7,8-dihydroxyflavone rescues spatial memory and synaptic plasticity in cognitively impaired aged...

Age-related cognitive deficits are highly prevalent health risksin the human population (Bishop et al. 2010), which maypresage development of age-related neurodegenerative dis-ease (Floyd and Hensley 2002; Chong and Sahadevan 2005;Mariani et al. 2007; Petersen and Negash 2008). The declineof learning and memory during aging involves changes inmultiple systems (Floyd and Hensley 2002). Neurotrophinsand their receptors, most notably Brain-derived neurotrophicfactor (BDNF) and tyrosine receptor kinase B (TrkB), areexpressed in brain areas exhibiting a high degree of plasticity.They are considered as important molecular mediators offunctional and structural synaptic plasticity and play essentialroles in memory formation and consolidation (Tapia-Aranci-bia et al. 2008; Minichiello 2009; Simmons et al. 2009;Waterhouse and Xu 2009). We and others have shown that theBDNF-TrkB system in the hippocampus is particularlysensitive to the aging process (Karege et al. 2002; Lom-matzsch et al. 2005; Silhol et al. 2005; Zeng et al. 2011),

which is consistent with age-related impairments of hippo-campus-dependent memory observed in humans, rats, mice,and other mammals in a wide variety of tests (Rapp andGallagher 1996; Driscoll et al. 2003; Rosenzweig and Barnes,2003). These findings suggest that the BDNF-TrkB signalpathway may be a viable therapeutic target (Tapia-Arancibiaet al. 2008; Zuccato and Cattaneo 2009). Jang and his

Received April 03, 2012; revised manuscript received May 29, 2012;accepted June 04, 2012.Address correspondence and reprint requests to Yan Zeng, Depart-

ment of Pathophysiology, School of Medicine, Wuhan University ofScience and Technology, Wuhan 430065, China.E-mail: [email protected] used: 7,8-DHF, 7,8-dihydroxyflavone; AI, aged-

impaired rats; AU, aged-unimpaired rats; BDNF, Brain-derivedneurotrophic factor; fEPSP, field excitatory post-synaptic potential; HFS,high-frequency stimulus; LTP, Long-term potentiation; TrkB, tyrosinereceptor kinase B.

*Department of Pathophysiology, School of Medicine, Wuhan University of Science and Technology,

Wuhan, Hubei, China

�Hanyang Affiliated Hospital of Wuhan University of Science & Technology, Wuhan, Hubei, China

Abstract

7,8-dihydroxyflavone (7,8-DHF) has recently been identified

as a potential TrkB agonist that crosses the blood–brain barrier

after i.p. administration. We previously demonstrated that 7,8-

DHF in vitro rescues long-term synaptic plasticity in the hip-

pocampus of aged rats. This study assessed the rescue effect

of 7,8-DHF in vivo on aging-related cognitive impairment in

rats, and further determined whether the effect of 7,8-DHF is

age dependent. Aged rats at 22 and 30 months of age were

pretested for spatial memory in Morris water maze. The aged-

impaired rats were retested twice during 7,8-DHF or vehicle

treatment, which started 3 weeks after the completion of the

pretest. In the 22-month-old rats, daily i.p. administration of

7,8-DHF for 2 weeks improved spatial memory. The

improvement in behavioral tests was associated with in-

creases in synapse formation and facilitation of synaptic

plasticity in the hippocampus, as well as the activation of

several proteins crucial to synaptic plasticity and memory. A

more extended treatment paradigm with 7,8-DHF was required

to achieve a significant memory improvement in the severely

impaired 30-month-old rats. Moreover, 7,8-DHF moderately

facilitated the synaptic plasticity, modified the density but not

number of spines in the hippocampus of the oldest rats. Taken

together, our results suggest that 7,8-DHF can act in vivo to

counteract aging-induced declines in spatial memory and

synaptic plasticity and morphological changes of hippocampal

neurons. The effect of 7,8-DHF is more pronounced in rela-

tively younger impaired rats than in those of more advanced

age. These findings demonstrate the reversal of age-depen-

dent memory impairment by in vivo 7,8-DHF application and

support the benefit of early treatment for cognitive aging.

Keywords: 7,8-dihydroxyflavone, aging, learning and mem-

ory, spines, synaptic plasticity, TrkB receptors.

J. Neurochem. (2012) 122, 800–811.

JOURNAL OF NEUROCHEMISTRY | 2012 | 122 | 800–811 doi: 10.1111/j.1471-4159.2012.07830.x

800 Journal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 122, 800–811� 2012 The Authors

colleagues (Jang et al. 2010) recently screened a chemicallibrary for compounds that activate TrkB in vitro anddiscovered a series of flavone derivatives with TrkB agonistproperties, most potently 7,8-dihydroxyflavone (7,8-DHF).7,8-DHF binds in vitro with high affinity to the TrkB receptorand provokes its dimerization and autophosphorylation,leading to downstream signaling cascade activation. Studieshave demonstrated that 7,8-DHF is the first drug to imitateBDNF actions and enter the brain with much more efficacythan the protein (Choi et al. 2010; Jang et al. 2010), making itan attractive candidate drug for memory impairment. We havereported that in vitro application of 7,8-DHF rescues long-term synaptic plasticity in the hippocampal slices of aged rats(Zeng et al. 2011). Other studies in vivo also indicate thatperipheral administration of 7,8-DHF can activate TrkB in thebrain, rescue memory impairment in young rodent models(Andero et al. 2011, 2012). However, the evidence is stilllacking for the effectiveness and time window of in vivotreatment with 7,8-DHF for aging-related cognitive deficits.

This study evaluated in vivo the long-term effect and agedependence of 7,8-DHF action on spatial learning andmemory, dendritic spine formation, and synaptic plasticity inthe aged Sprague-Dawley rats. Our results demonstrate thatchronic treatment with 7,8-DHF could significantly reversethe synapse loss and enhance the synaptic plasticity inhippocampus, and improve acquisition and retention of aspatial memory in cognitively impaired aged rats; the effectof 7,8-DHF is age dependent.

Methods

AnimalsSprague-Dawley male rats were obtained from Tongji animal center,Huazhong University of Science and Technology. Animals werekept at the vivarium before use under standard conditions: 12 h lightand 12 h dark; lights on at 6:00 a.m.; temperature: 22 ± 2�C; waterand food ad libitum. All rats were handled once a week and checkedfor health problems (tumors, body weight). Behavioral testing wasperformed during the daylight period. Animal care and experimentalprocedures were in accordance with National Institutes of Healthguidelines and were approved by the Institutional Animal Care andUse Committee.

Morris water maze testsThe Morris water maze task was performed as previously described(Adlard et al. 2008). We used a white tank with a 1.2-m diameter,virtually divided into four quadrants. The tank was made opaquewith the addition of non-toxic white paint and filled to a depth of0.5 M with water (22 ± 2�C). A circular 12-cm diameter platformwas submerged inside the tank, and four cue cards were placedaround the tank in each quadrant. The position of the platform andcue cards remained the same throughout the entire experiment. Allaged rats were subject to the pretest that separated them into twogroups: (1) those that learn on par with the young cohorts (i.e., aged-unimpaired rats) (AU) and (2) those that perform outside the range

of young rats, demonstrating impairment on the task (i.e., aged-impaired rats) (AI) (Gage and Bjorkhtnd 1986), 3-month-old ratsserved as young controls. Three weeks after the pretest, the AI ratsreceived daily treatment of either 7,8-DHF or vehicle for 34consecutive days and were retested twice for spatial memory duringthe period of treatment (Fig 1). During the pretest or retest week,rats were trained for six consecutive days for place discrimination,with four trials on each day (random quadrant entry; maximum120 s per trial), followed by a probe trial (one 90-s free swimwithout the escape platform present) 24 h later to assess the spatialmemory retention. After the probe trial, rats were subjected to avisible platform task, in which the escape platform was raised abovethe water line and the time taken to reach the platform was assessedduring two consecutive trials done in separate quadrants of the pool.Thigmotaxis time measured when rats swam at a distance within10 cm of the wall of the pool is an indicator of open space anxiety-related behavior (Wilcoxon et al. 2007). Data were analyzed usingthe EthoVision automated tracking system (EthoVision; Noldus,Wageningen, The Netherlands).

The 22-month-old group. A total of 56 aged (22-months old) and16 young (3-months old) rats were tested in the Morris water maze.Such significant impairment in performance was found in approx-imately 52% of the 22-month-old rats. In contrast, 29% of aged ratswere found to perform within, or better than, one standard deviationof the mean performance of the young rats (Gage and Bjorkhtnd1986). The aged-impaired rats were randomly allocated to the 7,8-DHF-treated (n = 15) or vehicle-treated (n = 14) group, whereas theaged animals selected to the aged-unimpaired group receivednothing (n = 16) served as AU groups.

The 30-month-old group. A total of 36 aged (30-months old) and16 young (3-months old) animals were screened for their ability toperform in the Morris water maze. Impairment in the water mazetask was found in over 90% of the 30-month-old group, using thesame criteria as for the 22-month-old rats. Of the impaired 30-month-old rats, 32 animals were randomly allocated to the 7,8-DHF-treated (n = 16) or vehicle-treated (n = 16) group.

Administrations of chemicals7,8-DHF was purchased from TCI America and prepared inphosphate-buffered saline containing 17% dimethylsulfoxide (Port-land, OR, USA). After the pretesting and a 3-week interval, rats indifferent age groups received daily i.p. injections (i.p. 5 mg/kg) of7,8-DHF or vehicle for 34 days (Fig. 1) and were retested in thewater maze. One day after the completion of all behavioral tests, allrats were anesthetized by CO2 and killed by rapid decapitation, andthe brains were removed from the skull without olfactory bulbs andpositioned in a cold matrix. Some brains were prepared for theelectrophysiological recording. The others were cut into twohemispheres, half for western blot and the other half for Golgistaining. At the time rats were killed, the young rats, referred to as‘young group’ throughout, were 5-months old. However, at the startof the behavioral testing, they were 3-months old, whereas the oldrats referred to as the ‘22-month-old group’ were 24-months old,and the ‘30-month-old group’ were 32-months old.

Golgi impregnation procedure and dendritic spine analysisHalf rat brains were processed using a Golgi impregnation stainingkit (FD Neurotechnologies, Ellicott City, MD, USA). Brains were

� 2012 The AuthorsJournal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 122, 800–811

Spatial memory and synaptic plasticity in rats | 801

washed, coronally sectioned (150 um) using a cryostat, mounted onadhesive microscope slides, and air-dried in the dark at 22–24�C.After 48 h, sections were rinsed, dehydrated, cleared of xylenes, andmounted onto ungelatinized glass slides. Slides were cover-slippedand allowed to dry before quantitative analysis. Bright-field imagesof pyramidal neurons in the hippocampus, cortex, and amygdalawere taken at 100x magnification using a Zeiss Axioplan (Zeiss,Decatur, GA, USA) microscope. Images were coded, and dendriticspines were counted using a computer-based Neurolucida software9.0 (MicroBright Field, Williston, VT, USA). For spine densitymeasurements, all clearly evaluable areas of 50–100 lm ofsecondary dendrites from each imaged neuron were used. Todetermine relative spine density, spines on multiple dendriticbranches from a single neuron were counted to obtain an averagespine number per 10 lm. For spine number measurements, onlyspines that emerged perpendicular to the dendritic shaft werecounted. The animal and neuron number was determined by the useof power calculations. Eight neurons in each section and eightsections per animal and six to eight animals were analyzed. Theaverage value for each region in each individual was obtained.These individual averages were then combined to yield a grandaverage for each region.

Electrophysiological recordingHippocampi were removed and cut into fine transverse slices(400 lm thick) using a vibrating slice cutter (VT1200S; Leica,Nussloch, Germany) in a well-oxygenated ice-cold ACSF (ACSFmedium: 124 mM NaCl, 3 mM KCl, 1.25 mM KH2PO4, 2 mMMgSO4, 26 mM NaHCO3, 1.8 mM CaCl2, and 10 mM glucose).Cut slices were recovered in oxygenated ACSF at 21–24�C for atleast 1 h before recording. Then slices were placed in a submergedrecording chamber and perfused with 29–30�C oxygenated ACSFat 2–3 mL/min. To record field excitatory post-synaptic potentials(fEPSPs) in hippocampal slices, the recording glass electrodeswere placed on the Schaffer collateral-CA1 pathway, a standardextracellular recording technique as previously described was used(Xie et al. 2000). Basal synaptic transmission was assessed usingthe synaptic input–output curve constructed by plotting fEPSPslopes versus presynaptic fiber volley amplitudes evoked by anincremental sequence of stimulus intensities (40–800 lA). Long-term potentiation (LTP) was induced using either a single high-frequency stimulus (HFS) or two high-frequency stimuli at 100 Hzfor 1 s and was expressed as the percent change of the fEPSP slopefrom its baseline value. The average amount of LTP recorded duringthe 60-min period after the HFS was used for comparisons betweengroups.

Western blot analysisThe levels of TrkB, phosphor-TrkB (p-TrkB), and several keyproteins that are crucial to synaptic plasticity and memory weremeasured in tissue lysates from entire hippocampus using apreviously described protocol (Zeng et al., 2010) with modifica-tions. Samples were snap frozen on dry ice and stored at )80�C untilsonicated. Tissues were homogenized in 50 mM Tris/HCl buffercontaining Protease Inhibitors Complete (Roche Molecular Bio-chemicals, Indianapolis, IN, USA) and Protein Phosphatase Inhib-itor Cocktail I and II (Sigma-Aldrich, St. Louis, MO, USA). Samplealiquots, each containing 20 lg protein, were electrophoresed on

12% SDS/PAGE. The proteins were transferred onto low-fluores-cent PVDF membranes (GE Healthcare, Pittsburgh, PA, USA),which were probed overnight at 4�C with the primary antibody andincubated for another 2 h with a HRP-conjugated secondaryantibody. The immunoreactive bands were visualized with ECL-plus fluorescence (GE Healthcare). Fluorescent signals wereacquired using the Typhoon 9410 Imaging System and quantifiedusing Imagequant 5.2 software (GE Healthcare). For loadingcontrols, all blots were reprobed with a b-actin antibody, and thesignal value for the band of interest was normalized to that of b-actinin the same lane. Primary antibody used to detect total Erk1/2 andphospho-Erk1/2 (p-Erk1/2) were from Cell Signaling (rabbitpolyclonal IgG; 1 : 500; rabbit polyclonal IgG; 1 : 200, CellSignaling, Danvers, MA, USA), total CREB and phospho-Ser133-CREB (p-CREB) were from Upstate/Millipore (rabbit polyclonalIgG; 1 : 1000; rabbit polyclonal IgG; 1 : 1000, Upstate/Millipore,Billerica, MA, USA), total CaMKII was from Upstate/Millipore(mouse monoclonal IgG; 1 : 2000), and phospho-Thr286-CaMKII(p-CaMKII) was from ABR (mouse monoclonal IgG; 1 : 1000,ABR, Golden, CO, USA), total and phospho-Ser831-GluR1(p-GluR1) were from Abcam (rabbit polyclonal IgG; 1 : 1000;rabbit polyclonal IgG; 1 : 500, Abcam, Cambridge, MA, USA), andTrkB and phospho-Tyr816-TrkB (p-TrkB) were from Abcam andMillipore (rabbit polyclonal IgG; 1 : 100; rabbit polyclonal IgG;1 : 50).

Statistical analysisData are presented as group means ± SEM. Two-way ANOVA testswere performed in all experiments using Origin 7.0 (OriginLab,Northampton, MA, USA). The Student Newman–Keuls test wasused for post hoc analyses, while Student’s t-test was used when anexperiment had only two groups to compare. The level ofsignificance for all analyses testing was set at p < 0.05.

Results

7,8-DHF improves the spatial learning and memory incognitively impaired 22-month-old aged ratsTo assess whether 7,8-DHF rescues the integrity of hippo-campus-dependent memory formation in 22-month-old AIrats, we used the Morris water maze paradigm. The AU rats,as well as the 7,8-DHF-treated and vehicle-treated AI ratswere retested twice during the period of treatment (Fig. 1). Inthe pretest, the 22-month-old rats all showed the ability tolearn the task, as evidenced by decreases in escape latencyduring training (Fig. 2a). There was a significant main effectof the training day (Fig. 2a, F(5, 39) = 3.535, p = 0.0098). TheAU rats performed significantly better than the two AI ratgroups (later to receive either 7,8-DHF or vehicle solution)(Fig. 2a, F(1, 43) = 4.0664, p = 0.0423), similar to the youngrats in this test (Fig. 2a, F(1, 28) = 0.9012, p = 0.2749). Oneday after the training sessions, memory retention wasevaluated by the probe test which quantitatively measuredthe time spent in the target quadrant of the swimming poolwhen the hidden platform was removed. AU rats spent

Journal of Neurochemistry � 2012 International Society for Neurochemistry, J. Neurochem. (2012) 122, 800–811� 2012 The Authors

802 | Y. Zeng et al.

comparable time in the target quadrant relative to that of theyoung group (Fig. 2b, p = 0.0672), whereas the AI ratsshowed no preference for the target quadrant indicating thedeficit of spatial recall (Fig. 2b, p = 0.0013 and 0.0027). TheAI rats also showed fewer platform crossings than the younggroup (Fig. 2c, p = 0.0053 and 0.0091), but the AU andyoung rats did not significantly differ from each other(Fig. 2c, p = 0.0598). In the visible platform tests, we did notobserve significant differences between aged and young ratsin escape latencies (Fig. 2d, p = 0.0842), or swimmingspeeds (Fig. 2e, p = 0.6712), indicating normal vision,motivation, and locomotor activity and coordination. Thig-motaxis (i.e., wall-seeking behavior) was also similar in agedand young rats, indicating a similar level of anxiety (Fig. 2f,p = 0.4576). The 22-month-old aged rats maintained theirswimming ability throughout the experiment.

In the first retest week, the AU rats and the 7,8-DHF-treated AI rats performed as well as the young rats did in thepretest to find the platform throughout the training period.However, the vehicle-treated rats showed significant impair-ment (Fig. 2a, F(1,28) = 7.6402, p = 0.0097), as measured bythe escape latency. There was a significant effect of trainingday (Fig. 2a, F(5, 9) = 6.06, p = 0.01) in the 7,8-DHF-treatedAI rats, significant effect of treatment (Fig. 2a,F(1,13) = 9.0312, p = 0.012), and significant interaction oftreatment and training day (Fig. 2a, F(7, 7) = 3.7954,p = 0.0497). Thus, the 7,8-DHF-treated AI rats performedsignificantly better than the vehicle-treated group and wereno longer significantly different in performance comparedwith the AU group. In the probe test, the AU rats and 7,8-DHF-treated AI rats spent more time in the target quadrant,whereas the vehicle-treated AI rats showed no preference forthe target quadrant (Fig. 2b, p = 0.0002 vs. young). 7,8-DHF-treated AI rats also crossed the previous location of thehidden platform more frequently than the correspondingvehicle group (Fig. 2c, p = 0.0063). Comparable motor andvisual function between various groups were observed in thevisible platform tests (Fig. 2d–f). These results suggest that7,8-DHF rescues both the acquisition of spatial memory andits retention in cognitively impaired 22-month-old aged rats.

In the second retest week after 10-day interval periodfollowing the first retest week, no significant differencesbetween retest weeks 1 and 2 in escape latency wereobserved in any of the groups (Fig. 2a). However, the

vehicle-treated AI rats showed significantly impaired perfor-mances (Fig. 2a, F(1, 28) = 4.20, p = 0.05) compared with theAU rats or the 7,8-DHF-treated AI rats. In the probe trial,both the AU rats and 7,8-DHF-treated AI rats performedsignificantly better than the vehicle-treated AI group (Fig. 2band c, p = 0.0047 and 0.0321 vs. young). Also, theintroduction of a visible platform on the last day in retestweek 2 did not have any clear effect (Fig. 2d). These resultssuggest that extended 7,8-DHF applications did not havefurther effect in cognitively impaired 22-month-old aged rats.

7,8-DHF treatment prevents the spatial learning andmemory from declining in cognitively impaired30-month-old aged ratsIn the 30-month-old group, three vehicle- and two 7,8-DHF-treated AI rats died during retest week 2. Thus, thirteenvehicle-treated, fourteen 7,8-DHF-treated AI, and 16 youngrats were used in the behavioral analysis. In the pretest, thetwo AI rat groups (later to receive either 7,8-DHF or vehiclesolution) reduced their escape latency over the six trainingdays (Fig. 3a, F(5, 21) = 4.039, p = 0.0092), but theyperformed much worse than the young control group(Fig. 3a, F(1, 41) = 7,2995, p = 0.0089). In retest week 1,the 7,8-DHF-treated rats did not differ in escape latency fromthe vehicle-treated AI rats, nor did they improve theirperformance relative to the pretest (Fig. 3a, F(1, 25) = 7.7792,p = 0.0087). In retest week 2, however, the mean perfor-mance of the 7,8-DHF-treated AI rats was significantly betteras a group compared with the vehicle-treated AI rats whenmeasured by escape latency (Fig. 3a, F(1, 25) = 7.7769,p = 0.0089). This was because of a decline in the perfor-mance of the vehicle-treated AI rats (Fig. 3a, F(1, 26) =7.7112, p = 0.0092 vs. retest week 1), whereas the perfor-mance of the 7,8-DHF-treatment AI rats remained stablebetween the two retests. As is shown in Fig. 3a, the 7,8-DHF- treated AI rats performed as well on the first day inretest week 2 as they did at the end of the previous retestweek, whereas the vehicle-treated AI rats showed a signif-icant learning deficit (p = 0.0134). This difference was alsoevident during the rest days in retest week 2. The spatialprobe trials showed that 7,8-DHF improved acquisition ofspatial memory in retest week 2 as measured by the timespent in the target quadrant (Fig. 3b, p = 0.0073) and theplatform crossings (Fig. 3c, p = 0.0025), whereas the vehi-

Fig. 1 Schematic drawing of the experimental plan. The rats were

screened for spatial memory performance in the pretest. The time

interval between the pretest week and the start of daily in-

traperitoneal injections (i.p.) of 7,8-dihydroxyflavone (7,8-DHF) or

vehicle was 3 weeks. Each testing took 7 days with the spatial

probe test and the visible platform placed on the last day of testing.

24 h after the completing of all behavioral testing, all animals were

killed.



cle-treated AI old rats showed no spatial memory of theplatform site in the pretest, nor in retests. There was aprogressive decline in swimming ability during the course of

the experiment in the AI rats (Fig. 3e, p = 0.0134). Thisdecline in swim performance could be because of age-relatedmotor impairments in combination with decreased somatic

(a)

(b) (c)

(d)

(f)

(e)



function in some of these animals. However, when intro-ducing a visible platform on the last day of the second retestweek, the vehicle-treated AI rats did not perform signifi-cantly differently from the 7,8-DHF-treated AI rats (Fig. 3d,p = 0.7857). These results suggest that 28 days of consec-utive 7,8-DHF treatment prevents the spatial learning andmemory from declining in cognitively impaired 30-month-old aged rats.

7,8-DHF treatment facilitates synaptogenesis and synapticplasticity in hippocampusBecause the synapse is widely assumed to be the cellularbasis for learning and memory (Martin et al. 2000), weassessed whether 7,8-DHF could facilitate the formation ofdendritic spines and therefore synapse numbers. No signif-icant reductions were observed in the density and number ofdendritic spines along individual dendrites of pyramidalneurons in the hippocampal CA1 region in the 22-month-oldAU rats compared with the young group (Fig. 4a–c,p = 0.0678 and 0.0912, respectively). However, dendriticspines were markedly decreased in 22-month-old AI rats incomparison with the young group (Fig. 4a–c, p = 0.0263 and0.0042, respectively). Conversely, spine density and numberin the hippocampus were significantly higher in 7,8-DHF-treated AI rats in comparison with vehicle-treated AI rats(Fig. 4a–c, p = 0.0321 and 0.0064, respectively). Theseresults suggest that 7,8-DHF facilitates synapse formationin 22-month-old aged-impaired rats.

As expected, the dendritic spines in hippocampus weresignificantly reduced in the 30-month-old AI rats comparedwith the young group (Fig. 4f–h, p = 0.0243 and 0.0023,respectively), 7,8-DHF restored spine density (Fig. 4f–h,p = 0.5325), but not spine number (Fig. 4f–h, p = 0.0337) inthis group. These results suggest that 7,8-DHF modifiessynapse structure in more advanced aged rats.

We next determined whether 7,8-DHF also facilitatessynaptic plasticity. We first performed electrophysiologicalrecordings on hippocampal slices prepared from 22-month-old rats after the completion of behavioral tests. Stable LTPof CA1 neurons induced by single train of high-frequency

stimulation (HFS) on the Schaffer collaterals was observed inAU rats, but impaired in vehicle-treated AI rats (Fig. 4d ande, F(1, 29) = 7.6, p = 0.01). A robust LTP was readilyinduced by one train of HFS in CA1 neurons of 7,8-DHF-treated AI rats (Fig. 4d and e, F(1, 28) = 7.6345, p = 0.0089).These data indicate that 7,8-DHF restored hippocampalsynaptic plasticity in 22-month-old aged-impaired rats.

We next obtained field potential recordings from CA1neurons of 30-month-old hippocampi. We found that twotrains of HFS was not able to induce LTP in vehicle-treatedhippocampal slices (Fig. 4i–j, F(1, 27) = 7.69, p = 0.0021),7,8-DHF treatment had a moderate effect on LTP inductionin the CA1 area, while it decayed in 30 min (Fig. 4i–j,F(1, 28) = 7,6645, p = 0.0034). These results indicate that7,8-DHF moderately restored hippocampal synaptic plastic-ity in 30-month-old aged-impaired rats.

7,8-DHF enhances the p-Trk B expression in hippocampusTo determine the ability of systemic 7,8-DHF to activate p-TrkB in vivo in aged rats, we examined TrkB and p-TrkB(at Tyr816) levels in the hippocampus (Fig. 5a and c).Western blot analyses showed significant decreases in TrkBand p-TrkB levels in the hippocampus in the 22-month-oldAI rats (Fig. 5b, p = 0.0074 and 0.0042, respectively) andthe 30-month-old AI rats (Fig. 5d, p = 0.0026 and 0.0037,respectively) compared with young controls. TrkB levels inneither 22- nor 30-month-old AI rats were changed by 7,8-DHF (Fig. 5b and d, p = 0.5672 and 0.6795, respectively),whereas p-TrkB levels were enhanced by 7,8-DHF treat-ment in the hippocampus of both 22- (Fig. 5b, p = 0.0013)and 30-month-old AI rats (Fig. 5d, p = 0.0212). Consis-tently with observations in synaptogenesis, synaptic plas-ticity, and memory formation, 7,8-DHF treatment increasedthe level of p-TrkB in 22-month-old AI rats to acomparable degree with that of the young group (Fig. 5aand b, p = 0.5212), but to a lesser degree in 30-month-oldAI rats relative to the young group (Fig. 5c and d,p = 0.0301). Altogether, given the ability to cross theblood–brain barrier, 7,8-DHF is able to activate p-TrkBin vivo in aged-impaired rats, especially in younger

Fig. 2 7,8-DHF improves spatial learning and memory in 22-month-

old aged impaired rats. (a) Hidden platform test. Four animal groups

were: Young, n = 16; AU, n = 16; 7,8-DHF + AI, n = 15; Vehicle + AI,

n = 14. The young group took only the pretest and the three others

took all three tests. The two aged-impaired rats (AI) groups per-

formed significantly worse than the aged-unimpaired rats (AU) and

young controls during the last five trial days (days 2–6) in the pretest

week. In the retest week 1, the 7,8-DHF-treated AI rats performed as

well as the AU and young groups did on the pretest, whereas the

vehicle-treated AI rats showed significantly longer latency. In retest

week 2, the vehicle-treated AI rats again spent significantly longer

time to find the hidden platform than the AU group. But, the 7,8-DHF-

treated AI rats did not show more improvement compared to that

during retest week 1. (b) Probe trials. AU rats and 7,8-DHF-treated AI

rats showed comparable task retention, while vehicle-treated AI rats

showed retention deficit on two retests. (c). Platform crossings. Both

AU rats and 7,8- DHF-treated AI rats displayed more platform

crossings compared to the vehicle-treated AI rats on two retests. (d).

The visible platform task. There were no significant differences be-

tween any groups for the latency to find the visible platform

throughout the three test weeks. (e). The swimming speed. There

was nosignificant difference in swimming speed among each animal

group throughout the three test weeks. (f). Thigmotaxis analysis.

Thigmotaxis was similar in aged and young rats throughout the three

test weeks. Error bars show SEM in all results. In all results,

*p < 0.05, **p < 0.01 compared to young rats in the pretest, and

compared to the AU rats in retests. #p < 0.05, ##p < 0.05 compared

to vehicle-treated AI rats..



(a)

(b) (c)

(d)

(f)

(e)

Fig. 3 Extended 7,8-DHF treatment improves the spatial learning and

memory in 30-month-old aged impaired rats. (a) Hidden platform test

for the two aged impaired and one young groups (Young, n = 16; 7,

8- DHF + AI, n = 14; Vehicle + AI, n = 13). The young group took only

the pretest. The two AI groups performed significantly worse than the

young controls during the all trial days in the three test weeks. How-

ever, in retest week 2, the vehicle-treated AI rats showed significantly

worse performances compared to that in retest week 1, while the 7,8-

DHF-treated AI rats performed similarly to that in retest week 1 and

significantly better on all days than the vehicle-treated AI rats. (b)

Probe trials and (c) Platform crossings. The young animals showed

acquisition of the task. No differences in the pretest and retest week 1

could be observed between the vehicle treated or 7,8-DHF-treated AI

rats. In retest week 2, only a few of the vehicle-treated rats swam over

the former platform site, whereas the 7,8-DHF-treated AI rats showed

some focused search. (d) Visible platform. Vehicle-treated AI rats

found the visible platform equally fast as the 7,8- DHF-treated AI rats.

(e). The swimming speed. The swim speed of the vehicle-treated AI

rats declined during the retest week 2. (f). Thigmotaxis was also si-

milar in two aged and young rats throughout the three test. *p < 0.05,

**p < 0.01 compared to young rats. #p < 0.05, ##p < 0.05 compared to

vehicle-treated AI rats.



impaired hippocampi. In turn, 7,8-DHF is able to act onTrkB-induced memory enhancement.

7,8-DHF treatments activate key proteins involved inlearning and memory in the hippocampusTrkB is known to activate several intracellular signalingcascades including Erk, CREB, and CaMKII (Cunningham

and Greene 1998).To further demonstrate activation of theTrkB signaling pathway in aged-impaired rats, we examinedthe activation of several proteins including Erk, CREB,CaMKII, and GluR1 by 7,8-DHF in the aged rats. Althoughtotal Erk1/2, CREB, CaMKII, and GluR1 levels were notconsistent with the behavior performance of aged-impairedrats or affected by 7,8-DHF treatment (Fig. 6b and c), the

(a)

(d)

(f)

(i)

(j)

(g) (h)

(b)

(e)

(c)

Fig. 4 7,8-DHF modifies dendritic spines

and facilitates synaptic plasticity in the hip-

pocampus of aged impaired rats. A-E pre-

sent results from 22-month-old groups. (a)

Images of Golgi staining of spine segments

from the apical dendritic layer of the CA1

region. (b) and (c) Quantitative analysis of

the four groups (Young, n = 8; AU, n = 8;

7,8-DHF + AI, n = 7; Vehicle + AI, n = 7).

The reductions in spine number and density

were reversed by treatment with 7,8-DHF.

(d) Superimposed sample sweeps from the

first 5 min (black) and last 5 min (red) of the

recording. Calibration bars: 1 mV/ 5 ms. (e)

LTP was successfully induced by a single

train of HFS in the CA1 region from Young,

n = 8; AU, n = 8; and 7,8-DHF + AI, n = 8;

but not Vehicle + AI, n = 8. 7,8-DHF but not

vehicle treatment restored LTP in AI slices.

(f-j) present results from 30-month-old

groups. (f) Images of Golgi staining of spine

segments from the apical dendritic layer of

the CA1 region. (g) and (h) quantitative

analysis of the three groups (Young, n = 8;

7,8-DHF + AI, n = 7; Vehicle + AI, n = 7).

The reductions in spine density but not

number was reversed by treatment with 7,8-

DHF. (i) Superimposed sample sweeps

from the first 5 min (black) and last 5 min

(red) of the recording. Calibration bars:

1 mV/ 5 ms. (j) fEPSPs recording in the

CA1 region from three groups (Young,

n = 8; 7, 8-DHF + AI, n = 7; Vehicle + AI,

n = 6). LTP was induced by two trains of

HFS in the CA1 region from 7,8-DHF-trea-

ted AI rats, but decayed to baseline by

30 min, whereas fEPSPs from young rats

remained potentiated until the end of the

recording. Two trains of HFS failed to in-

duce any fEPSPs potentiation in vehicle-

treated AI slices. *p < 0.05, **p < 0.01

compared to young rats. #p < 0.05,##p < 0.05 compared to vehicle-treated AI

rats.



levels of phosphorylation, highly active forms, of theproteins (p-Erk1/2, p-CREB, p-CaMKII, and p-GluR1) weresignificantly reduced in the hippocampus of 22-month-old AIrats (Fig. 6a and c, p = 0.0023; p = 0.0227; p = 0.0245;p = 0.0029). 7,8-DHF treatment restored these protein levelsin hippocampus to comparable levels of the young rats in 22-month-old group (Fig. 6a and c).

It is notable that both total (Fig. 6e and f, p = 0.0312;p = 0.0134; p = 0.0215; p = 0.0307) and phosphorylationprotein levels (Fig. 6d and f, p = 0.0023; p = 0.0312;p = 0.0037; p = 0.0319) in 30-month-old AI rats aredecreased in comparison with the young group. 7,8-DHFincreased the phosphorylation levels of p-CREB and p-CaM-KII, but not p-Erk1/2 and p-GluR1, to comparable levelsrelative to the young rats in the 30-month-old group (Fig. 6dand f, p = 0.0435; p = 0.0996; p = 0.3315; p = 0.0325)

(a)

(c)

(b)

(d)

(f)

(e)

Fig. 6 7,8-DHF activates the signaling molecules crucial for synaptic

plasticity in hippocampus. (a) and (b) Western blots from hippocampal

tissues of 22-month-old groups (Young, n = 8; AU, n = 8; 7,

8-DHF + AI, n = 7; Vehicle + AI, n = 7). (c) Significant decreases in

phosphorylation levels of Erk1/2, CREB, CaMKII, and GluR1 were

detected in the vehicle-treated 22-month-old AI rats, whereas the total

protein levels in these groups remain unchanged. 7,8-DHF treatment

increased phosphorylation of these proteins without altering the total

protein level of each in AI rats. (d) and (e) Western blots from hippo-

campal tissues of 30-month-old groups (Young, n = 8; 7,8-DHF + AI,

n = 7; Vehicle + AI, n = 7). (f) Significant decreases in both phos-

phorylation and total protein levels of Erk1/2, CREB, CaMKII, and

GluR1 were detected in the vehicletreated 30-month-old AI rats

compared to that in young rats. 7,8-DHF had a significant effect on

phosphorylation levels but not total protein levels in AI rats. However,

7,8-DHF could not increase the phosphorylation levels of Erk1/2 and

GluR1 to that of the young controls. *p < 0.05, **p < 0.01 compared to

the young controls. #p < 0.05 compared to vehicle-treated AI rats.

(a) (b)

(c) (d)

Fig. 5 7,8-dihydroxyflavone restores the level of TrkB phosphorylation

in aged impaired rats. (a) Western blots from hippocampal tissues of

22-month-old groups (Young, n = 8; AU, n = 8; 7, 8-DHF + AI, n = 7;

Vehicle + AI, n = 7). (b) Significant decreases in tyrosine receptor ki-

nase B (TrkB) and p-TrkB levels were detected in the vehicle-treated

22- month-old AI rats, whereas these protein levels in AU rats are in

comparison with young controls. The reduction in the p-TrkB level was

reversed by 7,8-DHF treatment. (c) Western blots from hippocampal

tissues of 30-month-old groups (Young, n = 8; 7,8-DHF + AI, n = 7;

Vehicle + AI, n = 7). (d) Significant decreases in TrkB and p-TrkB le-

vels were detected in the vehicle-treated 30-month-old AI rats in

comparison with young controls. The reduction in the p-TrkB level was

reversed by 7,8-DHF treatment. *p < 0.05, **p < 0.01 compared to

young rats. #p < 0.05, ##p < 0.05 compared to vehicle-treated AI rats.



without significantly altering the total protein levels (Fig. 6eand f, p = 0.0635; p = 0.0896; p = 0.9315; p = 0.7202).Thus, by enhancing phosphorylation of these two signalingproteins, 7,8-DHF increased the activity of TrkB downstreamsignaling cascades that are crucial for synaptic plasticity andmemory formation in the hippocampus.

Discussion

Behavioral flexibility, the ability to modify responses as aresult of changing task demands, is detrimentally affected byaging with a shift towards increased cognitive rigidity(Nieves-Martinez et al. 2012). Evidences suggest that age-related disturbances in the BDNF-TrkB system affecthippocampal-dependent memory and cognitive dysfunctions(von Bohlen und Halbach et al. 2006a,b; von Bohlen undHalbach O, 2010; Zeng et al. 2011). Herein, we have shownthat in vivo activation of TrkB by chronic applications of 7,8-DHF, a TrkB agonist, mimicking BDNF, improved thebehavioral performances and rescued spatial learning andmemory in cognitively impaired aged rats. Furthermore, 7,8-DHF treatment counteracted the loss of dendritic spinesand the impairment of synaptic plasticity in the hippocampusand activated a number of signaling proteins crucial tosynaptic plasticity and memory. Our results in this studydemonstrate that 7,8-DHF and/or TrkB agonists serve asuseful strategies to rescue spatial memory and synapticplasticity in cognitively impaired aged rats.

We previously demonstrated that 7,8-DHF in vitro rescueslong-term synaptic plasticity in the hippocampus of agedrats(Zeng et al. 2011). In this study, we have extended ourobservations to 22- and 30-month-old rats to determine thelong-term effect of 7,8-DHF in vivo on cognitively impairedaged rats, as well as the age limitations of the 7,8-DHF effect.We used water maze tasks to measure spatial memory that isvulnerable during the aging process (Moffat 2009). A total of52% of the 22-month-old rats displayed impaired placenavigation performance relative to the young control rats,whereas essentially all the 30-month-old rats were impaired.The aged-impaired rats were retested during daily 7,8-DHFor vehicle treatment. 7,8-DHF treatment significantly im-proved spatial memory parameter in 22-month-old AI rats inthe first retest week (after 14 days of consecutive injection).However, any significant effect by 7,8-DHF treatment in themuch more severely impaired 30-month-old AI rats was seenonly in the second test week (after 28 days of consecutiveinjection). While the vehicle-treated 30-month-old AI ratsshowed a progressive decline in the performance between thefirst and the second retest weeks, the performance of the 7,8-DHF-treated AI rats remained stable throughout thetreatment period. Furthermore, the 7,8-DHF treatment hadclear-cut effects on the platform-crossings measure in thespatial probe trial, which makes it likely that the effects in the30-month-old rats are related to improved spatial memory

performance, though the interpretation of these effects in theoldest animals was confounded by a progressive decline inswimming speed seen in the vehicle-treated AI rats. Whilethe difference between the vehicle- and 7,8-DHF-treated AIrats disappeared when the escape platform was made visible.The discrepancy between visible platform performance andswimming speed seem to reflect improved spatial memory in7,8-DHF-treated AI rats and impaired swimming ability inthe vehicle-treated AI rats. 7,8-DHF appeared to counteractthe progressive performance deficit in the 30-month-old rats.

The hippocampal function and structure involved in spatialmemory exhibit marked declines with aging in humans,monkeys, and rodents (Greene and Naranjo 1987; Walkeret al. 1988; Lee et al.1994; Rapp and Heindel 1994; Rappand Gallagher 1996; Driscoll et al. 2003; Erickson et al.2010). We previously demonstrated that a decrease inBDNF-TrkB in aged rats is related to the events leading tosuch age-related degenerative changes in the hippocampusincluding synaptic dysfunction and synapse loss (Zeng et al.2011). 7,8-DHF, via its action through TrkB receptors, hasprofound effects upon the spines of hippocampal neurons. Inthis study, the levels of TrkB and p-TrkB in hippocampuswere decreased in these two aged-impaired groups. Con-comitantly, TrkB- and p-TrkB-deficient AI rats displayed areduction in spine densities within the hippocampus, indi-cating that TrkB signaling plays a critical role in themaintenance of dendritic spines. 7,8-DHF is capable ofincreasing both spine density and number in apical dendritesof CA1 neurons of hippocampus in the 22-month-old AI rats,and only increasing the density but not number of spines in30-month-old AI rats. These results indicate that a morpho-logical response to 7,8-DHF can be obtained not only inyounger animals (22-months old) where spine loss occurs,but also in older impaired rats (30-months old), but the effectis more pronounced in younger impaired animals than inthose of more advanced age. In agreement with theobservations in spines, 7,8-DHF exerted a rescue effect onhippocampal LTP induction. 7,8-DHF treatment robustlyincreased LTP in the 22-month-old hippocampus, andmoderately increased LTP in the 30-month-old hippocam-pus. These results indicate that the hippocampus could be animportant target for 7,8-DHF improved memory enhance-ment. 30-month-old rats are more refractory than youngerones to synaptic plasticity and memory formation induced by7,8-DHF.

From a neuroscience perspective, the enhanced learningand memory, facilitated synaptic plasticity, and increaseddendritic spine density and number in the AI rats shouldengage several proteins that are critical to synaptic plasticityand memory, including Erk1/2, CREB, CaMKII, and GluR1.These proteins together are key signal transduction modulesinvolved in the control of neuronal plasticity (Alonso et al.2004; Thomas and Huganir 2004; Sanderson et al. 2008),local protein synthesis (Thiels and Klann 2001; Sweatt 2004),



formation of new synapses (Futter et al. 2005, Wayman et al.2006), and eventual formation of long-lasting memories(Benito and Barco 2010). Impairment of these TrkB-down-stream cascades may contribute to the synaptic and cognitivedeficits in cognitively impaired aged mice. Conversely,activation of TrkB via 7,8-DHF may enhance synaptic andcognitive function through increases in these signal proteins.We found that there were significant decreases in total proteinlevels of Erk1/2, CREB, CaMKII, and GluR1 in 30-month-old AI rats (Fig. 6e, f), whereas these levels in 22-month-oldAI rats are comparable to young controls. Moreover, 7,8-DHFfailed to bring up the phosphorylation of Erk1/2 and GluR1 tothe control level (Fig. 6d, f). These results may explain that7,8-DHF moderately restored synaptogenesis and synapticplasticity, and extended dosing of 7,8-DHF is required toimprove spatial memory in 30-month-old AI rats.

In conclusion, these results indicate that chronic treatmentwith 7,8-DHF can influence age-related decline in regards tospatial memory acquisition and retention in the water mazetask. The effects of 7,8-DHF on spatial memory performancein the aged rat may be mediated at least in part via an effecton the synaptogenesis and synaptic plasticity in hippocam-pus. The behavioral effect of 7,8-DHF is more pronounced inyounger impaired aged animals (22-months old) than in theolder impaired ones (30-months old). Taken together, ourobservations encourage the development and testing of 7,8-DHF, a selective agonist of TrkB, for human diseasesassociated with cognitive deficits and memory impairment.

Acknowledgement

This study was supported by start grant to Y.Z. from WuhanUniversity of Science and Technology.

Authors have no financial, personal or other conflict of interest todisclose.

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