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Short Communication

Reversible Changes in Hippocampal

CA1 Synapses Associated With

Water Maze Training in Rats

RUBE´ N MIRANDA,1_*_{EDUARDO BLANCO,}1_{AZUCENA BEGEGA,}1 LUIS J. SANTI´N,2AND JORGE L. ARIAS1

1_{Laboratory of Psychobiology, School of Psychology, University of Oviedo, Spain} 2

Psychobiology Area, School of Psychology, University of Ma´laga, Spain

KEY WORDS stratum radiatum CA1; hippocampus; spatial learning; synapses; electron microscopy

ABSTRACT Long-term memories seem to require protein synthesis to be estab-lished. This process can be related with synaptogenesis resulting in changes in the form or even in the number or proportion of synaptic contacts. Results from behavioral stud-ies assessing quantitative changes associated with different learning tasks are contro-versial. The aim of our work was to assess whether the number of CA1 hippocampal syn-aptic contacts can be modified after training in different tasks in the Morris water maze (MWM). We found transient changes in the synaptic density of the symmetric synapses associated with place learning. A reduced synaptic density of the symmetric synapses in the stratum radiatum of CA1 was found at 48 h posttraining, returning to control levels 72 h posttraining. The same effect was observed 1 h after training in a nonspatial task. Synaptic changes found in the CA1 shortly after water maze training suggest a possible participation of the hippocampus in the acquisition of nonspatial tasks together with a role in the short-term consolidation of spatial memory. As no changes were found in the total number of synapses counted, it is likely that subtle changes in synaptic efficacy than new synapse generation may be sufficient to support the acquisition and mainte-nance of new memories.Synapse 59:177–181, 2006. VVC2005 Wiley-Liss, Inc.

INTRODUCTION

Current models of memory consolidation (Dudai, 2004; Nader, 2003) assume that the storage of long-term memory (LTM) is associated with gene expres-sion, new protein synthesis, and synaptic remodelling. This synaptic remodelling can involve the modifica-tion of preexisting synapses or even the generamodifica-tion of new contacts and is proposed as a key process to explain the long-term maintenance of memories (Bai-ley and Kandel, 1993). Previous results from studies to assess the hypothesis that learning promotes mor-phological synaptic alterations are quite controversial. Specifically, there is little agreement about synaptic changes associated to spatial memory formation. On the other hand, there are strong evidences showing that a functional hippocampus is required for the acquisition of spatial tasks in the Morris water maze (MWM) (Eichenbaum et al., 1990; Morris et al., 1982). Different alterations in hippocampal synaptic transmission (e.g., long-term potentiation (LTP)) are

correlated with several behavioral deﬁcits (Jeffery, 1997). Maintenance of LTP and memory consolidation are related with an increase in multiple spine synap-ses at the hippocampus (Eyre et al., 2003; Toni et al., 1999). These evidences suggest that problems in long-term storage could be due to speciﬁc alterations in the synaptic physiology (i.e., LTP) as well as in the anatomy of synapses. However, it is not clear that in unaltered conditions, increases in synapse number are always correlated with the acquisition or consoli-dation of memories. Several methodological issues of ultrastructural studies in addition to the dynamic

Contract grant sponsor: MEC; Contract grant number: SEJ2004-07445/PSIC.

*Correspondence to: Ruben Miranda, Ph.D., Laboratory of Psychobiology, University of Oviedo, Plaza Feijoo, s/n 33003 Oviedo, Spain.

E-mail: [email protected]

Received 23 June 2005; Accepted 18 October 2005

DOI 10.1002/syn.20229

Published online in Wiley InterScience (www.interscience.wiley.com).

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nature of learning and memory processes have to be taken into account to explain quantitative electron microscopy data (Geinisman, 2000). Furthermore, re-gional differences may exist due to a differential par-ticipation of cellular assemblies in memory processes (Eyre et al., 2003; O’Malley et al., 2000; Ramirez-Amaya et al., 1999, 2001; Rusakov et al., 1997).

Together, these results suggest that hippocampal synaptic remodelling involving changes in the num-ber of synapses is likely to occur after training in spa-tial tasks. These changes appear to be transitory and evident at short posttraining periods. However, there is no consensus about the speciﬁc nature and time course of the diverse synaptic changes found in the different hippocampal subﬁelds. The main goal of this work was to assess whether quantitative synaptic changes occur in hippocampal CA1 area after spatial and nonspatial MWM training. We examined the stratum radiatum of CA1 (SR-CA1) in control rats and at different time intervals after training in two MWM tasks, using quantitative electron microscopy.

A total of 23 adult Wistar rats (300þ/50 g) were assigned randomly to the following groups: CC, caged control (n ¼ 8); SP-48, spatial hidden platform task sacrificed 48 h posttraining (n ¼ 4); SP-72, spatial hidden platform task sacrificed 72 h posttraining (n¼ 4); and N-SP, nonspatial visual fixedþcurtains task sacrificed 1 h posttraining (n¼5). Trained animals in the MWM were habituated to the maze in one day with 2 trials and then submitted to a 4-day task with 6 trials per day. The escape platform (hidden or visi-ble) was located in the same place over training in

both SP and N-SP tasks. N-SP animals were pre-vented from using room landmarks to navigate by placing black curtains around the pool. SP animals were submitted to a 1-trial probe test with no plat-form after the last training trial. The perplat-formance was measured as distance covered and escape laten-cies (Fig. 1). Behavioral analyses (repeated measures ANOVAs) revealed that acquisition of the SP task was gradual and animals mastered the task after two days of training (12 trials) (distance, days effect F(3,9) ¼ 6.333, P ¼ 0.013; Bonferroni posttest P < 0.05). On the other hand, the N-SP task was acquired faster, displaying short escape latencies since the first trials reaching asymptotic performance after one day of training (6 trials) (latency, group effect F(1,11) ¼ 8.947; P ¼ 0.012). The probe test confirmed (one-way ANOVA, F(3,4) ¼ 10.427; P ¼ 0.023) that rats recall the location of the hidden platform based on room landmarks (Bonferroni posttest P <0.05) (Fig. 1). No significant differences were found between SP-48 and SP-72 groups (t(5) ¼ 1.098; n.s.), validating

subse-quent comparisons of learning-induced synaptic changes.

For electron microscopy examination, all animals were transcardially perfused, under deep anesthesia, with a washing solution (0.12 M phosphate buffer, 0.02 Cl2Ca, and 8% sucrose) for 5 min, followed by

ﬁx-ative (glutaraldehydeþparaformaldehyde(1:1); 0.1 M; pH ¼ 7.3) for 15 min. After postﬁxation, coronal trimmed slabs (300 lm) of the left antero-dorsal hip-pocampus (Paxinos and Watson, 1988) were osmicated in a 1% osmium tetroxide-buffered solution for 90 min,

Fig. 1. Performance in the water maze. A: Mean distance covered during training. Animals trained in SP task displayed greater distances covered during training compared to N-SP ani-mals. Asymptotic performance is reached after 2 days by SP mals and after 1 day by N-SP ani-mals. B: Mean escape latencies performed. N-SP animals showed lower escape latencies during training compared to SP animals.

C: Probe test results of the SP group. Animals showed a signiﬁ-cant preference for the escape quadrant (D) compared to the rest of quadrants (Error bars ¼

SEM; *P < 0.05). No signiﬁcant differences in the mean percent-age of time spent in escape quad-rant were found between SP-48 and SP-72 groups.

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dehydrated, and embedded in araldite. Ultrathin sec-tions (70 nm thick) of silver-gray interference color were collected on 300 mesh copper grids, counter-stained with uranyl acetate and lead citrate, visual-ized in a Zeiss EM-109 transmission electron micro-scope, and photographed with a 35-mm black and white film camera. Synapses were counted in the SR-CA1 area at two different levels of the antero-dorsal hippocampus. For each level, systematic sampling of two different areas of the SR-CA1 was done taking a minimum of 18 photos per area. At least 36 fields were photographed in each level, resulting in a total of 72 fields per animal. Images were taken at 20,0003and, after conventional developing, negatives were digitally scanned at 1200 dpi (HP photosmart s20). Synapses were classified as asymmetric (type I) or symmetric (type II) (Gray, 1959). Only synapses identified inside the measure frame were counted (Fig. 2). The number of synapses per sampled area was calculated for each type of synapse and for the total. Sampling area was calculated as the sum of measure frames overlaid in the total of quantified images.

Overall, our results reﬂect a transient downregula-tion of the SR-CA1 symmetric synapses 48 h after completing a spatial task (SP) and 1 h after training in a nonspatial task (N-SP) in the MWM. On the other hand, no differences were found in asymmetric synapses and in the total number of synapses counted between groups.

In a previous study by Rusakov et al. (1997), no quantitative synaptic changes were observed in the molecular layer of DG and in the SR-CA1 5 days after water maze training in a 5-day protocol. Later, a transient increase in the number of dendritic spines in the middle molecular layer of DG was found after avoidance conditioning and after a 1-session spatial learning task. These changes were observed at 6 h posttraining returning to basal levels at 72 h post-training (O’Malley et al., 1998, 2000). Similarly, a transient increase of axo-spinous synapses was found in the same layer 9 h after the beginning of a spatial MWM training (Eyre et al., 2003). Together, these ﬁndings suggest that a speciﬁc time-window exists during which quantitative synaptic changes induced by training will be evident. This posttraining period seems to spread from 6 h to 48 h posttraining, although some variations may emerge depending on the type and amount of training as well as on the brain region. For instance, synaptogenesis in the stra-tum lucidum of CA3 was observed even 7 days after water maze training (Ramirez-Amaya et al., 2001).

Previous studies have stressed the importance of CA1 for memory processes (Zola-Morgan et al., 1986) and its relation with spatial learning and LTP (Tsien et al., 1996). Other authors have shown that enriched experience can promote increases in spines and syn-apse density in CA1, resulting in an improvement in spatial abilities (Moser et al., 1994; Rampon et al., 2000). However, no quantitative synaptic changes were observed 5 days after water maze training (Rusakov et al., 1997). More recent evidences suggest that learning-induced synaptic changes may occur at shorter posttraining periods (Eyre et al., 2003; O’Mal-ley et al., 2000).

Hence, we examined the number of asymmetric and symmetric synapses of the SR-CA1 at 48 h and 72 h after a 4-day training protocol in the SP version of the MWM. To account for nonspeciﬁc synaptic changes associated with water maze training, another group was trained in a nonspatial hippocampus-independent task and synapses were examined 1 h posttraining.

In agreement with previous anatomical and neuro-chemical data, in control conditions almost 90% of synapses were asymmetric (presumably excitatory) and 10% were symmetric (presumably inhibitory) in the SR-CA1 (Megias et al., 2001; Vizi and Kiss, 1998). One-way ANOVAs showed non signiﬁcant changes in the density of asymmetric synapses (F(3,17) ¼0.381; n.s.) and in the total number of synapses counted (F(3,17) ¼ 0.552; n.s.) after training. However, a reduction in the density of symmetric synapses were found at 48 h posttraining, but not at 72 h posttrain-ing (F(3,17) ¼ 5.703; P ¼ 0.007, Bonferroni posttest P <0.05). On the other hand, a signiﬁcant reduction in the percentage (F(3,17) ¼ 4.522; P ¼ 0.017) and density (F(3,17)¼5.703;P¼0.007) of symmetric

syn-Fig. 2. A: Quantified field in the SR of CA1. Asterisks indicate the location of synapses. Magnification 20,0003. B: Detail of an asymmetric synapse. Asterisk indicates presynaptic area. Arrow in-dicates the presence of postsynaptic density.C: Detail of a symmet-ric synapse. Asterisk indicates presynaptic area.

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apses was also found 1 h after training in the nonspa-tial task compared with controls (Bonferroni posttests P < 0.05) (Fig. 3). This change was concomitant to a signiﬁcant increase in the percentage of asymmetric synapses in the same group compared to CC (F(3,17)¼ 3.71; P ¼ 0.032, Dunnett posttest P < 0.05). These synaptic changes found shortly after nonspatial train-ing could be due to the enrichtrain-ing experience (Moser et al., 1994; Rampon et al., 2000; Sandi et al., 2003) in parallel to a general involvement of the hippocam-pus even in the processing of nonspatial information (Whishaw, 1998). Given that the posttraining period in which synapses were observed was different for the spatial and nonspatial tasks, it is difﬁcult to establish direct comparisons in terms of spatial vs. nonspatial memory structural correlates. Basically, the present design allows us to study synaptic changes associated with spatial memory consolidation (SP-48 and SP-72 groups) and whether similar changes can occur in the hippocampus shortly after water maze training (N-SP), related with the procedural dimen-sion of the task and not with hippocampus-dependent memory consolidation.

Our results showing no changes in the total num-ber of synapses counted are in agreement with earlier studies in different animal models and learning tasks and on spatial learning in MWM (Rusakov et al., 1997), and support the notion that memories follow-ing new learnfollow-ing may not necessarily involve a net synaptogenesis (Geinisman, 2000). Other authors have pointed out that greater neural remodelling involving new synapse formation appears to occur when animals require great effort to learn a task (Sandi et al., 2003, 2004). Moreover, studies on LTP suggest that neurons may homeostatically regulate input through spine number. This hypothesis predicts that more spines are

formed when neurons have less excitatory input, are maintained by optimal activation, and are lost when activation is too high (Harris, 1999).

The reduction in the proportion of symmetric syn-apses found in our study could reflect a subtle reor-ganization involving neurochemical changes of the hippocampal synaptic transmission. Discrepancies with studies demonstrating increases in the total num-ber of synapses after spatial learning (Eyre et al., 2003; O’Malley et al., 2000), may be explained by differences in the amount or schedule of training. Although spatial tasks can be acquired under both massed and spaced training schedules, it is well known that LTM is stron-ger after spaced training (Commins et al., 2003; Spreng et al., 2002) and performance can be improved due to overtraining correlated with CA3 mossy fiber synapto-genesis (Ramirez-Amaya et al., 1999). O’Malley et al. (2000) found a significant increase in spine density in the mid-molecular layer of DG after a 5-trial protocol at 6 h posttraining, and Eyre et al. (2003) used a 12-trial protocol carried out in 6 h, finding an increase in axo-spinous synapse density in DG 3 h later, whereas no changes were found 3 h after a 4-trial task or 24 h after the beginning of a 20-trial protocol fulfilled in 21 h. As opposed to the results obtained in CA3 (Ram-irez-Amaya et al., 1999, 2001), these evidences suggest that, at least in DG, synaptic changes occur only in short posttraining periods after massed training with few trials. Our findings, together with those of Rusakov et al. (1997), indicate that similar to DG, CA1 major synaptic alterations are likely to be found after massed training, since no changes in the total number of syn-apses exist after training with protocols taking 4 or 5 days, even when ultrastructural examination is done a few hours after training. Probably, subtle synaptic reorganization modifications could be due to the extended practice rather than to memory consolidation and, the difficulties in observing quantitative altera-tions associated with gradual learning, can be derived from the possibility that multiple waves of remodelling occur when training protocols comprise more than one session (Dudai, 2004; Eyre et al., 2003). Nevertheless, the functional role of quantitative synaptic changes after training with massed protocols that are not expected to promote a robust LTM remains unex-plained.

In summary, transient synaptic changes were observed in CA1 after water maze training involving a downregulation of the proportion of symmetric syn-apses, although the total number of synapses counted remains stable. This synaptic remodelling was ob-served in the spatial and nonspatial versions of the MWM shortly after training, suggesting a possible participation of the hippocampus in the acquisition of nonspatial tasks with a time-limited role in spatial memory consolidation. The absence of changes in the total number of synapses reﬂects that, under spaced

Fig. 3. Group comparison in the synapse density values for asymmetric and symmetric synapses. SP-48 and N-SP groups pre-sented a signiﬁcant decrease in symmetric synapses compared to CC animals (Error bars¼SEM; *P<0.05).

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training, physiological changes in synaptic efﬁcacy, instead of new synapse generation, may be sufﬁcient to support the acquisition and maintenance of new memories. Further studies are required to clarify the functional role of synaptogenesis in memory processes.

ACKNOWLEDGMENTS

The authors thank D. Carlos Villa and D. Fernando Jan˜ez for their expert technical support inside the electron microscopy facility of the University of Oviedo, and Dr. Sandra Rubio for her comments on the manuscript.

REFERENCES

Bailey CH, Kandel ER. 1993. Structural changes accompanying memory storage. Annu Rev Physiol 55:397–426.

Commins S, Cunningham L, Harvey D, Walsh D. 2003. Massed but not spaced training impairs spatial memory. Behav Brain Res 139:215–223.

Dudai Y. 2004. The neurobiology of consolidations, or, how stable is the engram? Annu Rev Psychol 55:51–86.

Eichenbaum H, Stewart C, Morris RGM. 1990. Hippocampal repre-sentation in place learning J Neurosci 10:3531–4.

Eyre MD, Richter-Levin G, Avital A, Stewart MG. 2003. Morphologi-cal changes in hippocampal dentate gyrus synapses following spa-tial learning in rats are transient. Eur J Neurosci 17:1973–1980. Geinisman Y. 2000. Structural synaptic modiﬁcations associated

with hippocampal LTP and behavioral learning. Cereb Cortex 10: 952–962.

Gray EG. 1959. Axo-somatic and axo-dendritic synapses of the cere-bral cortex: an electron microscopic study. J Anat 93:420–433. Harris K. 1999. Structure, development, and plasticity of dendritic

spines. Curr Opin Neurobiol 9:343–348.

Jeffery K. 1997. LTP and spatial learning–where to next? Hippo-campus 7:95–110.

Megı´as M, Emri ZS, Freund TF, Gulya´s AI. 2001. Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells. Neuroscience 102:527–540.

Morris RGM, Garrud P, Rawlings JNP, O’Keefe J. 1982. Place navi-gation impaired in rats with hippocampal lesions. Nature 297:681– 683.

Moser MB, Trommald M, Andersen P. 1994. An increase in dendritic spine density on hippocampal CA1 pyramidal cells following

spa-tial learning in adult rats suggests the formation of new synapses. Proc Natl Acad Sci USA 91:12673–12675.

Nader K. 2003. Memory traces unbound. Trends Neurosci 26:65–72. O’Malley A, O’Connell C, Regan CM. 1998. Ultrastructural analysis

reveals avoidance conditioning to induce a transient increase in hippocampal dentate spine density in the 6 hour post-training period of consolidation. Neuroscience 87:607–613.

O’Malley A, O’Connell C, Murphy KJ, Regan CM. 2000. Transient spine density increases in the mid-molecular layer of hippocampal dentate gyrus accompany consolidation of a spatial learning task in the rodent. Neuroscience 99:229–232.

Paxinos G, Watson C. 1998. The rat brain in stereotaxic coordinates. London: Academic Press.

Ramı´rez-Amaya V, Escobar ML, Chao V, Bermu´dez-Rattoni F. 1999. Synaptogenesis of mossy ﬁbers induced by spatial water maze overtraining. Hippocampus 9:631–636.

Ramı´rez-Amaya V, Balderas I, Sandoval J, Escobar ML, Bermu´dez-Rattoni F. 2001. Spatial long-term memory is related to mossy ﬁber synaptogenesis. J Neurosci 21:7340–7348.

Rampon C, Tang YP, Goodhouse J, Shimizu E, Kyin M, Tsien JZ. 2000. Enrichment induces structural changes and recovery from nonspatial memory deﬁcits in CA1 NMDAR1-knockout mice. Nature Neurosci 3:238–244.

Rusakov DA, Davies HA, Harrison E, Diana G, Richter-Levin G, Blis TVP, Stewart MG. 1997. Ultrastructural synaptic correlates of spatial learning in rat hippocampus. Neuroscience. 80:69–77. Sandi C, Davies HA, Cordero MI, Rodriguez JJ, Popov VI, Stewart

MG. 2003. Rapid reversal of stress induced loss of synapses in CA3 of rat hippocampus following water maze training. Eur J Neurosci 17:1–10.

Sandi C, Cordero MI, Merino JJ, Kruyt ND, Regan CM, Murphy KJ. 2004. Neurobiological and endocrine correlates of individ-ual differences in spatial learning ability. Learn Mem 11:244–252. Spreng M, Rossier J, Schenk F. 2002. Spaced training facilitates

long-term retention of place navigation in adult but not in adoles-cent rats. Behav Brain Res 128:103–108.

Toni N, Buchs PA, Nikonenko I, Bron CR, Muller D. 1999. LTP pro-motes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature 402:421–424.

Tsien JZ, Huerta PT, Tonegawa S. 1996. The essential role of hippo-campal CA1 NMDA receptor-dependent synaptic plasticity in spa-tial memory. Cell 87:1317–1326.

Vizi ES, Kiss JP. 1998. Neurochemistry and pharmacology of the major hippocampal transmitter systems: synaptic and nonsynaptic interactions. Hippocampus 8:566–607.

Whishaw IQ. 1998. Place learning in hippocampal rats and the path integration hypothesis. Neurosci Biobehav Rev 22:209–220. Zola-Morgan SM, Squire LR, Amaral DG. 1986. Human amnesia

and the medial temporal region: enduring memory impairment following a bilateral lesion limited to ﬁeld CA1 of the hippocam-pus. J Neurosci 6:2950–2967.