Neuroscience 209 (2012) 7483

REGIONAL DISSOCIATION OF PARADIGM-SPECIFIC SYNAPSE REMODELING DURING MEMORY CONSOLIDATION IN THE ADULT RAT DENTATE GYRUS
D. SCULLY, R. FEDRIANI, I. E. J. DESOUZA, K. J. MURPHY AND C. M. REGAN*
School of Biomolecular and Biomedical Sciences, Conway Institute, University College Dublin, Beleld, Dublin 4, Ireland

AbstractArising from studies on the amnesia that follows site-specic physical or chemical lesions, the acquisition and consolidation of certain behavioral tasks has been demonstrated to be associated with different hippocampal subregions. Although not absolute, spatial learning is reliant on the dorsal region of the hippocampus, whereas avoidance- and fear-conditioning tasks appear to be dependent on its more ventral aspects. Thus, if learning-associated synapse remodeling is a true feature of memory consolidation it must also follow these regional dissociations. We therefore determined if the learning-associated increases in synapse density that occur in the mid-molecular layer of the dentate gyrus at the 6-h post-training time and the frequency of polysialylated cells at the infragranular zone that occur at the 12-h posttraining time were dissociated to specic hippocampal subregions following training in either a massed water maze task or light dark passive avoidance response. Synapse remodeling was found to occur only in the dorsal hippocampus following spatial learning. We could not, however, discern any regional dissociation of neural remodeling following avoidance conditioning. These results point to strong associations between learning and specic groups of novel synapses during consolidation of spatial learning and avoidance conditioning paradigms. Crown Copyright 2012 Published by Elsevier Ltd on behalf of IBRO. All rights reserved. Key words: NCAM PSA, synapse number, spatial learning, avoidance conditioning.

Processing of information for long-term consolidation is dependent on the considerable neuroplastic potential of the hippocampal formation. One means by which the hippocampus expresses this plasticity is through activity-dependent alteration of synapse number and structure, a mechanism that has been consistently associated with learning in diverse species and paradigms (Bailey and Kandel, 1993; Ramrez-Amaya et al., 1999; Geinisman, 2000; Leuner et al., 2003; Marrone, 2007; Rekart et al., 2007; Holtmaat and Svoboda, 2009; Redondo and Morris, 2011; Ruediger et al., 2011). In neural networks, two types of synapse manipulation have been discerned: synaptic plasticity in which change in strength of existing synapses
*Corresponding author. Tel: 353-1-71-66-775; fax: 353-1-71-66-920. E-mail address: ciaran.regan@ucd.ie (C. M. Regan). Abbreviations: LTP, long term potentiation; PBS, phosphate buffered saline; PSA NCAM, polysialylated synapse-variant of the neural cell adhesion molecule.

occurs, and structural plasticity that involves the creation and pruning of synapses. Arising from this dual framework, the temporal persistence of synapse potentiation (or depression) in cell assemblies is now a widely accepted correlate of synaptic plasticity in memory consolidation (Redondo and Morris, 2011). Rapid formation and selective stabilization of cortical synapses as a correlate of motor learning consolidation has now been demonstrated (Xu et al., 2009; Yang et al., 2009; Roberts et al., 2010) and, within the molecular layer of the rat hippocampal dentate gyrus, transient increase in synapse number has been shown to occur at the 6 9-h post-training time, returning to basal numbers by 72 h, following training in both spatial and avoidance conditioning tasks (OMalley et al., 1998, 2000; Eyre et al., 2003). Learning-associated synapse remodeling has, in part, been related to the ongoing generation and incorporation of granule cells into the existing neural network (SchmidtHieber et al., 2004; Deng et al., 2010; Tronel et al., 2010). Integration of these newborn cells into the hippocampal neural network has been demonstrated to involve competitive synapse remodeling of their dendritic branches with the afferent axons of the perforant path that arises in the entorhinal cortex and terminates in the mid-molecular layer of the dentate gyrus (Toni et al., 2007). During the maturation of these newborn neurons the polysialylated synapse-variant of the neural cell adhesion molecule (PSA NCAM; Persohn et al., 1989; Doyle et al., 1992) may be expressed for a period of weeks following their birth (Seki and Arai, 1993, 1999), and this cell adhesion molecule has been proposed to play a role in their integration into the evolving neural network (Kee et al., 2007). Hippocampal PSA NCAM has also been demonstrated to be necessary for the effective consolidation of memory (Becker et al., 1996; Lopez-Fernandez et al., 2007; Seymour et al., 2008), and the frequency of granule cells expressing PSA NCAM in the infragranular zone of the dentate transiently increases at the 12-h post-training period (Fox et al., 1995; Murphy et al., 1996) when memory is committed to long-term storage (Rossato et al., 2009). The ability of PSA NCAM to modulate glutamatergic signaling through NR2B subunit-containing NMDA receptors (Hammond et al., 2006) further suggests polysialylated NCAM may play a role in ne-tuning the network during the nal stages of memory consolidation. Based mainly on studies on the amnesia that occurs following site-specic physical and chemical lesions, the acquisition and consolidation of diverse behavioral tasks has been demonstrated to be associated with different

0306-4522/12 $36.00 Crown Copyright 2012 Published by Elsevier Ltd on behalf of IBRO. All rights reserved. doi:10.1016/j.neuroscience.2012.01.020

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hippocampal subregions (Fanselow and Dong, 2010; McHugh et al., 2011). Spatial learning, for example, appears to be more reliant on the dorsal region of the hippocampus (Moser et al., 1993; Hock and Bunsey, 1998; Bannerman et al., 1999), an area that primarily receives afferents from sensory neocortical regions (Burwell and Amaral, 1998; Witter and Moser, 2006). In contrast, avoidance conditioning tasks have been found to be largely dependent on the more ventral aspects of the hippocampus (Czerniawski et al., 2009; Ambrogi Lorenzini et al., 1997; Maren and Holt, 2007; McHugh et al., 2004; Trivedi and Coover, 2004), which is preferentially innervated by the amygdaloid complex and hypothalamicpituitaryadrenal axis (Risold and Swanson, 1996; Petrovich et al., 2001). These distinctions between dorsal and ventral hippocampus, however, are not absolute, as under certain conditions the ventral hippocampus has been shown to also contribute to spatial learning (De Hoz et al., 2003; Ferbinteanu et al., 2003). Despite the considerable evidence that now exists to implicate hippocampal synapse remodeling in memory formation (Marrone, 2007), few studies have attempted to link this phenomenon to the subregions of the dentate gyrus that are believed to play a preferential role in the acquisition and consolidation of specic learning paradigms. We were interested, therefore, to determine if transient synapse formation and polysialylated cell frequency exhibited a dorsoventral separation within the dentate gyrus during hippocampus-dependent consolidation of tasks relying on spatial information (water maze paradigm) as compared with those processing other information, such as the anxiety-driven aspects of the passive avoidance paradigm.

diameter, 80 cm high, temperature 261 C) with a platform (11 cm diameter) submerged 1.5 cm below the water surface. Both the pool and the platform were constructed of black polyvinyl plastic and offered no intra-maze cues to guide escape behavior. The experimental room contained several extra-maze visual cues. During testing, the platform was hidden in the same quadrant 30 cm from the side wall. Each trial started with the rat facing the wall of the maze at one of three xed points A, B, or C arranged triangularly around the pool and separated by an angle of 120 degrees. The starting positions were used in sequence over the ve trials of each training session. Continuity in the pattern of starting positions was maintained over the different training sessions. The time taken by the rat to nd the hidden platform within a 60-s criterion period was dened as the escape latency time. On the rst trial, rats failing to locate the platform within the 60-s period were placed on it for 10 s. Passive control animals were swim-time matched to their trained counterparts in the absence of an escape platform. In the single massed training session the escape latencies were measured over ve trials with an inter-trial rest interval of 300 s A Mann Whitney U-test was used to compare rst and last trials, and a two-way ANOVA was used to compare time and synapse number. A single training session was employed, and separate cohorts of animals were killed at 1 h, 6 h, and 72 h for synapse analysis and at 0 h, 12 h, and 72 h for polysialylated cell analysis.

Avoidance conditioning paradigm

Animals were trained in a single-trial, step-through, light dark passive avoidance paradigm, as described previously (Fox et al., 1995). The training apparatus consisted of two chambers, the smaller illuminated chamber being separated from the larger dark chamber by a shutter that contained a small entrance. The oor of the training apparatus consisted of a grid of stainless steel bars through which a remotely controlled, scrambled shock (0.75 mA every 0.5 ms) of 5 s duration could be delivered when the animal entered the dark chamber. The animals were tested for recall of this inhibitory stimulus at dened post-training times (1 h, 6 h, and 72 h for synapse analysis and at 0 h, 12 h, and 72 h for polysialylated cell analysis) by placing them into the light chamber and recording their latency to enter the dark chamber. A criterion period of 300 s was used. All animals were killed immediately following their recall trial. Passive control animals followed the same procedure and were allowed to explore the passive avoidance apparatus for a matched period of time to their trained counterparts but received no foot shock. Between each training session, both compartments of the training apparatus were cleaned thoroughly to eliminate confounding olfactory cues. Values signicantly different from the control were determined using the MannWhitney U-test for non-parametric data and P-values of less than 0.05 were considered to be signicant.

EXPERIMENTAL PROCEDURES
Animal maintenance
Male Wistar rats (postnatal day 80) were obtained from Harlan (UK), maintained in the Biomedical Facility at University College Dublin and housed in pairs on a 12-h light dark cycle at 222 C, with ad libitum access to food and water. Animals were introduced, maintained, and handled in the test environment for 3 days before any study. During this period the animals were handled and their general behavior assessed in an open eld arena (808015 cm3) over a 5-min period for any anomalies in locomotor activity, rearing, and/or general behavior that might preclude an animal from the study were noted. Separate cohorts of animals were used for the water maze (n40) and passive avoidance (n35) paradigm, and no animals were precluded from the study based on anomalous behavior. All training was carried out in a quiet room, under low-level red illumination, between 07.00 and 11.00 h to minimize any circadian inuence. The experimental procedures were approved by the Animal Research Ethics Committee of University College Dublin, conformed to EU Council Directive 86 609 EEC, and were carried out in the Biomedical Facility within the parameters of the appropriate license issued by the Department of Health. At all times the number of animals used and their suffering was kept to a minimum.

Electron microscopy
Tissue xation and processing. Tissue was xed by transcardial perfusion using 0.12 M Srenson phosphate buffer (pH 7.2) containing a combination of 4% paraformaldehyde and 2.5% glutaraldehyde. Following xation the brains were immediately dissected and stored in double strength perfusate at 4 C overnight. Subsequently, the hippocampus was dissected from the brain, subjected to three 10-min washes in xation buffer, and placed in 1% osmium tetroxide in deionized H2O for 60 min. The tissue was then embedded in epoxy resin using standard procedures of dehydration with increasing concentrations of ethanol and propylene oxide and, subsequently, polymerizing the resin at high temperature. The resin block was then trimmed to reveal the dentate gyrus from which 2-m sections were obtained in a serial manner. In the rst instance, coronal sections were obtained in a rostro-caudal manner up to point 3.3 mm caudal in respect of bregma. Secondly, using the same brain tissue, horizontal sections

Spatial learning paradigm

The massed water maze spatial learning paradigm employed has been described in detail previously (Murphy et al., 1996). Briey, the water maze apparatus consisted of a large circular pool (1 m

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dorsal

Immunohistochemical detection of dentate NCAM polysialylated granule cells

caudal
Tissue collection and cryosectioning. Following sacrice at post-training times of 0 h, 12 h, and 72 h, the whole rat brain was immediately dissected, coated in Optimum Cutting Temperature (OCT) compound to provide an even freezing rate, and lowered into a Cryoprep freezing apparatus (Algen Inc.) containing dry ice-cooled n-hexane. The brains were then stored at 80 C until later analysis. Sections of 12 m, cut on a microM Series 550 cryostat at 12 C, were used immediately for all studies and none were stored frozen. Given the C-shaped structure and overhanging dorsal lip of the rat hippocampus, sectioning was performed in two planes (Fig. 1). As employed in the electron microscopy studies, coronal sections were obtained in a rostro-caudal manner up to point 3.3 mm caudal in respect of bregma. Secondly, using the same brain tissue, horizontal sections were obtained in a dorsoventral direction up to point 7.1 mm ventral with respect to bregma. These separate analyses were subsequently combined to describe the dorsoventral distribution of PSA-positive cells. Inter-animal consistency was maintained by dening the rostrocaudal and dorsoventral coordinates by reference to bregma, as described in a rat brain atlas (Paxinos and Watson, 2007). NCAM PSA immunohistochemistry. The cryosections were thaw-mounted onto 0.1% (w/v) poly-l-lysine coated glass slides, xed in 70% (v/v) ethanol for 30 min, washed twice for 10 min in a washing buffer of 0.1 M phosphate buffered 0.9% saline, pH 7.4 (PBS), and incubated overnight (20 h) in a humidied chamber at room temperature with anti-PSA (generous gift of Prof. G. Rougon; Rougon et al., 1986). This primary antibody was diluted 1:500 in an incubation buffer composed of PBS containing 1% (w/v) bovine serum albumin (Sigma Chemical Co., UK) and 1% (v/v) normal goat serum (Dako, Denmark) to eliminate non-specic staining. The sections were washed again and exposed for 3 h to uorescein-conjugated goat anti-mouse IgM (Calbiochem, UK) diluted 1:100 with incubation buffer. The sections received a nal wash before being mounted in Citiuor (Agar, UK), a uorescence enhancing medium. The staining pattern was observed with a Leitz DM RB uorescence microscope using an exciting wavelength of 495 nm and an emitting wavelength of 525 nm. Immunouorescence staining was specic as it was eliminated completely by omission of either the primary or secondary antibody and by pre-absorbing anti-PSA with colominic acid (1 mg/ml; Sigma Chemical Co., UK), which contains 2,8-linked homopolymers of sialic acid (Murphy et al., 1996). Where relevant, sections were counterstained by a brief exposure (25 s) to propidium iodide (50 ng/ml PBS), a uorescent DNA intercalating agent that was detected using an excitation wavelength of 552 nm and an emission wavelength of 570 nm. Photographs of sections were taken using the Zeiss AxioVision 4 system. Quantitative evaluation of dentate NCAM polysialylated cells. Quantitative image analysis was performed using the Leica Quantimet 500, a PC-based software package connected to the uorescence microscope by a high sensitivity CCD video camera. Each microscope lens was calibrated for length and area measurements using a 1 mm graticule. The total number of NCAM PSA-immunoreactive neurons on the right dentate granule cell layer/hilar border was counted in seven alternate 12-m sections, to preclude double counting of the 510 m perikarya. Cell counts were divided by the total area of the granule cell layer, which included all propidium iodide labeled cells, and multiplied by the average granule cell layer area, which was 0.150.01 mm2, and the mean value was calculated for each animal. These means were used to establish the meanSEM for each animal group. Parametric statistical comparisons were made using the Student t-test and P-values 0.05 were taken to be signicant.

rostral

hippocampus pp p
horizontal section

coronal section ventral

CA DG

CA DG

Fig. 1. Cartoon of hippocampal formation showing rostro-caudal and dorsoventral positions. Coronal sections were taken for analysis of dorsal hippocampal regions, and horizontal sections were taken for the more ventral aspects. CA, cornu ammonis region of the hippocampus; DG, dentate gyrus. For interpretation of the references to color in this gure legend, the reader is referred to the Web version of this article.

were obtained in a dorsoventral direction up to point 7.1 mm ventral with respect to bregma (Fig. 1). These separate analyses were subsequently combined to describe the dorsoventral distribution of synaptic density. The location of these sections was conrmed by referencing Toluidine Blue-stained sections (1% w/v in 1% aq. borax, pH 9) with a stereological atlas (Paxinos and Watson, 2007). The ultrathin sections used for electron microscopy were obtained using a 2 mm diamond knife, collected onto formvarcoated (0.4% w/v H2O) copper slot grids, and stained with uranyl acetate (5% w/v H2O) for 20 min and lead citrate (0.3% w/v 0.1 M sodium hydroxide) for 10 min. Average section thickness was 80 nm, as assessed using interference color patterns. Sections were examined in a Tecnai G2 Spirit BioTWIN electron microscope at an accelerating voltage of 120 kV. Images were recorded using a Megaview III CCD, analyzed using the analySIS programme (Soft Imaging Systems) and stored on CD for further analysis. Stereology. Quantication of ultrastructural features employed the double-disector unbiased stereological counting procedure (Sterio, 1984). An area of 9.593 m2 on adjacent sections was dened using a Gundersen frame. The number of objects present in the look-up but not reference section was estimated for each disector pair according to previously described rules (Gundersen, 1977). This was dened as the Q value (see below). For each animal, a total of 19 disector pairs were analyzed. Synapse density was then estimated using NvQ/ha(fra) in which Equation Nv is the density of objects (synapses) per unit volume, h is the thickness of the section, and a(fra) is the area of the Gunderson counting frame. Parametric statistical comparisons were made using the Student t-test and P-values 0.05 were taken to be signicant. To determine the relationship between synaptic density and learning ability (latency), these parameters were compared by Pearson correlation (GraphPad Prism 4 software; P0.05 indicated signicant correlation).

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A 300 Escap pe latency (s)

Passive avoidance paradigm

Water maze paradigm 90

Escap pe latency (s) 1 6 12 72

200 100 20

60

30

10 0

0 1 2 3 4 5

Post-training (h)

Trial number

Fig. 2. Behavioral response of rats exposed to learning paradigms. Panel (A) represents escape latency for animals exposed to the passive avoidance paradigm (n35) at indicated post-training time-points. Values are expressed as box plots. The box represents the rst and third interquartile range and the line denotes the median. All trained animals reached the criterion time of 300 s. Red box plots represent latency of passive animals to enter dark chamber. Panel (B) represents escape latencies of animals trained in the single session water maze paradigm (n40). Data points are meanSEM (bars) time taken to locate hidden platform. For interpretation of the references to color in this gure legend, the reader is referred to the Web version of this article.

All tissue collected for immunohistochemical and ultrastructural studies were evaluated by an observer who was blind to the experimental conditions.

RESULTS
Acquisition of behavioral paradigms At the various post-training times, animals were tested for recall of the passive avoidance task before being killed. Of this group, all animals achieved a latency time of 300 s (Fig. 2A; median value 300 s with interquartile range 0,0 s). Failure to reach the criterion time of 300 s dictated exclusion from the study. In contrast to those trained, the passive controls showed no hesitance in entering the darkened chamber upon reexposure to the testing apparatus (red box plots in Fig. 2A). This divergence in latency scores between the two groups was indicative of task acquisition (P0.05, MannWhitney non-parametric U-test). The water maze task was acquired by all animals as the swim latency times became signicantly reduced between the rst and the fth trial (Fig. 2B; P0.05, Mann Whitney non-parametric U-test). The passive control animals explored the water maze for a time matched to their trained counterparts but in the absence of the platform. Ultrastructural analysis of synapse density in midmolecular layer of dentate gyrus Electron microscopy was employed to quantify the ultrastructural alterations in the mid-molecular layer of the dentate gyrus where the main synaptic input comes from the entorhinal cortex. Within both dorsal and ventral aspects of this region, the majority of synapses were either axospinous or axodendritic in nature. Perforated synapses, which

exhibit a discontinuous postsynaptic density, were also present throughout the mid-molecular layer of the dentate gyrus. Further, no structural change in synapse prole was observed at any time examined after training in either paradigm (Fig. 3A, B). Change in the frequency of mature synapses, those with at least three presynaptic vesicles and an established postsynaptic density, was determined at increasing times post-training (1 h, 6 h, and 72 h) using the non-biased stereological disector method. In the dorsal hippocampus of rats trained in the spatial learning paradigm displayed a signicant increase in the density of simple synapses in the dentate mid-molecular layer at the 6-h post-training time was observed (Fig. 3C). This increase in synaptic density was specically associated with learning, as it was not present in swim time-matched passive controls (Trained: 2.170.06 vs. Passive: 1.820.12; P0.05; meanSEM, Students t-test). Synapse density at either the 1 h or 72 h post-training time was indistinguishable from that observed in naive animals, indicating that the increase observed at the 6-h post-training time was both transient and specic to the consolidation of the learning paradigm. Importantly, no increase in synapse density was observed in the ventral hippocampus at any of the time points analyzed (Fig. 3C), implying that the learningassociated synapse remodeling was restricted to the dorsal aspect of the hippocampus during the consolidation of the spatial learning paradigm. A signicant and transient increase in synapse density was also observed to occur in the dorsal aspect of the mid-molecular layer of the dentate gyrus at the 6-h post-training time following acquisition of the avoidance conditioning paradigm (Fig. 3D). The magnitude of this transient, time-dependent increase in dorsal synapse

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Spatial learning paradigm

Avoidance conditioning paradigm

PSD

vesicles PSD

vesicles vesicles

Spatial learning paradigm

Dorsal dentate midmolecular layer Ventral dentate midmolecular layer

Avoidance conditioning paradigm

Dorsal dentate midmolecular layer Ventral dentate midmolecular layer

2.5

3.0

*
20 2.0

** *

2.5

Synapses/m3

Synapses/m3
N 1 6 72 N 1 6 72

2.0 1.5 1.0 0.5 0.0 N 1

1.5

1.0 0.5 0.0

72

72

Post-training time (h)

Post-training time (h)

Spatial learning paradigm

Dorsal D ld dentate t t midid molecular layer 0.4 Ventral V t ld dentate t t midid molecular layer

Avoidance conditioning paradigm

Dorsal D ld dentate t t midid molecular layer 0.4 Ventral V t ld dentate t t midid molecular layer

Perforated syna apses/m3

Perforated syn napses/m3

0.3

0.3

0.2

0.2

0.1

0.1

0.0

0.0 N 1 6 72 N 1 6 72 N 1 6 72 N 1 6 72

Post-training Post training time (h)

Post-training g time ( (h) )

Fig. 3. Time-dependent frequency of synapse number in the mid-molecular layer of the adult rat dentate gyrus following training. Panels (A, B) illustrate the morphology of a simple synapse (left image) and perforated synapse (right image) at 6 h following training in a spatial learning paradigm (A) and avoidance conditioning paradigm (B). Panels (C, D) illustrate the temporal change in simple synapse density in both dorsal and ventral aspects of the dentate gyrus following training in spatial learning (C) and avoidance conditioning (D) paradigms. The inuence of training on perforated synapse number following training is similarly shown in panels (E, F). All values are meanSEM synapses per m3 (n5) and the asterisk indicates signicant difference (P0.05) with respect to passive control (lled column). N, naive; PSD, postsynaptic density.

frequency (Trained: 1.970.04 vs. Passive: 1.700.08; P0.05; meanSEM, Students t-test) was modest compared with the extent of change observed in the more ventral aspect of the dentate gyrus molecular layer following avoidance conditioning. Here, synapse density increased by approximately 50% at the 6-h post-training time above that observed in the naive animal (Fig. 3D). Moreover, this extensive remodeling was transient as

the learning-associated increase in synapse returned to the basal levels observed at the 1-h and 72-h post-training times. Finally, learning-associated change in synapse number appeared to be conned to simple synapses, either axodendritic and/or axospinous, as the frequency of perforated synapses, which accounted for approximately 10% of the total synapse population, showed no difference in

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either the dorsal or ventral regions during consolidation of either paradigm (Fig. 3E, F). Quantitative analysis of NCAM polysialylated cell frequency at the infragranular zone of the dentate gyrus Given a signicant number of polysialylated neurons exist at the infragranular zone of the dentate gyrus and that a transient increase in their frequency has been demonstrated to be necessary for the consolidation of both spatial and avoidance conditioning paradigms (Fox et al., 1995; Murphy et al., 1996; Seymour et al., 2008), we determined if the learning-associated activation of NCAM polysialylation exhibited the same regional dissociations observed for synapse remodeling during the consolidation of each task. Following spatial learning a signicant increase in polysialylated cells was observed to occur only in the more dorsal aspect of the dentate gyrus at the 12-h post-training time (Fig. 4A). Quantication of these polysialylated cells demonstrated this change to be learning specic, as it was not observed in the swim-matched control animals (Trained: 33.621.43 vs. Passive: 28.361.13 cells per U area; P0.05; meanSEM, Students t-test). Moreover, the increase was transient, as NCAM PSA-positive cell numbers returned to near-basal levels by 72 h (Fig. 4B). In contrast, a transient activation of polysialylated neurons was observed in both the dorsal and ventral aspects of the denate gyrus at 12 h following training in the passive avoidance paradigm (Fig. 4A, C). Quantication of polysialylated cell frequency in the dorsal dentate region revealed a signicant increase of approximately 30% at the 12-h post-training time (Trained: 36.82.1 vs. Passive: 28.7 1.3 cells per U area; P0.05; meanSEM, Students ttest). Although the density of polysialylated cells in the ventral dentate was substantially smaller than that observed in the dorsal dentate, these too exhibited a similar increase of about 40% at the 12-h post-training time (Trained: 20.480.43 vs. Passive: 14.520.41 cells per U area; P0.05; meanSEM, Students t-test). In both the dorsal and ventral aspects of the dentate gyrus the learning-associated transient increases in polysialylated cell frequency were transient, both returning to baseline level at the 72-h post-training time; however, the transient increase in respect of the 0 h time point was only signicant in the ventral dentate gyrus following avoidance conditioning.

DISCUSSION
Task-specic regional dissociations of synapse remodeling during memory consolidation In agreement with previous work demonstrating the dorsal hippocampus to be essential for spatial learning in both animals and humans (Moser et al., 1993; Maguire et al., 2000; Bannerman et al., 2004), we have found synapse remodeling, as evidenced by transient change in synapse formation and polysialylated cell frequency, to be restricted to the more dorsal aspect of the hippocampal formation. Moreover, such synapse remodeling, previously demonstrated to be associated with the consolidation of spatial

learning paradigms (OMalley et al., 2000; Eyre et al., 2003), was completely absent in the more ventral aspect of the hippocampus over the time frame examined. In contrast, synapse remodeling extended over the entire dorsalventral axis of the hippocampal formation following passive avoidance training, a task in which the learned response of avoiding the darkened chamber is based on both explicit spatial cues and anxiety associated with uncertainty of footshock. The preferential role of the ventral hippocampal formation in the neuroplastic response, as judged by the extent of synapse remodeling, observed during consolidation of the avoidance conditioning paradigm is consistent with its strong reciprocal connectivity with the amygdala and particularly the structures within the hypothalamicpituitaryadrenal axis (HPA; Risold and Swanson, 1996; Petrovich et al., 2001). As the HPA axis mounts highly plastic responses during the processing of sensory physiological information (Hatton, 1997; Theodosis et al., 2008), it is not surprising that there is a concomitant and substantial increase in synapse remodeling in the ventral hippocampus following training and that this possibly reects the greater demands required for processing the anxiogenic demands of the task. In humans, for example, sustained anxiety evokes signicant activation of anterior (ventral) hippocampus (Hasler et al., 2007). Passive avoidance learning, in contrast to spatial learning, appears to require synapse remodeling in both the dorsal and ventral aspects of the hippocampus, clearly indicating the importance of both spatial and emotional/ motivational components in task consolidation. Interestingly, animals with lesions that incorporate both dorsal and ventral aspects of the hippocampus have been found capable of learning certain tasks, but to persist in the learned response when it had become no longer appropriate (for review see Bannerman et al., 2004; Davidson and Jarrard, 2004). This form of inhibitory learning indicates that the hippocampus plays a more general role in information processing that includes the regulation of anxiety-related behaviors. The septo-hippocampal pathway plays a key role in generating this inhibitory response (Gray and McNaughton, 2000), and it should be pointed out that learning-associated change in NCAM PSA-mediated neuroplasticity within this system, which is located specically to the subtriangular septal zone, is indistinguishable during consolidation of either avoidance conditioning or spatial learning paradigms (Foley et al., 2003). This might, in part, explain the involvement of synapse remodeling across the entire dorsoventral axis of the hippocampus. Importantly, these studies further highlight the contention that the emotional/motivational role of the ventral hippocampus is more associated with anxiety than fear (for review see McHugh et al., 2004), as modulation of NCAM polysialylation state in this subregion of the hippocampus accompanies avoidance conditioning (these studies) but not context fear conditioning (Lopez-Fernandez et al., 2007). It should be noted, however, that the involvement of the ventral part of the hippocampus in context fear conditioning remains highly controversial (Anagnostaras et al., 2001; Kjelstrup et al., 2002; Rudy and Matus-Amat, 2005) but, clearly, it

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Dorsal Naive

Ventral Naive

Dorsal Spatial learning

Ventral Avoidance conditioning Dorsal Avoidance conditioning

Spatial learning paradigm

Dorsal dentate gyrus Ventral dentate gyrus

Avoidance conditioning paradigm

Dorsal dentate gyrus Ventral dentate gyrus

40

45

Polysialyla ated cells/au

ated cells/au Polysialyla

*
30

*
35 25

20

15

10

5 N 0 12 72 N 0 12 72

0 N 0 12 72 N 0 12 72

Post-training time (h)

Post-training time (h)

Fig. 4. Learning-induced increase of polysialylated granule cells at the infragranular zone of the rat hippocampal dentate gyrus. Panel (A) illustrates the distribution of polysialylated cells in the dorsal and ventral aspects of the dentate gyrus in naive animals and at 12 h following training in either a spatial learning or avoidance conditioning paradigm. The arrowheads indicate the distribution of polysialylated cells in the suprapyramidal blade of the denate gyrus and the inset shows a higher magnication of the cells in the infrapyramidal blade (Scale bar is 100 m). Panels (B, C) illustrate the temporal change in polysialylated cell frequency in both dorsal and ventral aspects of the dentate gyrus following training in spatial learning (B) and avoidance conditioning (C) paradigms. All values are meanSEM PSA-positive cells per unit area (n5) and the asterisk indicates signicant difference (P0.05) with respect to passive control (lled column). N, naive. For interpretation of the references to color in this gure legend, the reader is referred to the Web version of this article.

does not require change in NCAM polysialylation-mediated plasticity (Lopez-Fernandez et al., 2007).

CONCLUDING REMARKS
The present results point to strong associations between learning and specic groups of novel synapses during

consolidation of spatial and avoidance conditioning paradigms. Importantly, these studies directly show change in synaptic contact and are in contrast to those studies that use light microscopic imaging of Golgi-stained material (Kolb et al., 2008) or transcranial two-photon microscopy of neurons marked with a uorescent protein (Denk et al.,

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1990; Kelsch et al., 2010) to report change in learninginduced spine density. Secondly, change in synapse number was specic to learning as it was absent in the swimmatched controls employed in the spatial learning task or in animals exposed to context alone in the avoidance conditioning paradigm. Moreover, as change in synapse plasticity was restricted to the 6-h post-training time it is unlikely that this remodeling might have been induced by the retrieval process. Finally, the magnitude of the learning-associated synapse frequency increases observed during task consolidation is unlikely to have impact on brain functional coherence, as sequential training in spatial learning and avoidance conditioning tasks results in successful consolidation providing there is an inter-paradigm period of 12 h (Murphy and Regan, 1999). Similar conclusions have been derived from studies on human motor skill learning (Brashers-Krug et al., 1996). Moreover, activity-induced increases in synapse density are of the same order of magnitude to those observed following the induction of long term potentiation (LTP), which are without detriment to the electrophysiological parameters measured (Toni et al., 2001), and to the volumetric increase observed in the posterior hippocampus of people with a high dependence on navigational skills and who have not been associated with any behavioral anomalies (Maguire et al., 2000). These learning-associated synapses appear to be generated de novo during task consolidation, as there was no evidence of numerical change in perforated synapses, a mechanism of synapse splitting implicated in the formation in LTP (Toni et al., 2001; Mezey et al., 2004). The synapses observed at this post-training time appeared to be functional as all expressed fully elaborated postsynaptic densities and a signicant complement of presynaptic vesicles. This is not surprising as rapid and signicant increases in the frequency of mature hippocampal synapses, including simple and perforated forms and multiple synapse boutons, has been observed to occur within 30 min of change in neural activity (Toni et al., 2001). Moreover, our analysis of synapse density at increasing times following training has demonstrated the population of newly formed, learning-associated synapses to be transient. This transience, which we and others have reported previously (OMalley et al., 1998, 2000; Eyre et al., 2003), suggests that these synapses may undergo a selection process that allows the neural network to be remodeled into an alternative and stable synapse connectivity pattern that emerges in the 72-h post-training time. Finally, the learning-induced synapse formation was associated with memory consolidation as it occurred at the late 6 h posttraining time, as has been shown previously (OMalley et al., 1998, 2000; Eyre et al., 2003). This latter observation is quite distinct from the rapid spine formation in the cortex (1 h) that is linearly correlated with the number of successful trials in a reward-based motor reaching task (Xu et al., 2009). Given the strict temporal relationship between synapse remodeling within the dentate mid-molecular layer and the change in the frequency of polysialylated cells at the infra-

granular zone with arbors extending into the dentate midmolecular layer, it is tempting to suggest that NCAM PSA may play a role in the selection of supernumerary synapses within the evolving memory trace. The synaptic strength of synapses depends on postsynaptic NCAM expression (Schuster et al., 1998; Dityatev et al., 2000) and it is this isoform NCAM180 that is polysialylated during memory consolidation (Doyle et al., 1992). As NCAM PSA restrains synapse signaling through NMDA receptors containing GluN2B subunits (Hammond et al., 2006; Kochlamazashvili et al., 2010), possibly through the steric hindrance of receptorligand interactions (Rutishauser, 2008), this mechanism may regulate synapse elimination. The signicant correlation that exists between learning and number of activated polysialylated granule cell neurons (Sandi et al., 2004), further suggests the importance of NCAM PSA in providing the initiating molecular signal or tag for synapses to be selectively eliminated from the engram over the ensuing 23 days required for eventual memory consolidation.
AcknowledgmentsThis work was supported by Enterprise Ireland (SC/2003/0470) and Science Foundation Ireland (03/IN3/ B403/C). The authors wish to acknowledge helpful discussions with Dr. Sean Mulvany at the outset of this work. The technical advice of Dr. David Cottell of the Electron Microscopy laboratory is gratefully acknowledged. The authors declare no potential sources of conict of interest. All authors made substantial contributions to conception and design, acquisition, analysis, and interpretation of data. C.M.R. drafted and revised the manuscript and approved the nal version to be published.

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(Accepted 11 January 2012) (Available online 18 January 2012)