core nanocapsule was developed in order to contrast its therapeutic effects to free curcumin administration7.

Key words: Curcumin, nanocapsules, Aβ1-42, GSK-3β, Akt, BDNF, Neuroinflamation, synapse, Memory

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I. BACKGROUNDAlzheimer’s disease is a neurodegenerative disorder is defined by a progressive loss in cognitive function and memory3. The diagnosis with highest specificity for the disease is post-mortem examination. The disease is histologically characterized by a loss in neurons and synaptic integrity in the cortex, and subcortical regions1,2,6. Other symptoms include the formation of extracellular Aβ plaques, intracellular neurofibrillary tangles, astrocyte reactivity, and inflammatory responses3.

Curcumin is the active component of turmeric, a product that has been used as a spice and in Ayuverdic medicine for centuries in India2. The prevalence of the Alzheimer’s disease is among significantly lower in individuals who consume turmeric regularly2. This may be due to its anti-inflammatory, anti-oxidative and anti-amyloidogenic properties3. Calcium influx is able to trigger cell death in neuroblastula cultures interacting with amyloid beta protein plaques. Curcumin is able to diminish these effects in a drug-dependent fashion1. Curcumin is also known to up-regulate BAG2, a protein that has been implicated to have crucial influence in the clearance of tau neurofibrillary tangles2. Curcumin is a polyphenol with low water solubility and a short half-life, leading to decreased bioavailability in the plasma and tissues3. Furthermore, compound is not able to diffuse through the BBB and disperse throughout the brain efficiently. Therefore, curcumin is thought to show clinically significant therapeutic effects in individuals that consume high amounts of curcumin regularly.

Double-blind placebo-controlled randomized clinical studies suggest that oral administration of curcumin as large as 4g/day were not able to produce significant therapeutic effects in Alzheimer’s disease patients5. Furthermore, the biomarkers for plasma proteins such as Aβ1-40 and tau show no significant change in patients under curcumin treatment4-5.

Intra-peritoneal injections of curcumin is another possible method of delivery. Free curcumin injections require curcumin to be suspended in a solution containing carboxymethylcellulose due to its nonpolar properties7. The development of lipid-core nanocapsules may reduce the minimal curcumin dose required to induce clinically significant effects in Alzheimer’s disease patients.

II. MATERIALS AND METHODSGeneral preparation: Curcumin loaded lipid core nanocapsules (0.5mg curcumin/ml) and unloaded lipid core nanocapsules were prepared using the methods outlined by Jager and colleagues7. The characteristics of the nanocapsules were analyzed using laser diffraction and curcumin was analyzed by high performance liquid chromatography (HPLC) prior to use. Aβ1-42 were dissolved in a 0.1% ammonium hydroxide solution at a concentration of 1mg/

ml and stored at -20oC. Portions were withdrawn and allowed to aggregate for 72 hours at 37oC prior to infusion.

Animals: Male Wistar rats (300-350g) were housed in cages in a 22±1oC room with a 12 hour light-dark cycle. Food and water were provided ad libitum.

Surgery: Animals were anesthetized. Using a Hamilton syringe, 5ul of Aβ1-42 or the vehicle were intracerebrovetricularly (ICV) infused into each side of the lateral ventricles. Animals were allowed to recover for 4 days.

Drug administration: Control animals were randomly split into 3 groups while Aβ1-42 infused animals were split into 5 groups. Control animals were either left untreated or intraperitoneally (IP) injected with free curcumin (50mg/kg/day) in 0.5% carboxymethylcellulose (CMC) or nanoencapsulated curcumin (2.5mg/kg/day) daily for 10 consecutive days. Aβ1-42 infused animals were either left untreated, treated with vehicles (0.5% CMC or unloaded nanocapsules B-LNC), or treated with either drug treatment at the same dosage and schedule as to the controls given the same treatment.

Behavioral analysis: Behavioral analysis was conducted immediately after drug administration. Spontaneous alternation test (Y-maze) was employed for examining the spatial working memory and 3-day novel object recognition (NOR) test were utilized to test short-term and long-term recognition memory.

Biochemical assays: After sacrificing the animals, blood was immediately collected via a cardiac puncture and the brains are extracted. Hippocampi were immediately dissected on dry ice. Collected tissue was homogenized and used for a series of assays. Western blot analysis was used to measure the protein levels of the enzymes involved in neuroprotective pathways (phosphorylated Akt, Akt, BDNF), enzymes involved in the AD pathogenesis (phosphorylated GSK-3β, GSK-3β), marker for glial cell activation (glial fibrillary associated protein GFAP), presynaptic marker for synaptic integrity (synaptophysin), tau proteins (tau, phosphorylated tau), and β-actin. For measuring microglial activation (Isolectin B4 reactivity), hippocampal homogenates were treated with Isolectin B4 peroxidase conjugate prior to washing in the Western blot assay. Hippocampal inflammatory cytokines TNF- α (tumor necrosis factor-α) and IL-1β (interleukin-1β) levels were assessed using the immunosorbent assay. The activity of liver enzymes extracted from the blood plasma is analyzed using the LabMax240.

III. RESULTSThe laser diffraction analysis showed that the concentration of the curcumin in nanocapsules was 0.49mg/ml (98% drug recovery), suggesting the feasibility of creating nanoencapsulated curcumin.



Post-mortem analysis showed that Aβ infusion induced a series of AD pathology including synpatotoxicity (decreased levels of synaptophysin, a presynaptic marker of synaptic integrity), tau hyperphosphorylation (increased ratio of phosphorylated tau to tau proteins), and increased neuroinflammation (increased GFAP and Isolection-B4, markers of glial cell activation and microglial activation respectively; increased inflammatory cytokines TNF-α and IL-1β levels) in the hippocampus. All Aβ induced changes that were significantly different from the control levels and almost all of them were completely attenuated by both curcumin and nanoencapsulated curcumin treatments. Only the inflammatory cytokine levels were partially attenuated by both treatments.

In the search of possible mechanisms, the authors analyzed the ratios of the phosphorylated form of the signaling molecules Akt, and GSK-3β to its unphosphorylated form and the level of BDNF in the hippocampus. Phosphorylated Akt is the active form of Akt while the phosphorylation of GSK-3β results in its inactivation. Aβ infusion significantly decreased the post-mortem ratios of pAkt:Akt (decreased Akt activity), ratios of pGSK-3β: GSK-3β (increased GSK-3β activity), and the BDNF level. Both curcumin and nanoencapsulated curcumin treatment completely attenuated these changes. Based on these results, the authors hypothesized that both treatments treats Aβ induced pathologies by inhibiting (via phosphorylation) a key enzyme involved in Aβ induced pathogenesis (GSK-3β is involved in the production of Aβ and phosphorylation of tau proteins) and reversing the Aβ induced inhibition of Akt/BDNF mediated neuroprotective pathways.

IV. DISCUSSION AND FUTURE DIRECTIONSThe experiments by Hoppe and colleagues have demonstrated a substantial increase in the efficacy of curcumin treatment when it was loaded into nanocapsules. Across the various tests, the nanoencapsulated curcumin consistently outperformed or matched the improvements observed with free curcumin treatment, despite being given at a dose 20 times smaller.

Although data collected ranging from behavioural testing to inflammatory markers showed strong evidence that the nanoencapsulation process had increased the potency of curcumin, the basis of this improvement was left untested. Previous pharmakinetic experiments have found that nanoparticles improve drug delivery across the blood brain barrier8,9, but the authors in the current study did not verify this as being responsible for the observed effects. To confirm that nanoencapsulation increased curcumin drug delivery to the brain, liquid chromatography with tandem mass spectrometry could be employed10. Additionally, the effects of nanoencapsulation on curcumin half-life could be evaluated by measuring levels of its metabolite, tetrahydrocurcumin. To further comment on the authors’ methods, when measurements of synaptic integrity were made, it would have been preferable to use a more direct assay

The bulk of the experiments by Hoppe and colleagues (2013) examined the effects of curcumin and nanoencapsulated curcumin on Alzheimer’s disease (AD) using an Aβ infused rat model of AD. In terms of the behavior, rats infused with a single treatment of Aβ1-42 exhibited significant deficits in spatial memory and short-term/long-term recognition memory as measured by spontaneous alternation test and novel objection test (Figure 1) respectively. Both curcumin (50mg/kg/day IP) and nanoencapsulated curcumin (2.5mg/kg/day IP), but not the vehicles or either treatment, attenuated the Aβ induced deficits. Furthermore, both treatments of free and nanoencapsulated curcumin did not have any significant effects on the performance of control animals. Additionally, no significant changes were observed in the long-term body weight, serum liver enzyme levels, or mortality rate in controls animals, further signifying the safety of both treatments.

Figure 1. Adapted from Hoppe et al. (2013). Effect of 10-day consecutive daily IP injection of free curcumin (Cur) and nanoencapsulated curcumin (Cur-LNC) Aβ1-42 infused Wistar rats as compared to controls and Aβ infused animals treated with vehicles (CMC, B-LNC). The effect of the curcumin treatments was also examined in control animals. (A) Day 1: training session (B) Day 2: test for short-term recognition memory (C) Day 3: test for long-term recognition memory. One-way ANOVA analysis used with Newman-Keuls post hoc test. *Significant difference between novel object exploration and old object exploration (p<0.05) ** Significant difference (p<0.01).



such as spine density. Instead, the authors took a western blot of synaptophysin, a presynaptic marker, which resulted in synaptic health being confounded with neuronal loss.

It is worth noting the significant differences between the rat model and the human disease, and the implications this has with respect to potential for treatment. The model under study used non-transgenic rats, each given a single infusion of Aβ1-42. This is in contrast to the human condition, where Aβ1-42 is continually produced. Additionally, even though the infused rats exhibited hyperphosphorylated tau, they did not develop neurofibrillary tangles, which are a characteristic feature of Alzheimer’s Disease. Curcumin treatment started 4 days after the Aβ1-42 infusion. This is compared to the long 11-17 year preclinical stage11 in humans, before a probable diagnosis is given and treatment options are explored. With this in mind, it is expected that the treatment would be more beneficial for the rats than humans because there was less time for the disease to progress.

The PI3K/Akt and GSK-3β pathways have been implicated in a wide range of processes relevant to neurodegeneration including neuronal survival12, plasticity12, tau hyperphosphorylation13, microglial-inflammation13, inflammatory cytokines13, and synaptic and neuronal loss13. Based on the altered phosphorylation of Akt and GSK-3β observed in their experiments, the authors suggest that curcumin counteracts the Aβ1-42 effects through these pathways. Future studies could investigate specific proteins in these extensive pathways to further elucidate the mechanisms underlying curcumin’s effects. This could offer insight into potential therapeutic targets for neurodegenerative diseases.

In past attempts to translate curcumin treatment to human Alzheimer’s patients, clinical studies found oral administration of curcumin to be largely ineffective. It was suggested that one major barrier to seeing symptomatic improvements was limited bioavailability5,6. In this regard, nanoencapsulation could improve absorption and plasma concentrations of curcumin, as studies of other drugs have demonstrated enhanced oral bioavailability following nanoencapsulation14,15. This process could also overcome the issue of delivery across the blood-brain barrier9.

Another critical factor influencing the outcome of treatment is timing. The nature of Alzheimer’s disease as a progressive neurodegenerative disease means that the condition worsens as damage accumulates over time. Offering treatments earlier could more strongly attenuate cognitive and memory decline, and improve the quality of life in patients. The limitations of currently available diagnostic procedures delays treatment, and this has likely contributed to the lack of success in clinical trials of most potential therapies. Due to the safety and non-invasiveness of oral administration of curcumin, it is a viable candidate for prophylactic treatment. Taking a preventative approach to

treatment and starting well before any symptoms develop, this strategy could circumvent the shortcomings of diagnostics. Long-term studies could be conducted, having participants take pills containing nanoencapsulated curcumin to evaluate the efficacy of this approach.

The use of new and emerging technologies to address drug delivery issues is a field with much room for growth. The use of lipid-core nanocapsules has brought new hope for curcumin treatment in humans. From a broader perspective, nanoparticles could be used to non-invasively raise the bioavailability of any drug we desire to target the brain. These new drug delivery systems promise to overcome the blood-brain barrier, and open new frontiers in neuroscience.

REFERENCES1. Thapa, A. et al. Langmuir. 29, 11713-23 (2013).2. Patil, SP. Neurosci Lett. 554,121-5 (2013).3. Hoppe, J. et al. Neurobiology of Learning and Memory. 106, 134-144 (2013).4. Hishikawa, N. et al. Ayu.33, 499-504 (2012).5. Ringman, JM. et al. Alzheimers Res Ther. 4, 43 (2012).6. Baum, L. et al. J Clin Psychopharmacol. 28, 110-113 (2008).7. Jäger, E. et al. Journal of Biomedical Nanotechnology, 5, 130-140 (2009). 8. Mohanty, C., & Sahoo, S. K. Biomaterials, 25, 6597–6611 (2010).9. Tsai, Y. M., Chien, C. F., Lin, L. C., & Tsai, T. H. International journal of pharmaceutics, 1, 331–338 (2011).10. Ray, B., Bisht, S., Maitra, A., Maitra, A., & Lahiri, D. K. Journal of Alzheimer’s disease: JAD, 1, 61–77 (2011).11. Strobel, G., Zakaib, G.D., & Rogers, M.B. (2013) Coverage of AD/PD 2013. Retrieved from http://www.alzforum.org/new/pdf/ADPD2013Series.pdf12. Kitagishi, Y., Kobayashi, M., Kikuta, K., & Matsuda, S.Depression research and treatment, ---in press(2012).13. Hooper, C., Killick, R., & Lovestone, S. Journal of neurochemistry, 6, 1433–1439 (2007).14. Attili-Qadri, S. et al. Proceedings of the National Academy of Sciences of the United States of America, 43, 17498–17503 (2013).15. Zhou, H. et al. Journal of nanoscience and nanotechnology, 1, 706–710 (2013).

Copyright © 2013 Waldo Lefever, Ed (Zhuoliang) Li, Benjamin Ong, Human Biology Program



Neuroscience MattersRole of Diazepam binding inhibitor on GABAA Receptors:Treating epilepsy in mice modelsKamalpreet Mann1 & Victoria Marshe1


G protein-coupled receptors can be regulated by allosteric modulation, either in a positive or negative direction1. The inhibitory neurotransmitter gamma-amino butyric acid (GABA) has type A receptors which are allosterically modulated by benzodiazepines (BZs) which to its benzodiazepine-binding site1. BZs can either increase the duration and strength of inhibitory signals by prolonging the current through GABAA receptors, which is called positive allosteric modulation (PAMs) or it can be negative allosteric modulation (NAMs)1. With the discovery of benzodiazepine-binding sites on GABAARs, researchers speculated that there may be an endogenous molecule which mimics the effects of benzodiazepines. The subsequent discovery of diazepam-binding protein (DBI) hailed it the endozepine,

an endogenous benzodiazepine, which mimics the effects of exogenous benzodiazepines such as diazepam (Valium)1. Researchers elucidated the site of benzodiazepine binding as the reticular thalamic nucleus (nRT) of the thalamus which contains α3-subunits and is implicated at the root of absence seizures1. Using both α3 subunit-mutant mice (α3[H126]) and nm1054 mutant mice, which lacked a function DBI gene, researchers showed that a function α3 subunit and sufficient DBI-gene product are crucial for preventing synchronous oscillatory activity of the nRT1.

Key words: gamma-amino butyric acid (GABA); benzodiazepines (BZs); positive allosteric modulation (PAMs); diazepam-binding protein (DBI)


I. BACKGROUND As with many other types of seizures, absence seizures have been shown to result from synchronous neuronal firing within the thalamocortical (TC) networks1. As the thalamus begins producing synchronous oscillations, other neural regions are synchronized into the same pattern1. In the thalamus, the thalamic reticular nucleus (nRT) is important for gaiting sensory processing and regulating consciousness1. The nRT receives excitatory input from both corticothalamic and TC networks, while providing inhibitory input back to the cortex via TC networks1. Additionally, the nRT provides inhibitory inputs to its own sub-regions (intra-nRT inhibition) which is especially important in preventing synchronous oscillation of the thalamus1.

Intra-nRT inhibition within the thalamus is mediated via gamma-amino butyric acid (GABA)1. While neurons within the nRT express GABAA receptors which contain α3 alpha-subunits, other brain regions and other thalamic sub regions contain α1 alpha-subunits1. Studies have shown that mice with point mutations in the α3 alpha-subunit cannot bind benzodiazepines1. Classified as an endozepine, an endogenous substance which produces similar neurological effects as benzodiazepine, DBI was previously known as an intracellular transporter of acyl-CoA1. Produced by both neurons and glial cells, DBI has been shown to have allosteric effects on GABAA receptors1. Therefore, researchers hypothesized

that DBI acts as a positive allosteric modulator of GABAA receptors in the nRT, thereby reducing neuronal excitability and decreasing seizure activity1.

II. RESEARCHMATERIALS AND METHODSThe authors investigated the hypothesis by comparing inhibitory transmission, effects of benzodiazepines site blockade, and seizure profiles in wild type mice, α3(H126R) mice with a point mutation in alpha 3 (α3) subunit rendering it dysfunctional, and new mutation 1054 (nm1054) mice containing a 400kb deletion on chromosome 1 which includes the diazepam-binding inhibitor gene1. All mice were housed in 12:12 light dark photoperiod, given food and water as desired1.

α3(H126R) miceWild-type mice were compared with α3(H126R) mutant mice with a point mutation at the α3-subunit gene1. The α3-subunit gene is found on the X-chromosome. Therefore, the mutant mice were either homozygous females or hemizygous males1.

nm1054 miceA cross-between nm1054 heterozygote males with wild- type female mice produced heterozygous male and female progeny1. Then these mice were bred with each other and produced wild-

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type, homozygous, and heterozygous mutant mice. In the study nm1054 mice is a homozygous mutant mouse1. The study did not use any heterozygote mice, as the heterozygosity proved fatal1.

Brain Slice PreparationPentobarbital sodium was used to anesthetize the mice and killed by decapitation1. Horizontal thalamic slices were incubated and oxygenated in warm artificial cerebrospinal fluid for one hour1. Then performed the recording, after placing the slice in room temperature for fifteen minutes1.

RESULTSRole of α3 Subunits in Producing Postsynaptic InhibitionBefore testing their main hypothesis, researcher tested the assumption that α3 subunits are important for producing the primary inhibitory postsynaptic currents within the thalamus1. Using electrophysiological recordings, researchers examined both spontaneous inhibitory postsynaptic currents (sIPSCs) and laser-stimulated evoked inhibitory postsynaptic currents (eIPSCs)1. First, in comparing WT and α3(H126R) mutant mice, researchers showed that there was no difference in the number of channels mediating inhibition (Figure 1A)1. However, looking more closely, mutant mice showed lower amplitude sIPSCs (Figure 1B) and eIPSCs (Figure 1C)1. Additionally, mutant mice also showed faster decaying inhibitory currents (Figure 1D)1. Therefore, researchers determined that indeed α3 subunits are important for producing postsynaptic inhibitory currents1.

Figure 1. (A) WT and α3(H126R) mutant mice show no difference in the amount of ion channels; (B) α3(H126R) mutant mice show smaller amplitude sIPSCs; (C) α3(H126R) mutant mice show smaller amplitude eIPSCs; (D) α3(H126R) mutant mice show faster decaying inhibitory currents1.

Role of α3 Subunits in Benzodiazepine Binding in the NRTNext, researchers were interested in confirming their assumption that it is the α3 subunits of the nRT, and not those of other regions, which are specifically important for benzodiazepine binding. To test this, researchers applied Flumazenil (FLZ), a benzodiazepine-

site antagonist to the nRT and the ventrobasal (VB) nucleus of the thalamus of WT mice1. Results showed that applying an antagonist decreased the amplitude of inhibitory postsynaptic currents compared to control selectively in the nRT (which mostly displays α3 subunits) and not the VB nucleus (Figure 2A)1. FLZ reduced both sIPSCs and eIPSCs (Figure 2B)1. However, FLZ had no effect on α3-mutant mice, showing that these effects depend on the presence of a functional benzodiazepine-binding site (Figure 2C)1.

Figure 2. (A) FLZ decreased IPSCs selectively in the nRT; (B) FLZ reduced sIPSC and eIPSC duration and decay in WT nRT cells; (C) FLZ reduced had no effect on α3(H126R) mice1.

Figure 3. (A) Immunocytochemical staining of nRT shows nm1054 mutants have no DBI; (B) nm1054 mutants show reduced sIPSCs in nRT cells; (C) viral infection nm1054 cells with control vs. DBI-containing vector; (D) Injection of AAV-DBI-GFP into nRT of nm1054 mice increased sIPSC duration and showed decreased sIPSCs when treated with FLZ (GABAAR antagonist)1.

Role of DBI in the nRTUsing fluorescent DBI-antibody staining, researchers showed that thalami of nm2014 mutant mice show no DBI gene product in the nRT or VB nucleus, in comparison to WT mice (Figure



accurate IPSC potentiation1. In addition, the paper demonstrates that nucleus specificity within the thalamus is required for the observed effects results from nRT1. Finally, the authors showed that local PAM actions in the nRT can exert seizure- suppressive effects on TC1. While this model did provide the first functional evidence of endogenous benzodiazepine mimicking, there is still some key information missing before implementing this on humans1. One big concern is that researchers still need to determine whether it is DBI on its own, or is it the peptide fragment that is the benzodiazepine agonist, because their data shows that peptides of the DBI can act as PAMs1. Second, the data presented is not really that significant. When looking at the eIPSC traces across nRT cells recording from wild-type and α3(H126R) there is only a slight difference between the responses1. Therefore, other experiments should be done where the effect seen is larger.

SIGNIFICANCE OF THE WORKThis study is important because of two factors. Firstly this is the first study that has demonstrated that diazepam binding inhibitor can mimic the action of valium1. Secondly, this research can be immediately applied to the treatment for epilepsy, because it seemed to prevent seizures in mice1. Therefore, new therapies can be developed which have less side effects compared to valium. In addition it can also be used to cure anxiety or sleep disorders.

FUTURE DIRECTIONSFuture investigations may be interested in exploring the cause of the positive allosteric modulation associated with DBI. Currently, it is not known whether the full DBI protein or a DBI-fragment peptide, such octadecaneuropeptide (ODN), are responsible for the inhibitory effects1. Using viral transmission techniques, different DBI-protein fragments may be injected into the mice in order to observe their subsequent effects. Additionally, knowing which cells synthesize DBI products may lead to pharmacology methods of increasing the amount of gene product produced, either be increasing substrate supply or improving enzymatic activity. Lastly, naturally occurring pathologies are far more complex than simulations induced through pharmacological methods, seen here with pentylenetetrazol (PTZ). It is suggested that future research focus on subjects with naturally occurring neurological impairments such as epilepsy, BZ addiction, insomnia, or anxiety attacks. In such complex systems, the strength of DBI may be tested.

REFERENCES1. Christian, C. A. et al. Endogenous positive allosteric modulation of GABA (A) receptors by diazepam binding dsinhibitor. Neuron 78, 1063-1074 (2013).

3A)1. Additionally, nm1054 mutants show reduced amplitude of IPSCs compared to control (Figure 3B), thereby confirming that endogenous DBI-gene products are necessary for allosteric modulation of GABAA receptors in the nRT1. To confirm this, researchers used a viral vector to re-introduce a functional DBI gene into the nm1054 mutant genome1. Results showed that the injection of the functional gene led to increased DB-gene product in the nRT of nm1054 mutant mice (Figure 3C), and a subsequent increase in the amplitude of IPSCs compared to nm1054 injected with a control viral vector (Figure 3D)1.

Role of Endozepines in Modulating Seizure ActivityLastly, researchers were interested in finding evidence that increased DDBI-gene product could improve seizure activity1. Researchers focused on spike-and-wave discharges (SWDs) which were used to operationalize seizure activity1. Mice were injected with pentylenetetrazol (PTZ) which induced SWDs characteristic of absence seizures1. Compared to WT, both nm1054 and α3-mutant mice showed increased SWD activity (Figure 4A) and longer SWD duration (Figure 4B)1. Therefore, researchers concluded that both a functional α3 subunit and DBI-gene product are necessary to prevent synchronous oscillations of the nRT1.

Figure 4. (A and B) a3(H126) mutants injected with PTZ show more spontaneous SWDs and have longer lasting SWDs than control; (C and D) nm1054 mice injected with PTZ show more spontaneous SWDs and have longer lasting SWDs than control1.

DISCUSSIONChristian and colleagues showed functional evidence of endogenous benzodiazepine mimicking PAM actions and shows that DBI gene products mediate these effects. This was shown by nm1054 mice that displayed deficits in IPSC potentiation1. Furthermore, the mimicking effect was demonstrated by local viral transduction of DBI into the nRT, which displayed an





The Effect of Gut Microbiota on Anxiety and Mood Disorders in MiceCharlotte Chiarella-Redfern1 and Anastasiya Slyepchenko1

1Human Biology Department, Neuroscience, The University of Toronto. Toronto, Ontario CA.

The complex ecosystem of microorganisms living in our gastrointestinal tract has recently been found to play a key role in the communication of the gut-brain axis. Gut microbiota can communicate with the CNS through several bi-directional pathways resulting in effects on neural inflammation, metabolism, and neurochemistry. These changes can contribute to a wide range of diseases as well as influence brain function and consequently the organism’s behavior. There is a high comorbidity between gastrointestinal disorders and many neurological diseases, including stress-related psychiatric symptoms. This review paper will be looking at the gut microbiota and its possible role in regulating anxiety and mood disorders through communication with the CNS. A recent study performed by Neufeld K. M. and colleagues will be discussed, which found a reduction of anxiety-like behavior and changes in mRNA expression for proteins

related to stress and anxiety in the hippocampus and amygdala of germ free mice. This study was important in finding many ground breaking discoveries for neuroscience. Despite the presence of some shortcomings, it has opened the doors for many different future directions in the field and begun to pave a new path towards a future cure for many disorders. These results give us the potential to find a possible treatment for devastating psychiatric diseases like depression, bipolar disorders, and anxiety disorders that affect hundreds of millions worldwide today.

Key words: Microbiota; gut-brain axis; germ-free mice (GFM); anxiety-like behavior; mood disorders; elevated plus maze; serotonin (5-HT) receptor; NMDA receptor; BDNF receptor; amygdala; hippocampus

I. BACKGROUNDThe gastrointestinal tract is inhabited by a large range of microorganisms with a variety of compositions and a genome that consists of 150 times the genes as our own. The composition of this complex microbial ecosystem is partially determined by genetics and the rest by our environment in the first few years of life. This microbiota has recently become a target for the development of treatments for a wide variety of diseases due to the discovery of its role in the gut-brain axis by communicating with the CNS. The bi-directional communication of the gut-brain axis consists of neuronal pathways, including the vagus nerve, spinal cord and others, as well as humoural pathways, which include the endocrine system and the immune system2. Alterations to the gut microbiota can therefore influence brain function and behavior through these various pathways. There is often a link found between gastrointestinal symptoms, like diarrhea and abdominal pain, and changes in mood or emotion, like anxiety or stress. There is also a high comorbidity found between stress related psychiatric disorders like anxiety and gastrointestinal disorders, like irritable bowel syndrome1. These comorbidities explain why the study by Neufeld K. M. et al. hypothesized that altering gut microbiota in mice will have an effect on anxiety-like behavior3.

Anxious behavior is a common symptom of both mood and anxiety disorders. These disorders are highly comorbid and have a variety of symptoms that build on each other causing them to be devastating and disabling for patients. Mood disorders are

often characterized by changes in mood or extremes of mood, for example depression, mania, stress, and anxiety. Both mood and anxiety disorders are associated with numerous physical, affective, and cognitive symptoms. As previously mentioned, some of these psychiatric disorders like depression are affecting hundreds of millions of individuals worldwide, which consist mainly of individuals from the ages of 15-444. With the average age of onset around 30, this disabling disorder is greatly impacting society, since it affects individuals at an age where most people are moving up in their jobs, starting families, and significantly contributing to their community.

Several brain changes have been found in these psychiatric disorders which, as mentioned earlier, are hypothesized to be a result of alterations in the composition of gut microorganisms. These changes include a reduction in hippocampus and prefrontal cortex size, and a hyperactive amygdala, cingulate cortex, and HPA axis5. There disorders are devastating due to the lack of a known cure and the inefficiency of present treatments. Antidepressant medications are only effective in around 50% of patients and the same is true for psychotherapies4. With extremely high relapse rates for these disorders it is clear that the recent findings of this study are a huge breakthrough in the field of mood disorder treatments.

The study hypothesized that the existence of intestinal microbiota directly influences the CNS in terms of development and behavior.

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there was a significant difference between the GF mice and SPF mice when testing anxiety like behaviors with the EPM (Figure 1). The authors found that the GF mice had increased open arm exploration and were found to spend significantly less time in the closed arms of the maze. These mice also showed an increase in the number of times they entered the open arms but no difference in the number of closed arm entries. The number of open arm entries for these mice also stayed constant over the duration of the test whereas the SPF mice had a significant decrease in entries over time. This illustrates that the GF mice show decreased anxiety while the mice with a normal microbiota feel anxious in the open space and learn through conditioning not to enter the open arms of the maze during testing. These results can therefore lead us to conclude that the gut microbiota does communicate with the CNS resulting in influences on behavior. This also explains the link between mood changes like anxiety and many gastrointestinal symptoms.

Figure 1. Significant differences between the anxious behavior of GF mice and SPF mice in an EPM as shown by Neufeld K. M. et al. (2011).

Corticosterone levelsWhen looking at the corticosterone levels in the plasma of the mice there was a clear difference observed by the authors. The GF mice were found to have significantly higher levels of the hormone then the SPF mice showing communication with the gut-brain axis through humoural pathways by influencing the endocrine system. The alteration of gut microbiota was found to cause hormonal differences that could then communicate to the CNS through the HPA axis. Corticosterone is a steroid hormone produced from the

This paper shows how they were able to test this hypothesis using germ-free mice as animal models to show that removing the existence of gut microbiota will influence anxiety behaviors and mRNA expression in the hippocampus and amygdala. These observed changes can then be linked back to what is currently known about mood and anxiety disorders in hope of finding a new treatment approach.

II. REVIEWED RESEARCHMATERIALS AND METHODSNeufeld et al. examined anxiety behaviours, corticosterone levels, and the expression of mRNA in the hippocampus and amygdala in germ free (GF) and specific pathogen free (SPF) mice. These Swiss Webster mice were obtained from Taconic Farms Inc. at the age of 8 weeks, and were all female. The GF mice had a complete absence of microbiota in their system, and had a guaranteed germ-free shipment to the lab. Anxiety phenotypes had been tested by use of the elevated plus maze (EPM), which is an elevated maze with four arms. Two of these arms are closed, while two are exposed to open space. Open space induces anxiety in mice, so the anxiety levels had been determined by testing the amount of time spent in the open arms of the maze, as well as the number of entries into the arms of the maze. General locomotor activity of the mice had been tested by use of an open field test, using VersaMax software, as supplied by AccuScan Instruments. Corticosterone levels measurements were produced by use of a radioimmunoassay kit provided by MP Biomedicals. In order to analyze mRNA expression levels of BDNF, NR1, NR2A, NR2B, and 5HT1A, in situ hybridization had been used. The researchers used a BDNF riboprobe as provided by Dr J. Lauterborn and C. Gall from the University of California Irvine; a 5HT1A riboprobe as provided by Dr. Pat Levitt from the University of Vanderbilt; the NR1 receptor riboprobe had been designed through use of Primer 3 online software in the lab; finally, the NR2A, and NR2B receptor probes were acquired from the Allen Brain Atlas. Primer specificity for the NMDA subunit primers was confirmed by the use of BLAST, and the pGEM T-easy vector, as provided by Promega, was used to express PCR generated NMDA receptor cDNA. Data analysis was then performed through the use of GraphPad Prism (LaJolla, CA, USA)3.

MAJOR RESULTS AND DISCUSSIONElevated plus maze and open field testSignificant results were found while testing behavioral changes with the elevated plus maze (EPM), but none were found with the open field test (OFT). This means that the removal of commensal microbiota did not cause any changes in the general locomotor activity of the mice. Both the GF mice and SPF mice showed similar amounts of locomotor activity and entered the different quadrants of the OFT around the same number of times. These results may have been due to influences of prior testing since both the OFT and EPM tests were done in the same day. On the other hand,



adrenal gland and is known to play a key role in the body’s stress response3. It was therefore unexpected to find an increase in the GF mice since they exhibited less anxious behavior in the EPM test. This can possibly be explained by the fact that anxiety like behaviors in the EPM test may not be related to the same stress responses caused by corticosterone. However, these results still support the hypothesis that the gut microbiota is influencing the communication of the gut-brain axis through its various pathways to the CNS, in this case through the endocrine pathway.

Receptor mRNA expression in the hippocampus and amygdalaSignificant differences were found in the mRNA expression of the BDNF receptor, serotonin receptor, and one of the three NMDA receptor subunits.

The expression of BDNF mRNA was observed and compared in the hippocampus of the mice. The authors’ results found that there was a significant up-regulation of BDNF mRNA expression in the hippocampus of the GF mice but only in the dentate gyrus (DG) and not the CA1 or CA3 subregions3. This receptor is known to play a significant role in learning and memory by contributing to neuronal survival. Stress is known to reduce hippocampal BDNF expression, and many studies have shown the negative effects of high stress levels on learning and memory6. On the other hand, research has found that anti-depressant medications have the opposing effects and actually increase BDNF expression5. This shows that by altering the gut microbiota in mice they were able to influence brain functioning in similar ways as anti-depressant medications and resulting in behavioral changes. This indicates that the gut-microbiota may truly be an appropriate target for psychiatric disorders, like depression and other mood or anxiety disorders.

In contrast, the mRNA expression of the 5HT1A serotonin receptor did not increase, but rather showed a significant down-regulation in the GF mice. This difference was once again only observed in the DG and not in the other two subregions of the hippocampus. This result demonstrates the influence of microbiota in the regulation of mood and anxiety disorders through alterations in brain functioning. The 5HT1A receptor is one of the serotonin receptors most commonly known to regulate emotional behavior3. It is also known that serotonin acts as a mood regulator which explains why it is one of the main targets for antidepressant medications, for example the commonly used selective serotonin reuptake inhibitors (SSRIs)5. This links once again the effects gut microbiota and gut-brain axis to anti-depressant effects.

Finally, the mRNA expressions of three different NMDA receptor subunits were also observed. The NMDA receptor was most likely chosen by the authors due to its known role in synaptic plasticity for learning and memory. The three subunits used in this study were the NR1, NR2A, and NR2B subunits. This time however

the authors were looking at expression in a different part of the brain’s limbic system. Rather than looking at the hippocampus they observed expression in the amygdala, the emotional center of the brain. The results of this study showed a significant down-regulation in mRNA expression of the NR2B subunit in the central amygdala region of GF mice. This significant difference however was not found in the other regions of the amygdala or with the other two subunits of the NMDA receptor (NR1, NR2A)3. These results correlate with our knowledge of this receptor since the NR2B is the critical NMDA receptor subtype for the amygdala. Past research has found that NR2B antagonists block anxiety as well as amygdala-dependent fear learning7. This change in expression can therefore be linked back to the observed behavioral differences between the mice. It would be expected that the down-regulation of this subunit caused by the altered microbiome of the GF mice would result in similar behavioral effects as NR2B blockers. This explains the decreased anxiety-like behavior exhibited by these mice as well as decreased fear learning, shown by the constant number of open arm entries of GF mice in the EPM while the number of entries decreased in SPF mice. The authors were therefore able to support their hypothesis that the gut microbiota is in communication with the CNS resulting in an influence in brain function and consequently behavior. The effect of the gut’s microbial composition on behavior and psychiatric symptoms has been clearly illustrated by this study, leading us to the possibility of its use for the treatment of mood and anxiety disorder. The authors have found that the gut microbiota can influence our behavior through changes in hormone levels as well as influencing gene expression in different regions of the brain.

SIGNIFICANCE OF THE WORKAnxiety often feels incapacitating to those experiencing it, whether it be in the context of a mood or anxiety disorder or otherwise. The establishment of a clear link between the gut microbiota and the expression of anxiety-related receptors and behaviours provides a whole new spectrum of possible treatments and further research for mood and anxiety disorders. This study was the first to show a behaviour change due to the lack of gut microbiota, displaying a significant link to nervous system function. The researchers provided evidence of microbiota influence on hormone levels in the mice. The shift of serotonin receptor mRNA expression, in particular, shows a possible link to not only anxiety but mood disorders. The 5HT1A receptor mRNA downregulation in the hippocampus has been previously observed in patients with major depression8 and 5HT1A receptors are targeted by depression treatments through SSRI use5. The alterations in expression of BDNF, 5HT1A receptors and the NR2B receptor subunit provide possible bases of mechanisms of behaviour change in the mice, the details of which could be explored in further research. This study also provides us with an additional layer of information about the mechanisms and causes of anxiety and mood disorders, which have yet to be completely explained by other paths of research.



composition of the gut microbiota can actually cause depression and different symptoms of mood disorders. Similar research has already been done with obesity, resulting in a link between obesity in mice and the composition of the microbiome. Fecal microbial transplants have already been performed in humans to effectively treat some disease, for example Clostridium difficile infection (CDI), so it is known to be an effective and safe treatment that could potentially be accepted for clinical use9.

In conclusion, this ground breaking study along with the future possible studies in this field will get us closer to the treatment of anxiety and mood disorders. This will be done by altering the gut microbiota through one of the many possible techniques available, for example diet, probiotics, antibiotics, microbial pills, or fecal transplants. These treatments may finally lead us to the cure for several diseases that have a large impact on our society today.

REFERENCES1. Cryan J., and Dinan T. (2012). Mind-Altering microorganisms: the impact of the gut microbiota on brain and behaviour. Neuroscience, 13: 701-712.2. P. Bercik, S. M. Collins, and E. F. Verdu. (2012). Microbes andthe gut-brain axis. Neurogastroenterology and motility, 24:405-413.3. Neufeld, K. M., Kang, N., Bienenstock, J., & Foster, J. A. (2011). Reduced anxiety‐like behavior and central neurochemical change in germ‐free mice. Neurogastroenterology & Motility, 23(3):255-264.4. Nemeroff B. C. and Owens M. J. (2002). Treatment of mood disorders. Nature neuroscience supplement, 5:1068-1070.5. Frangou S. (2008). Brain structural changes in mood disorders. Psychiatry, 8(3):105-106.6. Kazlauckas V. et al. (2011). Distinctive effects if unpredictable subchronic stress on memory, serim corticosterone and hippocampal BDNF levels in high and low exploratory mice. Behavioural Brain Research, 218(1):80-86.7. Lopez de Armentia M. and Sah P. (2003). Development and subunit composition of synaptic NMDA receptors in the amygdala: NR2B synapses in the adult central amygdala. The Journal of Neuroscience, 23(17):6876-6883.8. López-Figueroa, A. L., Norton, C. S., López-Figueroa, M. O., Armellini-Dodel, D., Burke, S., Akil, H., ... & Watson, S. J. (2004). Serotonin 5-HT1A, 5-HT1B, and 5-HT2A receptor mRNA expression in subjects with major depression, bipolar disorder, and schizophrenia. Biological psychiatry, 55(3), 225-233.9. Borody T.J. and Khoruts A. (2012). Fecal microbiota transplantation and emerging applications. Nature reviews gastroenterology and hepatology, 9:88.

This work was supported by the Human Biology department, Neuroscience, at The University of Toronto. The authors thank Dr. Bill Ju for his inspirational lectures and motivation to explore neuroscience, and the students in HMB420 for feedback received in class on their presentation.

Address correspondence to: Charlotte Chiarella-Redfern and Anastasiya Slyepchenko Email:charlotte.chiarella.redfern@[email protected]

Copyright © 2013 Charlotte Chiarella-Redfern and Anastasiya Slyepchenko , Human Biology Program

FUTURE DIRECTIONSThere are a few critiques that can be done on this study since there were limitations found in the research as well as many unanswered questions. The authors of this study have simply started to pave the way for the use of microbiota as a future treatment of anxiety and mood disorders, but there is still a long ways to go before we get there. The study was only able to find a link between the alteration of gut microbiota with changes in behavior, hormone levels, and mRNA expression in the brain. There are therefore many possible future directions that can be taken from here to fix some of the study’s limitations and in hopes of leading us closer to the treatment of these psychiatric diseases and possibly others.

Firstly, this study had several limitations that may have influenced their results. The authors only looked at changes in the expression of mRNA in the hippocampus and amygdala however mRNA expression does not directly translate to protein expression. A change in mRNA expression does not mean that there is an equivalent up- or down-regulation of the protein expression for these receptors. A useful future study would therefore be to look at the difference in the actual protein expression of these receptors to give us sturdier evidence that there is a link between these changes with the difference in behavior observed. Another possible line of research stemming from this study could look at the expression of 5HT1A receptors in germ-free mice in different brain areas, such as the dorsolateral prefrontal cortex, an area frequently linked to higher cognitive functions and mood regulation, which are debilitated in patients diagnosed with major depression8. A final addition that could be made to this study in the future would be to test for depression in the mice rather than simply anxiety like behavior. This is important to create a clear link between the gut microbiota and actual mood disorders rather than simply anxious behavior. Depression could be induced in mice either through forced swim test or the tail suspension test to see how long the mice struggle before giving up and showing learned helplessness, a depressive behavior. Looking to see if it is easier to induce depression in SPF mice compared to GF mice would link the composition of the gut microbiome with an individual’s vulnerability to mood disorders like depression. This can therefore lead us not only to the use of gut microbiota as a possible cure for these disorders but also a preventative treatment, which is especially important due to the high relapse rates found in disorders like depression.

Secondly, a potential future direction to take from here would be to look at the effect of fecal transplants in mice to see if a specific composition of gut microbiota is linked with depression. This could be done by inducing depression in mice with the techniques previously mentioned and then transplanting their microbiota into perfectly healthy normal SPF mice, like the ones used in the study by Neufeld K. M. et al. If the SPF mice begin to show depressive behavior after transplantation than it will be clear that the





Changing Minds: The Creation False Memories Through Optogenetic Stimulation of the HippocampusAzubuogu Anudu1, Ruoshi Cui1, Chang Woo Park1, & Kaai Yee1


Optogenetics is a recent development in the field of neuroscience and with its wide range of applications it has become one of the most invaluable modern techniques for studying the physiological properties of the brain and other related structures. Memory is an intangible part of organisms that plays an important role in their survival, and as such this may lead them to rely heavily on their memory as an objective account previous events. Ramirez et al. (2013) performed an in-depth study of memory and how it could be altered or tampered with in order to produce false memories. Using optogenetic stimulation of channelrhodopsin-2 (ChR2) transfected into the dentate gyrus (DG) and CA1 memory engram cells of their c-fos-tTA transgenic mice, were able to by activating a previously formed contextual memory engram while simultaneously delivering footshocks. Subsequent experimentation also revealed that false memories induced by

optogenetic recall activated the same downstream brain regions as legitimate fear memories. Finally, Using a conditioned avoidance paradigm, it was found that the artificial induction of retrieval of false memories could also influence the behaviour of the mice. The authors then observed results obtained from the photic stimulation of the dentate gyrus and the CA1 separately. It was found that the pairing of footshocks with photic stimulation was capable of inducing false fear memories only when the mice received photic stimulation to the dentate gyrus but not the CA1. This suggests a difference in the capability and the modality in which these two hippocampal regions encode context.

Key words: Optogenetics; false memories; hippocampus; dentate gyrus (DG); CA1; amygdala

I. BACKGROUND The manipulation of memory continues to be an interesting topic in neuroscience, especially with the advent of new technologies. Memory is a powerful tool that allows organisms to learn and adapt. The flaw, however, lies in how memory is encoded. Many factors can influence and alter memories, affecting both behavior and recall. Prior experiments have demonstrated how easily false memories can be induced through cued recall, causing subjects to remember things that were not present2. The internal mechanisms of memory are not fully understood, but there is a general consensus that the hippocampus plays an important role in memory formation, particularly episodic memory1. Exploring how external stimuli and neurophysiology interact can yield important insights.

A critical role of the hippocampus is in fear conditioning, where it is believed to be responsible for the retrieval of contextual cues3. But beyond that not much is known about the exact molecular activity underlying fear memory retrieval. Recent research though has localized the contextual memory-engram cells to a subpopulation of granule cells in the dentate gyrus (DG)1. Activation of CA1 neurons has also been identified in contextual fear memory retrieval3. CA1 neuron activation occurs only with contextual fear association, but not in cued fear association3. The specificity in CA1 neuron activation is compelling evidence for the role of the hippocampus in contextual memory.

One of the most prominent techniques for modulating specific neurons is optogenetics. Optogenetics utilizes light sensitive proteins such as channelrhodopsin-2 (ChR2) to activate individual neurons4. The precision and speed of optogenetics make it an extremely versatile tool for testing the functions of chosen pathways. Optogenetics can be applied to the research of memory by targeting the DG and CA1 neurons. In order for the DG and CA1 neurons to respond to optogenetic stimulation they need to express ChR21. To achieve this c-fos-tTA transgenic mice, which display ChR2 expression in the DG and CA1 neurons are used1. These mice also exhibit mCherry, which is used a fluorescent marker1.

Combining optogenetics with fear based contextual tests provides a way to study how the underlying mechanism of these memory engrams, as well as how they can be altered. The manipulation of memory also entails the manipulation of behavior, making the study topic somewhat controversial. In spite of that the ability to artificially generate false memories has important implications in the future of memory research.

II. REVIEWED RESEARCHMATERIALS AND METHODSMiceTransgenic mice with the c-fos gene were used; c-fos drives the expression of the tetracycline trans-activator (tTA), which allows the expression of channelrhodopsin-2 (ChR2) within the DG

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activity across all groups were found. When replicated with exploration of context C instead of context A, the experimental group froze significantly more in context C instead of context A. The entire procedure was replicated in the CA1 region instead of the DG, but no significant freezing activity the experimental group, the control group, or any other treatment groups were found (Fig H – N).

Figure 1. Initiation of a false contextually specific memory. (A – E) c-fos-tTA mice were injected with the vector complex AAV-TRE-ChR2-mCherry in the DG. After taking off Dox, the mice were allowed to explore context A, and their engram cells were labeled with mCherry (red). After labeling they were either taken back to context A (A and C) or taken to the novel context C (B and D), and the amount of c-fos expression after 24 is shown (green). The overlaps of c-fos mCherry cells between the two contexts are shown in E. (F) Contextual fear specificity was tested with the exploration after taking off Dox, fear conditioning, and freezing measurements after return to original or new contexts. (G) The experiment was replicated with other conditions. (H – N) Themethods in (A – G) were replicated in CA1.

Figure 2. Interference between two CS and expression of c-fos in the BLA and CeA.(A) Light activated fear vs. genuine fear was compared in light-on and light-off epochs. (B) Freezing levels with light activation in a completely novel context (context D) are displayed. (C) The amounts of c-fos expression in the BLA and CeA during neutral recall, false recall, and recall in a neutral context is shown. (D – F) c-fos expression of neutral, false, and contextually different recalls are shown (green).

and the CA1 through the activation of the tetracycline response element (TRE) in the adeno-associated virus (AAV). The mCherry acts as a fluorophore to label the engram cells. Doxycycline was used to inhibit the AAV vector from expressing itself until the start of the experiment.

Context SpecificityThe degree of engram cells overlapping between context A and context C within the DG of the mice was first evaluated. The mice were tested for false contextual fear memory outlined in Fig. 1F. Different treatment groups including mice that expressed mCherry without ChR-2, mice that expressed ChR2-EYFP instead of ChR2-mCherry, mice that expressed ChR2-mCherry with no light activation, and mice that expressed ChR2-mCherry with light but were given non-simultaneous foot shocks were also tested for freezing. The same paradigm was replicated with context C exploration instead of context A and the amount of freezing was measured. The above methods were also replicated in the CA1 region.

Test For Interference / Quantification of c-fos Expression in the BLA and the CeAThe effect of light activated DG cells in context A vs. genuine fear of context B with no light were tested in light-on and light-off epochs. The method was replicated in the CA1 region. Whether memory recall occurred with the stimulation of engram cells of DG and CA1 in a distinct context (context D) was investigated with light-on and light-off epochs. During the recall of both a false and genuine memory contexts, the c-fos level within the basolateral and the central amygdala were measured and compared to c-fos levels in the recall of a neutral context.

Behavioral Test of False Fear MemoryA conditioned place avoidance paradigm (CPA) was conducted. With exploration and fear conditioning, and after 24 hours the mice were taken back to the chambers and the preference for each chamber was measured through time spent in the chambers. This paradigm was conducted both in DG and CA1 implanted mice. EYFP marker was used in the paradigm in order to test for independent activation of engram cells within the two test chambers.

RESULTSContext SpecificityA minimal overlap between the engram cells of context A and context C (~1%) was found within the DG, but a significant higher overlap of the engram cells was found within the CA1 region (~30%) (Fig. 1A - E). The DG experimental group froze significantly more in context A after conditioning but did not exhibit significant freezing in the novel context C (Fig. 1F). No significant increase in any other groups (Fig. 1G). EYFP instead of mCherry was also used as a label and the same results of freezing



Test For Interference / Quantification of c-fos Expression in the BLA and the CeAThe experimental group with light activation froze significantly less in the light-off epoch than the control group with no light activation. During the light-on epoch, the freezing of the experimental group increased while the freezing of the control group decreased (Fig 2A). No significant difference in freezing was found in any other test groups. When replicated in the CA1 region, the mice did not elicit any significant increase in freezing in either the experimental group or the control group. In a completely novel context (context D), the light activated experimental group froze significantly more, but no freezing was observed in the control group with the mCherry only (Fig 2B). When replicated in the CA1 region, no significant increase in freezing was observed in either light-on or light-off epochs. When the levels of c-fos were measured in the BLA as well as the CeA, there was a significant increase in gene expression in both regions during recall of false and genuine fear memories (Fig. 2C - F).

Behavioral Test for False MemoryThere was significant preference for the unlabeled chamber in the experimental group, but no significant preference in the control group (Fig. 3A – D). EYFP indicated statistically independent DG engram cells activation within the hippocampus of the mice between the two chambers (Fig. E – K), and when the method was replicated in the CA1 region, no particular chamber preference was observed (Fig. L – M).

Figure 3. Behavioral translation of false fear memory.(A) CPA paradigm. (B) Foot tracks traced with mice in different treatments: mCherry only (top), ChR2-mCherry labeled left (middle), and ChR2-mCherry labeled right (bottom). (C – D) Quantifications of chamber preference ratios between ChR2-mCherry and mCherry only. (E – K) c-fos levels in the DG were labeled with EYFP and the quantification of population of c-fos EYFP+ cells is shown in K. (L – M) The same CPA paradigm tested in CA1 region.

DISCUSSION AND SIGNIFICANCEIn this present study, Ramirez and colleagues have shown that they were able to manipulate memory formation through optical stimulations of the memory engram cells in the hippocampus using optogenetics. False memories were able to be created by having the cells artificially reactivated in the dentate gyrus (DG) region of the hippocampus, functioning as the conditioned stimulus (CS) in the fear-conditioning model. As shown in the results, light stimulation plays a critical role in these experiments as the experimental groups of mice showed significant freezing after conditioning when they were placed back in the context A with the light stimulation. In addition with the mice group that only had expressing mCherry alone without any light activation, there was no significant increase in freezing. Similarly, they demonstrated that false memories were able to lead to fear behavior in mice using the CPA paradigm. When the chambers were labeled, either left or right, the mice with ChR2-mCherry significantly showed preference to the unlabeled chamber; whereas mice with only mCherry showed no preference. This showed conditioned avoidance of the labeled chamber and the ability to recognize it from false fear memory.

The relationship of the false and genuine memories is not clearly understood as to the specific regions and circuits that it works through within the hippocampus. However, in this experiment, they demonstrate the possible interaction between the two memories by simultaneously making both conditions of memories available. By having both the artificially induced fear contextual memory and the genuine contextual cues, they either competed for the foot shock fear stimulus or the false memories interfered with the genuine memories. Furthermore, they illustrated that both false and genuine memory elicited increase levels of c-Fos expression in both the basolateral and central amygdala. These results suggest that there is no notable difference between the artificially light activated false memory and the natural activations of genuine memory.

Although they were able to successfully create false contextual memories by reactivating the engrams cells in the dentate gyrus, they failed to reproduce any significant results using the exact same procedures for CA1, a region that has been implicated in the formation of fear contextual and associative memories5. The authors suggest that the negative results of the CA1 could be due to lesser reliance on spatial activation of the cells and increasingly more towards temporal encoding within the trisynaptic circuits of the hippocampus1.

Memory is highly susceptible to modifications and renders it to be very misleading6. Previous studies have shown strong evidence for positive activities within the hippocampus during both false and genuine memory recalls7. However, more research needs to be done to be able to distinguish the specific regions within the hippocampus that produces the false and genuine memories.



then it could also play a role in the induction of the fear memories themselves. In their paper, Ramirez et al. (2013) discussed how false memories are constantly being formed on a daily basis in human life be in the forms of source misattribution, déjà vu and many others. This implies that in even in humans our memories are subject to alteration just like in rodents. Future studies in the field of mood disorders such as depression could involve asking the subjects to recall particularly troubling memories from their past, allowing for c-fos expression within a selective neuronal population. If optogenetics is one day approved for human use then we could selectively express NpHR within these neurons in a similar fashion to Ramirez et al. (2013), and then inhibit these neurons using photic stimulation. This could in theory suppress the memory traces, and by preventing access to such depressing memories researchers could alleviate some of their symptoms. On the other hand, the same experiment could be performed using ChR2 stimulation to enhance the more enjoyable memories in the depressed patients. The study by Ramirez et al. (2013) was focused on fear memory but it is well know that other forms of memory such as classical conditioning, procedural memory and working memory exist. False memory induction of these types would provide neuroscience with an even deeper insight into how memory works. Finally, other experiments could study the effect of photic stimulation of ChR2 and NpHR on the CA1 and dentate gyrus cells simultaneously during false memory recall and induction. This would give a much more holistic view of the roles these areas play in the induction of false memories.

REFERENCES1. Ramirez, S., Liu, X., Lin, P., Suh, J., Pignatelli, M., Redondo, R.L., Ryan, T.J., &Tonegawa, S. Creating a False Memory in the Hippocampus. Science,341, 387-391 (2013).2. Carneiro, P & Fernandez, A. Retrieval dynamics in false recall: Revelations from identifiability manipulations. Psychonomic Bulletin & Review, 20(3), 488-495 (2013).3. Hall, J., Thomas, K.L., &Everitt, B.J. Cellular Imaging of zif268 Expression in the Hippocampus and Amygdala during contextual and Cued Fear Memory Retrieval: Selective Activation of Hippocampal CA1 Neurons during the Recall of Contextual Memories. The Journal of Neuroscience, 21(6), 2186-2193 (2001).4. Kasparov, S., &Herlitze, S. Optogenetics at a crossroads? Experimental Physiology, 98(5), 971-972 (2013).5. Ji, J., &Maren, S. Differential roles for hippocampal areas CA1 and CA3 in the contextual encoding and retrieval of extinguished fear. Learn Mem.15(4), 244-51 (2008).6. Olszewska, J., &Ulatowska, J. Encoding strategy affects false recall and recognition: Evidence from categorical study material. AdvCogn Psychol., 9(1), 44-52 (2013).7. Cabeza, R., Rao, S.M., Wagner, A.D., Mayer, A.R., &Schacter, D.L. Can medial temporal lobe regions distinguish true from false? An event-related functional MRI study of veridical and illusory recognition memory.ProcNatlAcadSci U S A, 98(8), 4805-10 (2001).8. Pagliardini, S., Janczewski, W. A., Tan, W., Dickson, C.T., Deisseroth, K., & Feldman, J.L. Active expiration induced by excitation of ventral medulla in adult anesthetized rats. The Journal of Neuroscience, 31(8),

III. LIMITATIONS AND FUTURE DIRECTIONSAlthough the study by Ramirez et al. (2013) was quite astounding in its findings and methodology, there is still much room for improvement on their experimentation practices.

First, recovery from optrode insertion usually takes about 3-4 weeks meaning that the authors had to let the mice recover for this period of time while they were on doxycycline8. Within a period of 4 weeks, even if the utmost care is taken, accidents are bound to happen. Even with the doxycycline preventing c-fos expression in their hippocampi the authors could not control the physical and mental effects that prolonged doxycycline use had on the rodents’ recovery. Doxycycline, although a widely used antibiotic also has some negative side effects if used for long periods of time; these include nausea, headache, inflammation of the joints, stomach cramps amongst many others9. In addition to impairing the recovery process of the mice, these side effects may have also confounded the results obtained from the behavioural tests false memory induction. If a mouse is in pain or cannot focus then its context discrimination abilities may not be at their best. Currently there are man-made drugs such as T-5224 that are capable of inhibiting c-fos expression without giving any of the side effects of doxycycline10. One could also try to make use of halorhodopsin (an inhibitory cation channel-sensitive to yellow light) transfection into the hippocampus to inhibit all of the neurons there thereby preventing c-fos expression (while obviously bringing its own unique side effects such as temporary retro- and anterograde amnesia)11. In addition to this, research has shown that providing the rodents with a physical and mentally stimulating environment will help elevate their mood and hence hasten the recovery process (via psychological methods)12. Diets high in fibre such as fruits and vegetables in combination with correct doses of painkillers (e.g. morphine) help reduce the recovery time and prevent the mice from reopening their implantation site with their scratching13.

Second, though the results of this study seemed very convincing and were indeed statistically significant they do not indicate causality. The authors were able to show that compared to controls a subset of ChR2+ mice showed false fear memory induction but in reality this was less than half (20-30%) of the treatment group mice meaning that the vast majority did not show this false fear memory induction. More stringent criteria for significance as well as higher percentages of successful false fear memory inductions are needed before causality can be inferred from the results.

Future researchers in this field of neuroscience now have a solid foundation from which they can make their own discoveries. One study could observe if the same type of false fear memory induction could also occur by transfecting ChR2 into the amygdala and directly trying to induce fear via reactivation of this region in novel contexts. It seems plausible that since the amygdala was activated following the retrieval of both genuine and false fear memories



2895-2905 (2011).9. Bryant, S. G., Fisher, S. & kluge, R. M. Increased frequency of doxycycline side effects. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy, 7(4), 125-129 (1987).10. Aikawa, Y., Morimoto, K., Yamamoto, T., Chaki, H., Hashiramoto, A., Narita, H., Hirono, S., &Shiozawa, S. Treatment of arthritis with a selective inhibitor of c-Fos/activator protein-1.11. Deisseroth, K. Optogenetics. Nature Methods, 8, 26-29 (2010).12. George, M.G., Scott, D.S., Turner P.S., Gregg, M.J. The effect of psychological factors and physical trauma on recovery from oral surgery.Journal of Behavioural Medicine.3(3), 291-310 (2008).13. Kehlet, H., & Wilmore, W.D. Multimodal strategies to improve surgical outcome. The American Journal of Surgery, 183(6), 630-641 (2002).

Address correspondence to: Kaai Yee, Email: [email protected] © 2013 AzubuoguAnudu, Ruoshi Cui, Chang Woo Park, and Kaai Yee, Human Biology Program



Understanding obsessive compulsive disorder: Optogenetic stimulation of the orbitofrontalcortex induces OCD-like behavior in miceRuoshi Cui1, Chang Woo Park1, Azubuogu Anudu1, and Kaai Yee1


Ever since the introduction of obsessivecompulsive disorder (OCD) into the publication of the DSMIV in 1994, many interests in the OCD studies have continued to expand1,2. With much evidence directed to the dysregulation of the corticostriatothalamocortical (CSTC) circuits, the authors of this study were interested in the use of optogenetics as a neuromodulator medium to artificially induce OCD in transgenic mice (specifically on the orbitofrontal cortexventromedial striatum projection) similarly found in OCD

patients with hyperactivation in these circuits. In addition, they were also interested if it was possible to reverse the repetitive behaviors of OCD symptoms using a serotonin reuptake inhibitor, fluoxetine, as it is the only known therapy for OCD3. The results of this study show the possible pathophysiological mechanisms mediating compulsive repetitive behaviors and they also validate effectiveness of fluoxetine as an OCD drug treatment3.

BackgroundObsessivecompulsive disorder or OCD is characterized by repetitive behaviors aiming to reduce thoughts that produce uneasiness, fear, and apprehension2. Such behaviors include excessive washing, repetitive checking, and preoccupation with inappropriate thoughts related to subjects such as sexuality or violence. Although the exact pathophysiological mechanism of OCD remains uncertain, there have been many affirmation of studies and researches with the implication of dysregulated corticostriatothalamocortical (CSTC) circuits4. Researchers believe that there is an intimate connection between OCD-like behaviors and the hyperactivity of glutamatergic projection from the orbitofrontal cortex (OFC) to the ventromedial striatum (VMS)5. Previous research has speculated the SSRI fluoxetine drug therapy reduced OCD-like behaviors. Ahmari et al. (the authors) attempted to induce OCD-like symptoms through optogenetics, and reduce the symptoms again by using fluoxetine.

Optogenetics is defined as a neuromodulatory technique that combines optics and genetics in order to control the activity of individual neurons in living tissue6. One of its biggest advantages is the ability to control the activity of freely moving animals in vivo. The mechanism of optogenetics, therefore, is the insertion of fast lightactivated channels that allow electrical events within the cortex to be precisely manipulated both spatially and temporally. Because of it’s spatial acuity, the authors were able to activate specific corticostriatal circuits, making optogenetics an elegant method to test their hypothesis. There are two major subtypes of opsins that affect the neurons through inhibition or excitation. Inhibitor opsins include NpHR, eNpHR, Arch Activators such as ChR2, ChR1, VChR1, and SFO are used to specifically excite neurons, and in the experiment ChR2 opsin was used6,7.

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Figure 2. Persistent Photic stimulation in ChR2 transgenic mice led to an increase in grooming behavior when compared to controls3.

Figure 3. Daily stimulation of the OFCVMS pathway leads to increased rate of firing of neurons in the circuit3.

Figure 4. Effect of long term fluoxetine treatment on grooming behavior. After 2 weeks of fluoxetine treatment, there was a reduction in the levels of grooming in the ChR2 mice to control levels. Once fluoxetine treatment stopped for 1 week (washout) there was a return of the grooming behavior3.

Major ResultsI. ChR2 was selectively expressed in the OFC as shown by Cfos (an immediate early gene) + EYFP coexpression3.Ahmari and colleagues tagged ChR2 with EYFP in order to aid in the visualization of the ion channels in vitro. With the ion channels in the desired location, optogenetic stimulation certainly targeted the intended group of neurons3.

II. Chronic photic stimulation of the OFC led to an increase in grooming behavior and an increase in the level of EPSPs generated in the VM3.This supports the author’s idea that with an increase in the OFCVMS circuit activity, there would be an increase in repetitive behaviors commonly seen in OCD. Chronic stimulation of the OFCVMS circuit pathway resulted in an induction of repetitive grooming behaviors as well as an increase the rate of firing of the circuits. This behavior persisted for 2 weeks after stimulation ceased while acute stimulation, on the other hand, did not give the same increases. This suggests that some form of LTP may have taken or there may have been removal of serotonin from the synapse1,8. It must be noted that this stimulation specifically induced grooming behavior and not many of the other forms OCD may take such as prepulse inhibition and open field anxiety. This implies that there may be other similar pathways in the brain mediating these function3.

Figure 1. Location of DIOChR2 injections. Left, blue shows Cre expression selectively in the hippocampus and surrounding cortex. Right, green patch indicates the the inversion of the ChR2EYFP in the Creexpressing glutamatergic orbitofrontal cortex neurons3.

III. Fluoxetine treatment reversed the effects of the photic stimulation in ChR2 mice3.Control mice that received vehicle (phosphate buffered saline) instead of fluoxetine did not show any signs of recovery in their grooming or firing rate of neurons. As expected, only the longterm(2week) use of fluoxetine was able to give a reduced rate of grooming and neuronal firing since fluoxetine requires some time to take its synaptic effects (general feature of SSRIs)3. The authors also stopped fluoxetine treatment for one week (washout) and found that the grooming and increased neuronal activity returned. This shows that they were indeed looking at a model of OCD in mice since treatment is done via fluoxetine, a drug known to treat OCD in humans3.



ConclusionsAhmari et al. demonstrated that OCD-like behaviors are correlated with the CSTC circuit. The grooming behaviors triggered by repeated hyperactivation of OFCVMS projections were persistent as well as treatable with fluoxetine, indicating a similarity with naturally induced OCD. What was missing from these findings was a definitive causal link between CSTC circuit dysregulation and OCD. “Though genetic and pharmacologic manipulations of norepinephrine and dopamine can lead to transient increases in repetitive behaviors, the interventions were not limited to specific circuits”3. The benefit of optogenetics is the ability to target specific neural circuits.This experiment took advantage of said specificity to target the CSTC circuit, but failed to provide information on other circuits that may be related to OCD.

Optogenetics is a powerful tool, but there is much that can be done to improve the technique. Currently it is extremely invasive, requiring the neural implantation of stereooptrodes. Future advances in the technology should be directed towards less invasive delivery methods. Of particular interest are ‘Receptors Activated Solely by Synthetic Ligands (RASSLs)’, and ‘Designer Receptors Exclusively Activated by a Designer Drug (DREADDs)9. RASSLs and DREADDS are both noninvasive and due to the prevalence of Gprotein coupled receptors (GPCRs) are able to target large neuronal populations9. One disadvantage, however, is that the drugs that are used to activated these receptors can sometimes have undesired sideeffects9. Optogenetics still has the advantage in precision though, providing faster control over neural activity, as well as better spatial resolution. Ideally future research

could explore a type of hybrid between optogenetics and these engineered GPCRs that take advantages of both technologies, while eliminating the disadvantages. And with improved tools researchers may finally be able to activate all the circuits involved in OCD, and properly understand its pathophysiology.

References1. Arora, T., et al. Oxcarbazepine and fluoxetine protect against mouse models of obsessive compulsive disorder through modulation of cortical serotonin and CREB pathway. Behav. Brain Res. 247, 146152 [2013].2. Leckman, F., et al. Obsessivecompulsive disorder: a review of the diagnostic criteria and possible subtypes and dimensional specifiers for DMSV. Depression & Anxiety. 27(6), 50727 [2010].3. Ahmari et al. Repeated CorticoStriatal Stimulation Generates Persistent OCD-Like Behavior. Science. 340, 12341238 [2013].4. Bourne, K., et al. Mechanisms of deep brain stimulation for obsessive compulsive disorder: effects upon cells and circuits. Front. Integr. Neurosci. 6(29), 114 [2012].5. Milad, R. & Rauch, L. Obsessivecompulsive disorder: beyond segregated corticostriatal pathways. Trends Cogn. Sci. 16(1), 4351[2012].6. Yizhar O., et al. Optogenetics in neural systems. Neuron. 71(1), 934 [2011].7. Nagel, G., et al. Channelrhodopsin2, a directly lightgated cationselective membrane channel. Proc. Natl. Acad. Sci. 100, 13940-13945 [2003].8. Subrià, M., et al. Brain structural alterations in obsessivecompulsive disorder patients with autogenous and reactive obsessions. PLoS One. 8(9), e75273 [2013].9. Nichols, C.D. & Roth B.L. Engineered Gprotein Coupled Receptors are Powerful Tools to Investigate Biological Processes and Behaviors. Front. Mol. Neurosci. 2(16), [2009].



Abnormal neuronal circuit development in Christianson Syndrome: Deficit of cation exchanger of NHE6 distributes overacidification resulting in branching defects associated with BNDF/TrkB signalingJia Yan Zhang1, Dehi Joung1, Ingrid Quevedo1, and Luisa Garzon1


Christianson syndrome (CS) is a rare novel autism-related disorder known to be caused by a mutation in the X-linked endosomal Na+/H+ exchanger 6 (NHE6) gene. It was known previously that patients with CS typically manifest symptoms such as severe to profound neurodevelopmental delay and other physical features

similar to Angelman’s syndrome. The authors of this study were interested in examining and exploring the cellular mechanisms underlying this disease in connection with a mutation in the NHE6 gene.

BackgroundChristianson Syndrome (CS) is a genetic disorder that primarily affects the nervous system. Similar to other neurogenetic disorders, some of Christianson Syndrome’s characteristic features include

intellectual and developmental delays, brain atrophy, aphasia, ataxia, microcephaly, autistic features, and more1.

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demonstrated that both full length human NHE6 and functional domain express equally, in other words, deficit in the NHE6 cation exchanger domain is unrelated to protein stability or trafficking. Thus, deficit in the cation exchanger NHE6 protein leads to impairment of axonal branching not due to deficit in protein stability.

III. Loss of NHE6 increased number of distally located low pH endosomes and overacidification of early endosomesNHE6 (Na+/H+ exchanger 6) aids in regulating endosomal acidity within neurons resulting in low pH endosomes mainly at and near the soma and high pH endosomes within the dendrites. The researchers hypothesized that loss of NHE6 would result in protons being sequestered within the endosomal compartment resulting in an increase in the acidification of distally located endosomes. Using a low pH tracker the researchers found that within NHE6 mutant neurons low pH endosomes were found at great distances away from the soma compared to control. This finding suggests that NHE6 has an important role in regulating endosomalpH.

IV. BDNF can alleviate branching defects observed in NHE6 mutant NeuronsIt was then hypothesized that this increase in acidification would lead to deficits in axonal and dendritic branching. The BDNF/TrkB signaling pathway was found to have decreased leading to branching deficits. Leupeptin (a reversible protease inhibitor) when added before BDNF partially increased TrkB levels suggesting that the observed decrease in signaling was perhaps a result of increased degradation of TrkB.

It was then hypothesized that application of high exogenous levels of BDNF would alleviate the branching abnormality. BDNF was applied to wild-type and mutant cultures and examined five days later for its effects on axonal and dendritic branching. Results showed that BDNF increased axonal and dendritic branching within mutant cells, almost reaching wild-type levels. Altogether the data indicated that loss of NHE6 resulted in a decrease in BDNF/TrkB signaling which could be overcome through exogenous BDNF application.

ConclusionsAs demonstrated by the results, there is no doubt that NHE6-associated endosomes play a critical role in the arborization of axons and dendrites. Failures in axonal and dendritic branching will significantly impair neuronal connectivity as shown both in vitro and in vivo, and these deficits will eventually lead to a decrease in the strength of the overall circuit, and to fewer functional synapses. Even tough there was a significant increase in the degradation of TrkB, NHE6 seems to have an attenuating effect on TrkB signaling. It is definitely not an absolute blockage because Ouyang and colleagues where able to rescue the neuronal growth by turning up the signaling pathway.

CS is caused by a mutation in the Na+/H+ exchanger 6 (NHE6) gene, which encodes the NHE6 protein responsible for modulating the luminal pH of endosomes2. Luminal pH plays a critical role in the regular functioning of endosomes and downstream endosomal signaling pathways. It has been previously established that within the endosome, a vacuolar H+-ATPase (V-ATPase) enzyme is in charge of acidifying the lumen by pumping protons across the plasma membrane3. On the other hand, the NHE6 protein opposes V-ATPase by exchanging Na+ and H+ ions down their concentration gradient, thus allowing protons to leak out of the endosomal compartment2. Therefore, V-ATPase and NHE6 proteins act together to closely monitor the acidity and alkalinity of endosomes.

Though much investigation remains needed for endosomal biology within neurons, it is known that one of the downstream signaling pathways is the BDNF/TrkB pathway4. BDNF is a member of the neurotrophin family that is important for cell proliferation, neuronal growth and maturation, neural plasticity, and neuronal survival and resilience5. When BDNF binds to TrkB receptor, TrkBautophosphorylates its kinase domain and triggers a series of signaling pathways that eventually leads to the activation of CREB and transcription of genes necessary for neuronal survival5. Consequently, a dysregulation of endosomal activity could cause degradation and death of neurons leading to pathological disorders.

Major ResultsI. NHE6 mutant hippocampal neurons causes reduction in dendritic and axonal branches.Enriched NHE6 expressions were found at hippocampal axonal and dendritic branching points. The researchers hypothesized that ablating NHE6 protein would lead to inadequate axonal and dendritic branching. By generating mouse hippocampal cultures of NHE6 null and WT transfected with GFP-vector in vitro, the researchers found great decrease in axonal branch point compared to WT. Also, basal and apical region of pyramidal neuron in CA1 and CA3 stained by Golgi-Cox revealed significant reduction in number of axonal branch in vivo. These results indicate that NHE 6 protein axonal and dendritic branching process overtly depends on NHE6 protein.

II. Functional domain of NHE6, the protein cation exchanger, is critical for axonal and dendritic branching.It was then hypothesized that axonal and dendritic branching deficit in NHE6 null neuron would be rescued by cation exchanger function. NHE 6 knock out neuron with full-length human NHE6 containing cation exchanger function was significantly rescued; however, impairment of branching in mutant was not rescued in the presence of cation exchanger-deficient NHE6 protein (E255Q/D260N). The NHE 6 functional domain, cation exchanger, was determined by amino acid alignment. However, western blot



Figure 1. Proposed model by Ouyang and colleagues. NHE6 gene affects TrkB/BDNF signaling pathway for neuronal cell growth by accelerating the acidification of the endosomal compartment, increasing degradation of TrkB and attenuating its signal9.

Observations seen in this study are also relevant to other conditions such as autism, wherein NHE6 may be actually be down-regulated for certain subcategories6 . However, there are also some other forms of autism that may result from too much branch growth7. The issue would then become how to determine whether a child diagnosed with autism has too much or too little neural branching. Researchers also noted that TrkB levels in response to leupeptin treatment were partially and not completely rescued in the mutant. One plausible explanation could be that the mutant was producing less TrkB due to genetic modification.

Because of the mechanism proposed by the researchers, there is a plausible future direction for potential interventions in CS and related forms of severe autistic disorders. In fact, there are already drugs capable of delivering doses of chemicals that increase or mimic BDNF in the body. But, nevertheless human clinical trials are still required. Similar studies have already been carried out involving TrkA pathways, and their results have revealed those presented in this paper by showing that NGF signaling is down-regulated at low pH8. It would be interesting to see if just there is also a reduction in ligand affinity between TrkB and BDNF due to over acidification and to examine if the stability of the receptor itself is affected.

Although the reasoning behind using the hippocampus instead of cortex to study the effect of NHE6 mutation is not mentioned within the paper, one postulation is that compared with the cortex, changes in hippocampus may happen in a shorter period of time, as hippocampus is the region in which short-term memory and thus new synapses are first formed, and only with repeated stimulation would the cortex form new synaptic connections. Finally, other endosomal processes are likely to be involved in the cellular pathology underlying CS, and there are other potential consequences yet to be explored that result from NHE6 mutation.

References1. Gilfillan, G.D., , et al. SLC9A6 mutations cause X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome. Am. J. Hum. Genet. 82, 1003–1010 [2008].2. Ohgaki, R., van IJzendoorn, S.C., Matsushita, M., Hoekstra, D., and Kanazawa, H. Organellar Na+/H+ exchangers: novel players in organelle pH regulation and their emerging functions. Biochemistry 50, 443–450 [2011].3. Mindell, J.A. Lysosomal acidification mechanisms. Annu. Rev. Physiol. 74, 69–86 [2012].4. Reichardt, L.F. Neurotrophin-regulated signalling pathways. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 1545–1564 [2006].5. Chao, M.V., and Lee, F.S. Neurotrophin survival signaling mechanisms. J. Alzheimers Dis. 6(Suppl), S7–S11 [2004].6. Schwede, M., Garbett, K., Mirnics, K., Geschwind, D.H., and Morrow, E.M. (2013). Genes for endosomal NHE6 and NHE9 are misregulated in autism brains. Mol. Psychiatry. Published online March 19, 2013.7. Wayman GA, Bose DD, Yang D, Lesiak A, Bruun D, Impey S, Ledoux V, Pessah IN, Le PJ. (2012). PCB-95 Modulates the Calcium-Dependent Signaling Pathway Responsible for Activity-Dependent Dendritic Growth. Environ Health Perspect 120(7): 1003–1009.8. Harrington, A.W., St Hillaire, C., Zweifel, L.S., Glebova, N.O., Philippidou, P., Halegoua, S., and Ginty, D.D. (2011). Recruitment of actin modifiers to TrkAendosomes governs retrograde NGF signaling and survival. Cell 146, 421–434.9. Ouyang Q, Lizarraga SB, Schmidt M, Yang U, Gong J, Ellisor D, Kauer JA, Morrow EM. (2013). Christianson Syndrome Protein NHE6 Modulates TrkBEndosomal Signaling Required for Neuronal Circuit Development. Neuron 80, 1-16.



Neuroscience MattersCerebral organoid model recapitulates early human brain development in-vitro: with applications to primary microcephaly Benjamin Ong1, Waldo Lefever1, and Zhuoliang (Ed) Li1


IntroductionIn the past, the study of neurodevelopment has been mostly limited to cell cultures and animal models. When it comes to human neurodevelopmental disorders, these models are limited in their application due to their inability to parallel certain brain structures during development. One particular area for concern is the subventricular zone (SVZ) due to its role in neurogenesis. It is here that progenitor populations give rise to neurons, which migrate into the organized layers that will become the cortex. Humans have an inner fiber layer, which is absent in mice, dividing the SVZ into an inner and outer portion. Within the outer SVZ in humans, there are many outer radial glia (oRG) and intermediate progenitor cells, which proliferate to amplify the neuronal precursor population1. The emergence of this region with oRG is thought to have been critical in the evolution of a larger and more complex brain2.

Advances in our understanding of stem cells has led to the development of a new in vitro technique in which organoids, three-dimensional tissues that resemble mini-organs, are grown. One major appeal for this new approach is the ability to use human induced pluripotent stem cells to generate the organoids. While growing ‘mini-human brains’ has tremendous potential as a model for in vitro studies, it remains unknown how well these cerebral organoids could recapitulate human neurodevelopment and diseases. Previous experiments had cultured isolated neural tissues, but various aspects of tissue formation and organization, including the oRG, were not described, encouraging a more comprehensive assessment of the model3.

Microcephaly is a neurodevelopmental disorder clinically characterized by a head circumference that is three or more standard deviations below average. Microcephaly can be broadly categorized as either primary or secondary. Primary microcephaly refers to prenatal abnormalities, resulting in smaller than expected brain size at birth. Secondary microcephaly refers to stunted brain growth after birth4. There are many potential causes for microcephaly, including genetics, environmental factors, and metabolic disorders. For the purposes of this study, the authors investigated CDK5RAP2, an autosomal recessive gene known


to cause primary microcephaly in humans5. Previous attempts to study CDK5RAP2 and related genes with mouse models were difficult because the severity of the disorder could not be replicated, and the co-occurrence of uncharacteristic symptoms like anemia and hematopoietic tumors were observed6,7,8. It has been hypothesized that the pathogenesis may involve fewer rounds of replication, or a reduced population of proliferating neuron precursors. The genetic mutation may disrupt normal function of mechanisms pertinent in the outer SVZ, which would explain the phenotypic difference between species.

Major ResultsAnalysis of discrete brain region development in cerebral organoidsIn order to study the heterogeneity and regionalization of cerebral organoids, Lancaster et al performed histological analysis using distinct biomarkers. Organoids collected at 16 days of differentiation were stained with forebrain (PAX6), and hindbrain (PAX2) biomarkers. Forebrain and hindbrain regions were clearly segregated by the biomarkers. Other areas targeted included the ventral cortex, dorsal cortex, retina, choroid plexus and hippocampus.

Although all of the organoids formed a dorsal cortex, only 34% of specimens expressed ventral forebrain identity. Organoids that developed ventral forebrain contained interneurons with calretenin positive (CR+) neurites protruding to the dorsal cortex. However, specimens lacking ventral forebrain fail to develop CR+ neurites as well as dorsal cortex CR+ interneurons. These observations suggest that interneurons first develop in the ventral forebrain and migrate to the dorsal cortex afterwards. Thus, this is an example where ventral regions of the organoid are able to influence the development of dorsal structures.

Recapitulation of dorsal cortical organizationThe preplate is a structure that serves as the precursor of the cortical plate during embryonic development. The preplate biomarker TBR1 was used to verify proper development in the organoid. Radial glial cells (RG) electroporated with GFP were analyzed through live imaging. The RGs engaged in interkinetic mitotic nuclear migration (IKNM), a characteristic of neurogenesis that occurs in the ventricular zone during cortical development9.

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The SVZ of human and mouse organoids were contrasted through histological analysis. The IFL was only found in human organoids, which suggest a distinction in dorsal cortex development between these species.Additionally, while the mitotic spindle orientation of RGs in the mouse organoids was mostly horizontal (parallel to the ventricular surface), the human organoids exhibited vertical and oblique orientations. It was previously found that that the orientation of mitotic spindles affects the outer radial glial cell population size10. This phenomenon could account for the interspecies difference in cortical size.

In another set of experiments, Lancaster and colleagues were able to show that the neurons in the cerebral organoids are functional usingcalcium dye imaging. Basal calcium oscillations were observed and recorded. Furthermore, the calcium oscillations were shown to be positively affected (larger spikes) by the infusion of exogenous excitatory neurotransmitter glutamate and were negatively affected (inhibited) by the infusion of the depolarization blocking tetrodotoxin.

Cerebral Organoids model human microcephalyInduced pluripotent stem cells, (iPS), were derived from a microcephaly patient and controls. The iPS from both groups were segregated into two sets of organoid cultures. Bright-field and histology images illustrate a significant decrease of neurons and progenitor zones at 30 days of differentiation in organoids derived from the microcephaly patient. It has been hypothesized that the pathogenesis may involve early differentiation of neural progenitors, depleting progenitor pools and hence reducing the total number of cortical neurons in later stages of neurodevelopment. Histological analysis illustratesa significantly greater proportion of matured neurons in the microcephaly organoid model at 22 days (early stage) of differentiation.Premature differentiation observed in the microcephaly model supports the hypothesis stated above.

CDK5RAP2 mutations were analyzed through exon sequencing in the microcephaly patient. The phenotype of this mutation leads to CDK5RAP2 protein loss. CDK5RAP2 protein was electroporated into microcephaly organoid models at 12 days of differentiation. There was a significant increase of RG progenitor cells and neurons in microcephaly organoid models exposed to CDK5RAP2 in contrast to their control. These results demonstrate that the microcephaly phenotype can be rescued when CDK5RAP2 is administrated.

Conclusions and Future Directions The cerebral organoid model engineered by Lancaster and colleagues is the first model that was able to recapitulate many aspects of the developing human brain that were not captured by previous models3 such as the formation of discrete brain regions, dorsal cortical organization, rudimentary cortical layer organization, interkinetic nuclear migration, and the formation of

functional cerebral cortical neurons. In a second set of experiments, Lancaster and colleagues were able to apply the cerebral organoid in practice and model primary microcephaly. Furthermore, the group was able to observe the neurodevelopmental causes of the CDK5RAP2 variant of primary microcephaly and was able to reverse the condition using a gene therapyapproach.

Although the cerebral organoid model is novel, unique, and possesses much potential, there are many severe limitations. The cerebral organoid model was only partially successful at recapitulating the complex layer organization found in the human brain. Furthermore, the organoid model failed to recapitulate the shapes of many brain structures. It could be hypothesized that the current limitations associated with the model is the result of the poor circulation to the inner core of the cerebral organoid that ultimately limits the size of the organoid to a maximum thickness of 4mm, a size that is insufficient for proper brain development.

To counteract the poor circulation and the size limitation, future researchers should utilize a scaffold wired with a series of semipermeable tubes. The scaffold is a support structure for which the iPS can be grown upon. Scaffolds can be obtained by perfusing out all of the cells in a dissected organ11 or engineered from biomaterials12. iPScan be placed at different regions of the scaffold. As the isolated iPS cultures grow on the scaffold, they will eventually converge into a large structure that will hopefully become the developing brain. Previous researchers12 have made a functional bladder by growing iPS cultures on a bioengineered bladder scaffold. Although the brain and its development are substantially more complex, the possibility is still present. Additionally, the internal semipermeable tubes can act as a makeshift circulatory system that provides the required nutrients for all the iPS cultures along the scaffold. In addition, the internal tubing allows mechanically controlled infusion of a wide range of neurotrophins such as BDNF and NGF at specific times to further mimic the development of the human brain.

The cerebral organoid model is at its infancy. Constrained by many limitations, the cerebral organoid is only able to model simple genetic-based neurodevelopmental disorders with observable physical abnormalities at its current stage. However, the cerebral organoid model is not without a future. If some of its limitations can be overcome, the cerebral organoid model could be a viable tool for a wider range of fields, including pathology, developmental biology, pharmacology, and toxicology.



7) Barrera, J. A., Kao, L. R., Hammer, R. E., Seemann, J., Fuchs, J. L., &Megraw, T. L. (2010). CDK5RAP2 regulates centriole engagement and cohesion in mice. Developmental cell,6, 913–926.8) Pulvers, J. N. et al. Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline. Proc. Natl Acad. Sci. USA 107, 16595–16600 (2010).9) Kudoh, Y., &Iimura, O. Slow continuous hemodialysis--new therapy for acute renal failure in critically ill patients--Part 1. Theoretical consideration and new technique. Japanese circulation journal 10, 1171–1182 (1998).10) LaMonica, B. E., Lui, J. H., Hansen, D. V., &Kriegstein, A. R. (2013). Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex.Nature communications 4,1665.11) Goh, S.A. et al. Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering. Biomaterials34, 6760-6772 (2012).12) Atala, A. et al. Tissue-engineered autologous bladders for patients needing. Lancet367, 1241-1246 (2006).

References 1) Hansen, D. V., Lui, J. H., Parker, P. R., &Kriegstein, A. R. (2010). Neurogenic radial glia in the outer subventricular zone of human neocortex. Nature, 7288,554–561.2) Lui, J. H., Hansen, D. V., &Kriegstein, A. R. (2011). Development and evolution of the human neocortex. Cell, 1, 18–36.3) Eiraku, M., &Sasai, Y. (2012). Self-formation of layered neural structures in three-dimensional culture of ES cells. Current opinion in neurobiology, 5, 768–777.4) Woods, C. G. (2004). Human microcephaly. Current opinion in neurobiology, 1,112–117.5) Kaindl, A. M., Passemard, S., Kumar, P., Kraemer, N., Issa, L., Zwirner, A., Gerard, B., Verloes, A., Mani, S., &Gressens, P. (2009). Many roads lead to primary autosomal recessive microcephaly. Progress in neurobiology, 3, 363–383.6) Lizarraga, S. B., Margossian, S. P., Harris, M. H., Campagna, D. R., Han, A. P., Blevins, S., Mudbhary, R., Barker, J. E., Walsh, C. A., & Fleming, M. D. (2010). Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors. Development (Cambridge, England),11, 1907–1917.



Engineering cognitive facilitation and reversing cognitive impairment: Application of the MIMO-model to a neuroprosthetic in nonhuman primates

Ailya Jessa1, Kamalpreet Mann1, Stephany Francisco1, Michelle Hayano1, and Victoria Marshe1


IntroductionHistorically, the idea of a neural prosthesis and artificial intelligence have been considered to belong in the realm of science fiction. However, over the past few years a number of labs around the world have successfully created prostheses in the rodent brain1. This paper is ground-breaking because this is the first time a neural prosthesis has been successfully used in non-human primates and has not only been used to repair cognitive deficits, but also has been shown to improve cognitive function1.

The area of the brain examined in this study is in the prefrontal cortex (PFC). The PFC has previously been shown to have a very specific minicolumnar organization and an important role in cognition3. A multi-electrode recording array was used to determine the specific area in the PFC used in the delayed matched sample (DMS) task. Once the area was determined, patterns were recorded from this region and were replicated with the multi-input multi-output (MIMO) based prosthesis. The MIMO device was designed to recognize distinct patterns in the brain and differentiate between patterns associated with correct answers and incorrect answers on the DMS task. In this study, the experimenters were able to model cognitive dysfunction in

the area of the PFC involved with the DMS task and then used the prosthetic to restore the cognitive performance and actually showed that they could improve cognition.

Major ResultsObserving Neuronal Firing First, researchers began by testing their original assumption that the L2/3 supragranular layer and L5 infragranular layer minicolumnar organization of the PFC plays a role in cognitive tasks involved in the delayed match of sample task (DMS) task. To determine which neurons were firing during the DMS task, a multi-electrode array device was implanted in the supragranular and infragranular minicolumns of the PFC. During this task, subjects were shown an image on a screen, and after a variable delay, they had to select an appropriate match from variety of different images. Upon selection of correct image, the subjects received a reward. Simultaneously, electrophysiological recordings of output from L2/3 and L5 layer pairs were recorded from 2 seconds prior to match response onset to 2 seconds after match response (Figure 1). Subsequently, recordings showed that at response onset, the L2/3 and L5 neuronal pairs were selectively activated, firing at an increased rate.

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Figure 1: Electrophysiological recordings show selective minicolumnar pair firing at match onset (0s) as shown in (a) and similarly in another pair (b)2.

Figure 2: Application of different electrical stimulation paradigms did not create the same cognitive facilitation as the original MIMO model2.

Integration of the MIMO model In this study, researchers used the recordings to make a computational model that predicted L5 activity based on input from L2/3 layer neurons. Based on the subjects’ decision about the matching image, the output from the computational model was used to electrically stimulate L5 layer neurons. After application of the MIMO model to trials, researchers found that for every subject, MIMO-derived stimulation generally enhanced the match response performance compared to control levels, but this was not consistent across individuals. An improvement was seen in cognitive workload, executive function, attention and working memory. Also, it was shown that the MIMO model was especially effective in improving performance on more difficult tasks (e.g. trials with more images and longer retention latencies).

Figure 3: Scatterplot showing neuronal synchronization for the first half of the recovery trials, and de-synchronization for the latter 60 trials2.

Figure 4: MIMO model application reduced cognitive impairment and restored cognitive function to control and further enhanced performance above normal2.

Validating the MIMO modelIn order to determine whether the MIMO paradigm was responsible for the facilitation of match response performance, researchers applied other stimulation paradigms to observe their effect. These included: (a) random electrical stimulation; (b) sub-threshold MIMO stimulation; (c) MIMO stimulation prior to match onset (prior to 0s); (d) MIMO stimulation after match onset (after 0s); and (e) randomly scrambled MIMO stimulation. Subsequently, as shown in Figure 2, none of these alternate paradigms produced the same kind of facilitation as the original MIMO model.

Inducing and Reversing Cognitive ImpairmentIn order to create a control, subjects completed 60 trials without impairment and 60 trials with cocaine-induced impairment. The control trials suggest a high correlation between the firing of L2/3 and L5 layer neuronal pairs due to firing synchronization.



patients who have memory deficits such as Alzheimer’s patients7. A prosthesis could also potentially be used to alter the production of neurotransmitters and possibly reverse damage after a stroke. If successful in humans, the possibilities for treatment could be endless. It could also have implications for cognitive enhancement in normal individuals. However, it is also important to examine the long term effects of having the implant in the brain and whether it could be harmful to the individual. Overall, if researchers find an effective way of implanting the device in the human brain, this tool could revolutionize the field of rehabilitation in individuals with neurological deficits.

References1. Berger T. W. et al. A hippocampal cognitive prosthesis: multi-input, multi-output nonlinear modeling and VLSI instrumentation IEEE Trans. Neural System Rehabilitation Engineering [2012].2. Hampson, R.E. et al. Facilitation and restoration of cognitive function in primate prefrontal cortex by a neuroprosthesis that utilizes minicolumn-specific neural firing. J. Neural Eng.9, 1-17 [2012]. 3. Buxhoeveden, D. P. et al. The minicolumn hypothesis in neuroscience Brain 125 935–51 [2002].4. Lebedev, M.A &Nicolelis, M.A. Toward a whole-body neuroprosthetic. Prog. Brain Res, 19447-60 [2011].5. Ackerman, E. How smart dust can be used to monitor human thought. Forbes [2013]. 6. Berger, T. W. et al. Restoring lost cognitive function. Ieee Engineering in Medicine and Biology Magazine. 24, 30-44 [2005]. 7. Zada, G et al. Neural prosthesis for recovery of impaired cognitive function: bridging the gap between concept and reality. World Neurosurg. 79, 3-4 [2013]. 8. Laczko, J. Modeling of human movements, neuroprostheses. IdeggyogySz, 64 229-23 [2011].

The impairment condition show a de-synchronization of firing in the neuronal pairs which is suggestive of cognitive impairment. When the MIMO model neuroprosthetic was applied, not only did the MIMO model effectively reverse the effects of cognitive impairment, but it also enhanced cognitive performance to levels similar to those in the original control plus MIMO-facilitation trials (Figure 3).

ConclusionsHampson and colleagues were thorough in confirming their assumptions of the inter-laminar relationship between layer L2/3 and L5 neural firing and its role in decision-making. Their results have shown that the MIMO model effectively delivered the appropriate firing patterning based on the mathematical analysis of the MIMO model during both baseline and cognitive impairment trials. In addition, this study was unique from prior studies that developed prosthetics that normally target the sensory input and motor output pathways or prosthetics that directly effect corticospinal and thalamo-motor systems4. Here the researchers applied a neuroprosthetic model that bypassed the sensory-motor pathways and rather influenced cognitive decisions that govern motor movements via the PFC5.

While the MIMO model effectively enhanced the performance in decision-making via layers L2/3 and L5 neurons, there are still issues that need to be assessed before implementing this prosthetic device to future clinical trials. First, the DMS task is limited to testing certain cognitive processes. The MIMO model should be examined with other cognitive tasks and in other inter-columnar pathways. Second, the animals were placed under strenuous procedures. Using a non-invasive and wireless apparatus such as neural dust can potentially reduce the amount of trauma and infection6.

The next steps in future research would be to test animals with natural cognitive impairment due to neurological disorders rather than pharmaceutically inducing impairment. Also, researchers may want to explore why each primate showed different degrees of cognitive improvement following prosthesis stimulation. In future trials, researchers should measure the depth of cortical stimulation with MIMO model signalling to see how deep MIMO signals can reach. As well, if researchers can test whether MIMO model outputs can be used to inhibit or interfere with overactive connections in certain disease pathologies. Instead of strengthening signalling of impaired circuitry as shown in this study, hindering the over-activity of defective circuitry that result in detrimental symptoms.

The ultimate goal of future research is to use cognitive neural prostheses in humans. If accomplished, neural prostheses can be used to treat a wide variety of disorders. For instance, if implanted in the hippocampus, the prosthesis can improve memory in



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