Molecules, Behavior, and Insular Regulationaliceabr/mol_behav_insular_reg.pdf · * tetanic...

Molecules, Behavior, and Insular Regulation

By Bradly Alicea

NEU 820, Summer 2008

Introduction (1.6)Introduce molecular approaches to cognition:

* processes at the cellular and molecular

level (synaptic modification).

* high-throughput data collection.

* introduce a multi-scalar approach

to cognition (from genes to behavior

using a simple mechanism, pathway).

* understand how regulation at the molecular

level ~ changes associated with environmental

challenges, learning and memory.

Introduction to Physiological Regulation:Homeostasis: first-order regulation (simple feedback mechanism produces response that keeps system stable).

* molecular regulation = organizing principles of the ‘internal milleu’.

http://www.molcellcog.org/

Introduction (2.6)Learning, Memory, and Adaptation (3 stage model):

Acquisition – getting

information from the

environment.

Consolidation – storing

information in a long-term

physical medium (synapse).

Recall – retrieving information

From physical medium.

* initially, memory is stored

in short-term (working)

memory.

* reinforcement reduces

susceptibility (interference),

strengthens memory trace.

Exercise and taste aversion (non-declarative). Insula = classical conditioning.

* interference: where one memory signal interferes with another (or protein synthesis interfered with).

* time-course = minutes (ultrastructural changes) to hours (synaptic modification).

Introduction (3.6)

#1: Input specificity- Induced, maintained one synapse at a time (stimuli do not have direct network effects).

#2: Associative - when weak stimulation of one pathway won’t induce LTP, strong stimulation of associated pathway = LTP in both.

#3: Cooperative - weak stimulation of manyconvergent pathways (singleLocation on post-synaptic membrane) = LTP.

Three phases of LTP:

A)induction: stimulus produces a large concentration of calcium in post-synaptic cell, depolarization magnitude important in transient activation (protein kinases play a role).

B) maintenance: persistent activation of CaMKII and Protein Kinase C (PKC).

C) expression: phosphorylation of AMPA receptors, transported back to postsynaptic membrane. Provides feedback mechanism via two mechanisms:

i) increases number of receptors

ii) increases efficacy of receptors (phosphorylated)

LTP = a candidate mechanism for (not necessarily concurrent with) memory consolidation.

Introduction (4.6)

Long-term changes at molecular scale: neuropeptides,messenger RNAs, growth factors all produced in soma (nucleus),transported to synapse (in vesicles).

Neurotransmistter release: during depolarization, content of vesicles released into synaptic cleft.

Receptors, ion channels on postsynaptic surface. Molecules releasedfrom presynaptic cell either taken up by postsynaptic cell or not.

Sustained activity in pre- and post-synaptic cells = LTP, LTD. Activates molecular pathways (e.g. ERK). AMPA receptors in postsynaptic membrane phosphorylated, enhanced function.

What is a gene, and how is it expressed?Gene = promoter region, coding region (non-contiguous exons).

Transcription factor = biomolecule binds to promoter, activates gene.

RNA polymerase = produces gene product. Basis for mRNA, miRNAs, and proteins (growth factors, neuropeptides).

Introduction (5.6)

Kinase/Phosphotase equilibrium: Phosphorylation = addition of a phosphategroup to a biomolecule. Introduction of this complex to structure ‘activates’ molecule (biologically active).

Kinases: enzymes involved in ‘forward’phosphorylation. Switchesreceptors and downstreammolecules ‘on’.

Phosphotases:enzymes involved in ‘reverse’(de) phosphorylation. Switches receptors and downstreammolecules ‘off’.

Changes conformationalstructure of proteins, activatessignal transduction pathways.

http://www.chinaphar.com/1671-4083/25/977.htm

http://content.answers.com/main/content/wp/en/thumb/6/64/260px-Reversible_phosphorylation.PNG

http://jkweb.berkeley.edu/external/pdb/2001/tgf_beta_R1/irk_

phosphorylation.jpg

http://www.scq.ubc.ca/wp-content/uploads/2006/07/phosphocascades.gif

Introduction (6.6)High-throughput methods:

Whole-genome methods (DNA):Pyrosequencing (Roche-454): sequencing using pulses of light! “Sequence by Synthesis”;chemoluminescent enzyme identifies complementary strand as it is assembled on primer sequence.

Shotgun: DNA broken into small segments, produces “reads” (fragments that overlap). Overlappingfragments used as sequence data, rest of bases inferred. n-fold coverage.

HumanHap 550 Bead Chip (Illumina): genome-wide scan, detects 550k single-nucleotidepolymorphisms (SNPs - single-base differences), not sequences per se. Used in disease research.

Gene product methods (RNA):Microarray (Affymetrix): tile a glass array with multiple primer sequences, hybridize with DNAfragments. Each cell floureseces according to relative presence of gene (measure of expression level).

RNA-seq (Illumina, Solexa): transcripts converted to cDNA, fragmented and sequenced using high-throughput methods (sequence across genome).

RT-PCR: two reactions, convert transcripts to cDNA, amplify DNA fragments using PCR. Compare specificgene products against reference (18S, Actin). Quantitative (qRT-PCR).

Molecular Learning, Memory, and

Adaptation (1.5)

Taste aversion:* glossopharengeal nerve from palate.

* projects to NTS (along with vagus nerve, which is involved in fear conditioning and sympathetic regulation).

* projects to thalamus, then to insula and frontal operculum.

Sympathetic control:* pathway from insula to the Prefrontal Cortex (sympathetic “state” information).

* right insular cortex = cardiac “representation”

* stimulation = tachycardia, increased blood pressure.

* control via ipsilateral basolateral amygdala.

http://www-psych.stanford.edu/~lera/psych115s/notes/lecture11/images/tastepath1.jpg

Bogousslavsky, J. (2003). Acute Stroke Treatment. Taylor-Frances, New York.


Adaptation (2.5)Postsynaptic response in insular neurons:

* new taste stimulus, conditioned stimulus, upregulationof acetylcholine, activates PKC, ERK pathways (long-termmemory).

* unconditioned stimulus, afferents from basolateralamygdala, upregulate glutamate, intracellular calcium.

Behavioral interference (Learning and Memory,13,571-574, 2006):

* two novel tastes introduced at separate intervals(anterograde/retrograde).

* input of a second novel taste (if familiar) may interferewith consolidation of first novel taste.

* rats remember last novel taste they consume, memorytrace vulnerable for 10h.

* process of learning (not retrieving new tasteinformation) disrupts consolidation of former knowledge.

Bermudez-Rattoni, F. (2004). Molecular mechanisms of taste-recognition memory. Nature Reviews

Neuroscience, 5, 209-217.


Adaptation (3.5)Alberini, C.M., Taubenfeld, S.M., and Garcia-Osta, A. (2005). CREB and the CREB C/EBP-dependent

Gene Expression Cascade in Long-term Memory. Cell Science Reviews, 2(2).

http://www.cellscience.com/reviews6/CREB_long-term_memory.html

Hypothesis: Stimulus >> immediate-

early genes (c-fos) >> cyclic AMP >>

CREB (cAMP response binding protein

activation) >> memory consolidation.

Take-home message: time-limited CREB-

dependent consolidation mechanism.

1) inject HC neurons with CREB

antisense (3’-5’), bind up CREB.

* prior to training, impair retention

up to 48hr after; 24hr after training,

no effect.


Adaptation (4.5)2) In mouse HC, CREB KO = spatial, contextual,

and cued L&M deficits.

3) Timecourse of CREB cascade :

* CREB activation vs. C/EBPβ (CREB cascade takes 24h to induce expression of C/EBPβ).

* co-occurrence of CREB activation and PKA

phosphorylation (in HC, peak at 0-30 minutes and

3-6 h post-stimulus).

CREB is a transcription factor (protein that effects expression of downstream gene).

CREB activation dependent on phosphorylation at Ser-133 residue (to initiate cascade).

http://www.mcb.mcgill.ca/~hallett/GEP/

PLecture6/2Hybrid_files/image002.jpg


Adaptation (5.5)

Jones et.al, Journal of Neuroscience, 19, RC36, 1999. Long-term potentiation (LTP) in vivo in insular cortex:

* synaptic plasticity, contributes to neuronal connectivity.

* connections between the basolateral amygdala (BLA) and insula (central gustatory area) in rat brain.

Range of molecular changes after novel taste learning in insula (tyrosine phosphorylation of NMDA receptor, increased PKC and ERK 1/2 (kinase) activity:

* tetanic stimulation of BLA pathways into insula induced a stable potentiation

* blockade of NMDA and Ach receptors attenuates novel taste learning (even during tetanus).ERK 1/2 required for LTP in insula, or a by-product of LTP?

* 30 minutes after LTP, ERK 1/2 upregulation (significant vs. contralateral insula).

* injection of a MEK inhibitor (MEK activates ERK 1/2) 30 min. before tetanus successfully blocked insular LTP.

Experimentally uncover/identify nervous

system regulatory mechanisms (1.4)#1: Amnestic Agents.

Two ways to induce amnestic state in experimental animals:* Weingarten et.al (1984). Memory consolidation: psychobiology of cognition. L. Erlbaum and Assoc.

1) Electroconvulsion, hypothermia. Disrupt neural transmission before consolidation

(passive storage state).

2) Antimetabolites (block protein synthesis). Also prevent conversion of memory into a

passive storage state.

Example:

Hydroxylamine: efficacious up to three weeks after training of passive avoidance or

water maze navigation. Other functions and subsequent learning unaffected, and no

return of erased memory trace.

* Reinis, S. Journal Inflammation Research, 2(5), 216-222, 1972.


system regulatory mechanisms (2.4)#2: Stress Response.

Cornett et.al (2000). J. Applied Physiology, 89, 1432-1436. Ischemic exercise and

the muscle metaboreflex:

* interstitial metabolites accumulate and stimulate muscle afferents.

* evokes muscle metaboreflex and rasises arterial blood pressure.

* greatest reflex response at 60% maximum voluntary contraction.

As tension increases, factors aside from insufficient blood flow = effect on

metabolites, magnitude of reflex response.

* low-tension = slow twitch, fatigue-resistant. High-twitch = fast-twitch, fast-fatigue.

* fast and slow twitch motor units recruit metabolites, muscle reflex.

* slow twitch recruitment = less metabolic product over time (developed tension).


system regulatory mechanisms (3.4)

#3: Response to Exercise Stimulus.

Williamson et.al (2003). J. Applied Physiology, 94, 1726-1734.

Static Handgrip Exercise: created a tension that triggers expression of metabolites. Due to

reflex response, this activity can maintained.

* how much work does the brain do after the exercise experience ends?

Post-SAG cuff occlusion of working arm (maintains muscle metaboreflex). After muscle

activity, sustained 15mm/Hg blood pressure increase.

* during SHG, cerebral blood flow increases 4-10% in right inferior posterior insula,

increases 6-10% in left inferior anterior insula.

* not found during post-exercise circulatory occlusion.


system regulatory mechanisms (4.4)#4: Induce pre- and ultrastructural changes: Swimming in Lebistes reticulatus (Palacios-Pru and Colasante, Journal of Neuroscience Research, 1983, 1988).

“ultrastructural” changes in neuro-muscular junction (NMJ) in fin muscle due to sustained training.

* fish exposed to inertial hydrodynamic forces such as current, need to maintain attitude (i.e. roll, pitch)

* 30-40 minutes of training: nearly complete depletion of synaptic vesicles (83%), nearly all axon terminals exhibited sprouting (76%).

* rest after training, gradual return to pre-training phenotype (80 minutes).

* maintainance of homeostasis: to maintain stable neurotransmitter release (in scenario of perpetual exercise without rest periods), system may either adapt or utilize another mechanism.

Role of Insula in Sensory Feedback

Integration (1.4)Emotional memory: another form of fast (one trial) memory, involves basolateral amygdala.

Pathway from periphery to insula to basolateral

amygdala, plays role in consolidation:

* Zif 268 (Egr1) = immediate early gene

(positive regulator).

* CaN = calcineurin (phosphotase, negative

regulator).

Immediate Early Genes (IEGs) =

* transcription activated within minutes

after extracellular stimulation (expression

low or nonexistant in non-stimulated

cells). Can encode transcription factors

(promote expression of downstream

genes).


Integration (2.4)Baumgarten et.al (2008). Control of the establishment of aversive memory by calcineurin.

Nature Neuroscience, 11, 572-578.

Aversive Memory

CaN

Zif268

Normal Initial Consolidation

Aversive Memory

KO

Zif268

Stronger Initial Consolidation (robust)

+

+

+

- - -

+ +

+ +

LEFT: normal activation and expression.

RIGHT: CaN knockdown, driven overexpression of Zif268.

* robustness = initial strength of memory.

* Zif 268 (Egr1) = immediate early

gene (positive regulator).

* CaN = calcineurin (phosphotase,

negative regulator).


Integration (3.4)

Morris, Trends in Cognitive Science, 6(8), 317-319 (2002).

Does your heart pound because you feel afraid, or do you feel afraid because your heart is racing?

Pure Autonomic Failure (PAF): disorder ofperipheral nervous system.

* do not increase heart rate, blood pressure,show skin conductance changes with emotionalstimuli (sympathetic activity).

* role of insula – supports representations of peripheral autonomic arousal, provides input to conscious awareness of emotional states.

PAF – lack feedback, normal insular function during fear conditioning.

* you feel afraid because your heart is racing (see figure).


Integration (4.4)Three types of “ception”:

* proprioception (forces from muscle, joints.

* exteroception (inputs from vision, audition).

* interoception (third leg of sensory reception).

Interoception, mediated by insula, also affects other two legs:

* adaptive (learning) response in insula – organizing center of consciousness involving all inputs?

* insula = point of emergence for consciousness??

How does this fit together (1.5)?

Emergent phenomena:

Collective activity of many individual components = coordinated, macrostate behavior.

Emergence of Cognition:

Collective activity, modification of synapses = interaction of neurons = coherent regulatory

mechanisms in specific brain regions.

Top-down (constraints): genotype, sensory stimuli, other aspects of cognition, behavior.

Bottom-up (information): synaptic interactions, neuronal interactions, molecular and

anatomical cross-talk.


Examples of social behaviors fromphysiological, cellular-level mechanisms:

* East African cichlids (Haplochromis burtoni):

Social rank affects endocrine release, phenotype, subsequent social rank.

Hofmann and Fernald (2000). Journal of Neuroscience, 20(12), 4740-4744.

* Side-blotched lizards (Uta stansburiana):

Transitive hierarchy, influenced by genetic/endocrine factors (difference between males and females).

Alonzo and Sinervo (2001). Behavioral Ecology and Sociobiology, 49, 176-186.

* Fire ants (Solenopsis invicta):

GP-9 polymorphism, affects phenotype = social rank.

Bourke (2002). Trends in Genetics, 18(5), 221-223.


High-throughput cognitive neurobiology:

How do we monitor, measure activity at multiple scales simultaneously?

* develop methods to measure many genes, synapses, etc. in parallel.

* use computational methods to interpret,

analyze results, discover patterns.

* establish regularities (e.g. from

traffic flow to traffic jams).

* record instances of emergence in

real brains.

* interactions will form activational

networks (ongoing work - fMRI). Dosenbach et.al, (2007). Distinct brain networks for adaptive and stable task control in humans.

PNAS 104(26), 11073–11078.


What do Genetic Regulatory Networks (GRNs) and metabolic pathways do for

Behavior and cognition?

* genes up- and down-regulated given the current, cumulative state of the

internal millieu.

* produces, maintains pools of mRNA (transcripts), regulatory mechainsms,

and proteins.

* affects metabolism, hemodynamics, and electrical activity either directly or

indirectly.

* current measures of brain activity/function (PET, EEG, fMRI) measure these

variables (and are ultimate driving forces behind “brain function”).


IBM BlueBrain Project: http://bluebrain.epfl.ch/page17871.html

* create transgenic neurons (~30 cell lines).

* each line expresses only a specific ion

channel developmentally (genetic knockout

method).

* yield different firing patterns (alone and in

a population).

* differential behavior from different cell types

(analogue with evolution).

* Blue Brain: towards a better artificial

intelligence…..

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