Seralynne D. Vann, 1 * Jonathan T. Erichsen, 2 Shane M. O Mara, 3 and John P. Aggleton 1 INTRODUCTION HIPPOCAMPUS 21: (2011)

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1 HIPPOCAMPUS 21: (2011) Selective Disconnection of the Hippocampal Formation Projections to the Mammillary Bodies Produces Only Mild Deficits on Spatial Memory Tasks: Implications for Fornix Function Seralynne D. Vann, 1 * Jonathan T. Erichsen, 2 Shane M. O Mara, 3 and John P. Aggleton 1 ABSTRACT: It is now clear that the integrity of the fornix is important for normal mnemonic function. The fornix, however, is a major white matter tract, carrying numerous hippocampal formation afferents and efferents, and it is not known which specific components support memory processes. Established theories of extended hippocampal function emphasize the sequential pathway from the hippocampal formation (i.e., subicular complex) to the mammillary bodies and, thence, to the anterior thalamus, as pathology in each of these structures is implicated in anterograde amnesia in humans and spatial memory deficits in rats. The specific importance of the hippocampal formation projections that just innervate the mammillary bodies has, however, never been tested. This study isolated these specific projections in the rat by selectively cutting the descending component of the postcommissural fornix. Two successive, cohorts of rats with these tract lesions were tested on working memory tasks in the water-maze, T-maze, and radial-arm maze. Disconnecting the descending postcommissural fornix had only a mild effect or sometimes no apparent effect on the performance of these spatial memory tasks, even though tracing experiments confirmed the loss of hippocampal formation-mammillary projections. One implication is that the spatial deficits found in rats following standard fornix lesions are only partly attributable to the loss of projections from the hippocampal formation to the mammillary bodies. Perhaps more surprising, the behavioral impact of cutting the descending postcommissural fornix in rats appeared appreciably less than the effect of either mammillary body or mammillothalamic tract lesions. The present experiments show that the mammillary bodies can still effectively support spatial memory in the absence of their dense subicular complex inputs, so revealing the importance of the other afferents for sustaining mammillary body function. This new evidence for independent functions shows that the mammillary bodies are more than just a hippocampal relay. VC 2010 Wiley-Liss, Inc. KEY WORDS: diencephalon; medial temporal lobe; amnesia; rat INTRODUCTION The hippocampal formation connections carried in the fornix are vital for learning and memory, as demonstrated by both clinical and animal 1 School of Psychology, Cardiff University, United Kingdom; 2 School of Optometry and Vision Science, Cardiff University, United Kingdom; 3 School of Psychology, Trinity College Dublin, Ireland Additional Supporting Information may be found in the online version of this article. Grant sponsor: Wellcome Trust grant; Grant number: #081075; Grant sponsor: BBSRC David Phillips Research fellowship; Grant number: BB/ B501955/1. *Correspondence to: Dr. Seralynne Vann, School of Psychology, Cardiff University, CF10 3AT, United Kingdom. vannsd@cardiff.ac.uk Accepted for publication 14 February 2010 DOI /hipo Published online 7 May 2010 in Wiley Online Library (wileyonlinelibrary.com). studies (e.g., Gaffan and Gaffan, 1991; D Esposito et al., 1995; Olton, 1977; Aggleton et al., 2000; Vann et al., 2008). The fornix carries numerous projections both to, and from, the hippocampal formation, raising the important question of which fornical pathways are most critical for memory. The number of potential candidates is high given that lesions of the fornix can result in a widespread functional-related hypoactivity (Vann et al., 2000), leaving the need to target systematically the various hippocampal formation connections that comprise this tract. One direct approach is to disconnect specific hippocampal formation connections within the fornix. As the fornix descends into the septal region, the tract splits into two components: the precommissural fornix passes rostral to the anterior commissure, while the postcommissural fornix passes behind. The precommissural fornix innervates the prefrontal cortex and ventral striatum, while fibers in the postcommissural fornix either turn caudally to run directly into the anterior thalamus or form a compact bundle that descends through the posterior hypothalamus to reach the mammillary bodies (Raisman et al., 1966; Poletti and Creswell, 1977). This study examined the importance of this latter, descending tract. The fibers in this tract originate in the subicular complex and entorhinal cortex, rather than the hippocampus proper (Swanson and Cowan, 1975; Ishizuka, 2001; Aggleton et al., 2005b). Interest in the hippocampal formation (i.e., subicular complex) projections to the mammillary bodies arises from the proposal that the mammillary bodies and anterior thalamic nuclei form part of an extended hippocampal memory system (Delay and Brion, 1969; Gaffan, 1992; Aggleton and Brown, 1999). This idea is supported by the density of the hippocampal formation projections that terminate throughout the mammillary bodies and the fact that the separate components of this system (hippocampal formation? fornix?mammillary bodies? anterior thalamic nuclei) have all been linked to anterograde amnesia and all impair spatial memory when damaged in animals (Dusoir et al., 1990; D Esposito et al., 1995; Aggleton and Brown, 1999; Aggleton et al., 2000; Vann and Aggleton, 2004; Aggleton et al., 2005a; Cipolotti et al., 2008; Tsivilis et al., 2008; Vann et al., 2008; Vann et al., 2009). Particularly striking is the recent VC 2010 WILEY-LISS, INC.

2 946 VANN ET AL. discovery that in patients who have suffered from colloid cysts the volume of the mammillary bodies is consistently correlated with scores on various tests of recall (Tsivilis et al., 2008). Not surprisingly, the mammillary bodies have typically been seen as a hippocampal relay to the anterior thalamus via the mammillothalamic tract (Papez, 1937; Barbizet, 1963; Aggleton and Brown, 1999). This overview of mammillary body function accords with evidence that damage to the mammillothalamic tract, following strokes in humans (Van der Werf et al., 2000; Van der Werf et al., 2003) or following lesions in rats (Vann and Aggleton, 2003), disrupts memory. Likewise, the impact of mammillary body lesions and mammillothalamic tract lesions can appear equivalent (Vann and Aggleton, 2003). A major shortcoming with this model of temporal lobemedial diencephalic interactions is that the importance of the direct projections from the hippocampal formation (i.e., subicular complex) to the mammillary bodies remains untested. While numerous studies in rats have described the behavioral impact of complete fornix lesions, surgeries targeting the postcommissural fornix remain exceptionally rare (Henderson and Greene, 1977; Thomas, 1978; Tonkiss et al., 1990; Tonkiss and Rawlins, 1992). Furthermore, in all of these previous instances the surgeries cut the fornix at the level of the septum, thus disconnecting multiple regions including the septum and thalamus, in addition to the mammillary bodies. This study targeted just that component of the postcommissural fornix that reaches the mammillary bodies, and assessed this selective disconnection using spatial tasks sensitive to complete fornix lesions and mammillary body lesions. As these are novel surgeries, much of the first disconnection study (Experiment 1) was replicated (Experiment 2). The effectiveness of the lesions was assessed by the injection of retrograde tracers into the mammillary bodies and anterior thalamic nuclei to determine whether, as planned, the descending postcommissural fornix surgeries disconnected the hippocampal formation-mammillary body projections while sparing the projections from the hippocampal formation to the anterior thalamic nuclei. In addition, cholinergic assays were used to determine whether, as intended, the septal inputs to the hippocampus were spared. In Experiment 3, rats with complete fornix lesions helped to confirm the sensitivity of the behavioral tasks. MATERIALS AND METHODS The study consisted of three experiments. The first two experiments examined the effects of descending postcommissural fornix lesions (dpfx1 and dpfx2) designed to disconnect the mammillary bodies from their subicular complex inputs, while the third experiment examined the impact of complete fornix lesions (Fx3). Because many of the behavioral manipulations were identical across the three experiments, the relevant information is combined. Training occurred in the same sequence as the tasks are reported. Subjects and Surgery Apparatus and behavioral training Total subjects for experiments 1 3 were 64 male, pigmented rats (Dark Agouti strain; Harlan, Bicester, UK) weighing between 215 and 268 g at the time of surgery. Animals were housed in pairs under diurnal light conditions (14 h light/10 h dark), and testing was carried out during the light phase. Animals were given free access to water throughout. All experiments were carried out in accordance with UK Animals (Scientific Procedures) Act, 1986 and associated guidelines. Animals were deeply anesthetized by intraperitoneal injection (60 mg/kg) of sodium pentobarbital and then placed in a stereotaxic headholder (David Kopf Instruments, Tujunga, CA) with the nose bar at The scalp was then cut and retracted to expose the skull. All lesions were made by radiofrequency using an RFG4-A Lesion Maker (Radionics, Burlington, VT). For the postcommissural fornix lesions (dpfx1, n 5 14; dpfx2, n 5 14), the electrode (0.7 mm tip length, 0.25 mm diameter) was lowered vertically and, at each site, the tip temperature was raised to 608C for 15 s. The anteroposterior (AP) and lateromedial (LM) coordinates, relative to ear-bar zero, were: (i) AP mm, LM mm; the depth was 28.2 mm from the top of the cortex. For the fornix lesions (Fx3, n 5 10), the electrode (0.7 mm tip length, 0.25 mm diameter) was lowered vertically, and at each site the tip temperature was raised to 758C for 60 s. The coordinates, relative to ear-bar zero, were: (i) AP mm, LM mm; (ii) AP mm, LM mm; the depths, from the top of cortex, were 23.7 mm and 23.8 mm, respectively. A total of 26 animals served as sham surgical controls (Experiment 1, Sham1, n 5 8; Experiment 2, Sham2, n 5 10; Experiment 3, Sham3, n 5 8). These rats received exactly the surgical procedures as the animals receiving lesions except that the electrode was not lowered as deep in order to avoid damage to the tracts; for the postcommissural fornix shams (Experiments 1, 2) the probe was lowered to 7.0 mm from the top of cortex while, for the fornix shams (Experiment 3), the probe was lowered to 2.0 mm from the top of cortex. No radiofrequency lesion was made. At the completion of all lesion and control surgeries, the skin was sutured and an antibiotic powder (Acramide: Dales Pharmaceuticals, Skipton, UK) was applied topically. Animals received subcutaneous injections of 5 ml glucose-saline and given paracetamol and sucrose in their drinking water for three days postsurgery. All animals recovered well following surgery. Behavioral training began two weeks after surgery, when pre-surgery body weight was fully restored. Delayed-Matching-to-Place in the Water-Maze (dpfx1, dpfx2, Fx3) In this task, the rat has to locate a hidden platform that remains in a constant position within a session, but changes positions across sessions. Consequently, trial 2 provides a measure of one-trial place learning.

3 POSTCOMMISSURAL FORNIX LESIONS AND MEMORY 947 Apparatus The water-maze (200 cm diameter, 60 cm deep) was made of white fiberglass and mounted 58 cm above the floor. The pool was filled with water ( C) made opaque by the addition of nontoxic emulsion (Opacifier, Chesham Chemicals, Harrow, UK). An escape platform (10 cm diameter, 2 cm below water surface) could be placed in the pool. The pool was in a room measuring cm 2. Lighting was provided by four floor-mounted spotlights (500 W) and the room contained salient visual cues such as geometric shapes and high contrast stimuli on the walls. There was a curtain hanging from the ceiling around the pool that could be opened or closed. Swim paths were tracked with a video camera suspended directly above the pool. The animals were transported between the holding room and water-maze in an opaque, aluminum traveling box. They were also placed in the opaque holding box in between each trial. Data were collected and analyzed on-line with an HVS image analyzer connected to a computer that used Water-maze Software (Edinburgh, UK). Procedure For this working memory task ( delayed matching-to-place ) in the water-maze, food and water were available ad libitum. Twelve platform positions, which varied in their distance from the pool perimeter, were used along with eight possible start positions. Animals received two days of pretraining with four swims a day. For pretraining, the curtain was drawn closed around the pool and both the start position and platform position were changed for every forced swim. Each swim was terminated when the animal either located the submerged platform or after 120 s had elapsed. If the animal had not located the platform at the end of 120 s it was guided there by the experimenter and then had to remain on the platform for 30 s. For the actual training, the curtain was removed from around the pool, i.e., room cues were visible. The location of the platform remained constant across the four trials of a given session but varied between sessions. The same start position was used for the first two trials of each session but was then varied for the remaining two trials. This arrangement made it possible to match distances for Trials 1 and 2, which best tests one-trial learning. Each trial terminated when the animal had either located the platform or 120 s had elapsed. The animals were then left on the platform for 30 s. The next trial began almost immediately afterwards, giving an ITI of about 15 s. The two cohorts of rats with postcommissural fornix lesions were tested for 16 sessions while the fornix lesion group was tested for 12 sessions. Reinforced Spatial Alternation in the T-Maze (dpfx1, dpfx2, Fx3) This test of spatial working memory takes advantage of rats spontaneous avoidance of places that have just been visited. In Experiments 1 and 2 (dpfx1, dpfx2), task difficulty was first increased by adding more proactive interference, e.g., increased trials per session, massed trials (Stages 2, 3, respectively). Next, intra-maze and extra-maze cues were set in conflict (Stage 4). Each stage followed immediately after the previous one. Rats with complete fornix lesions (Fx3) were only tested on Stage 1 given the severity of their deficit. Apparatus Testing was carried out in a modifiable four-arm (crossshaped) maze. The four arms (70 cm long, 10 cm wide) were made of wood while the walls (17 cm high) were made of clear Perspex. At any time, one of the arms could be blocked off to form a T-shaped maze. Aluminum barriers could be positioned approximately 25 cm from the end of each arm to create a start area. For this experiment, the location of the start arm remained constant such that the T-maze was in the same orientation throughout testing. The maze was supported by two stands (94 cm high), and was situated in a rectangular room ( cm 3 ) with salient visual cues. Procedure Animals were food deprived to 85%, or above, of their freefeeding bodyweight. Each animal was given seven sessions of 5 min pre-training. For this, the stem of the T-maze was closed off from the arms and the animals were placed in the arms and stem, separately. This procedure meant that the animals were habituated to the maze and learnt to find food rewards at the end of the arms but did not learn other task demands. Following pre-training, task acquisition began. Stage 1 (Sessions 1 8; dpfx1, dpfx2, Fx3) At the start of each acquisition trial, which consisted of two stages, two food pellets (45 mg; Noyes Purified Rodent Diet, UK) were placed in each food well and an aluminum block was placed at the neck of the T-maze, thus closing off one arm. As a consequence, on each sample run, the animal was forced to enter the open arm where it was allowed to eat the food at the end of the arm. The animal was then picked up and placed in the start box (facing away from the maze) for a delay of 10 s, during which the aluminum block was removed. While the animals were not deliberately disorientated, they were rotated as a natural consequence of replacing them in the maze. The door to the start arm was then opened and the animal allowed a free choice between the two arms of the T-maze. On this choice run, the animal was considered to have chosen the correct arm if it had alternated i.e., had entered the arm not previously entered on the sample run." The rat would then be allowed to eat the food reward before being returned to the holding box. If the animal made an incorrect choice, i.e., returned to the arm visited on the sample run," the rat was confined to that arm for 5 s before being returned to the traveling box. The rats were tested in groups of four with each animal having one trial in turn so that the intertrial interval (ITI) was about 4 min. The animals received six trials a session for a total of eight sessions.

4 948 VANN ET AL. Stage 2 (Sessions 9 14; dpfx1, dpfx2) Rats received six sessions of continuous alternation." At the start of each of these sessions, the rat was forced to either the right or left arm, and this initial sample run was rewarded with two pellets. This sample run was immediately followed by 10 consecutive massed trials in which the correct choice was always the opposite arm to the one chosen by the rat on the previous trial; the ITI was 15 s and there were no correction trials. Stage 3 (Sessions 15 20; dpfx1, dpfx2) The continuous alternation procedure remained, but the number of trials increased to 16 per session. Stage 4 (Sessions 21 24; dpfx1, dpfx2) The continuous alternation continued with 16 trials per session, but after each arm choice the maze was rotated by 908, either clockwise or anticlockwise. As a consequence, the previous choice arm became the start arm for the next trial. This procedure was to control for the use of intra-maze cues such as odor trails. Animals continued to be rewarded for selecting the arm in the opposite place (defined allocentrically) to that chosen by the rat on the previous trial. Stage 5 (Sessions 25 26, dpfx1; sessions 25 28, dpfx2) This stage was the same as Stage 3 where animals received 16 continuous alternation trials per session. Radial-Arm Maze (dpfx1) This test of spatial working memory again takes advantage of rats spontaneous avoidance of places that have just visited. By rotating the arms of the maze mid-trial (where a trial consists of visiting eight different arms) and placing the remaining rewards in the arms now occupying the unvisited locations on that trial, it is possible to nullify the use of intra-maze cues to solve the task. Only rats from Experiment 1 were tested. Apparatus Testing was carried out in an eight-arm radial maze. The maze consisted of an octagonal central platform (34 cm diameter) and eight equally spaced radial arms (87 cm long, 10 cm wide). The base of the central platform and the arms were made of wood, whereas panels of clear Perspex (24 cm high) formed the walls of the arms. At the start of each arm was a clear Perspex guillotine door (12 cm high) attached to a pulley. The maze was positioned in a room ( cm 3 ) which contained salient visual cues such as geometric shapes and high contrast stimuli on the walls. Procedure Animals were maintained on restricted feeding at 85%, or above, of their free-feeding body weight. Pretraining for the radial-arm maze began 10 days after the completion of testing in the T-maze and involved two habituation sessions where the animals were allowed to explore the maze freely for 5 min with the guillotine doors raised and food pellets (45 mg; Noyes Purified Rodent Diet, UK) scattered down the arms. The animals were then trained on the standard radial-arm maze task (see below). A time limit of 10 min was placed on each session. Animals were tested until they had completed 15 sessions in Stage 1 and six sessions in Stage 2. Stage 1 (Sessions 1 15) was the standard working memory version of the radial-arm maze task (Olton et al., 1978) where the animals optimal strategy was to retrieve the reward pellets from all eight arms without re-entering any previously entered arms. At the start of a trial, all eight arms were baited with two food pellets. The animal would make an arm choice and then return to the central platform, and all the doors were closed for about 10 s before they were opened again, permitting the animal to make another choice. This continued until all eight arms had been visited or 10 min had elapsed. Only trials where animals made a minimum of eight arm choices were included in the analyses. The number of sequential choice responses was calculated, which is when the animals successive choices involve immediately adjacent arms in a constant direction. It is measured by giving the animal a score of 11 (clockwise) or 21 (anticlockwise) if the arm is immediately adjacent to previous choice and 0 for any other arm choice. A higher absolute score would therefore reflect the use of a sequential response strategy (Olton and Samuelson, 1976; Ennaceur and Aggleton, 1997). Stage 2 (Sessions 16 21) tested for the possible use of intramaze cues in performing the task. The start of the trial was as before but after the animal had made four different arm choices it was removed from the maze. The animal was placed in a traveling box that had an aluminum top, base and sides ( cm 3 ) which was also in the testing room. The maze was then rotated by 45 8 (clockwise/anticlockwise on alternate sessions) and the remaining food pellets removed and repositioned so that they were placed back in their same allocentric locations but the actual arms had changed. The animal was then returned to the central platform after the 60 s that it took to rotate the maze, and the session continued until all reward pellets had been retrieved. Stage 3 (Sessions 22 24) was the same as Stage 1 as the animals performed the radial-arm maze task without rotation of the maze. Histological Procedures On completion of the three experiments, rats were deeply anesthetized with sodium pentobarbital (60 mg/kg, Euthatal, Rhone Merieux, UK) and transcardially perfused with 0.1 M phosphate buffer saline (phosphate buffered saline (PBS)) followed by 4% paraformaldehyde in 0.1 M PBS (PFA). The brains were removed and postfixed in PFA for 4 h and then transferred to 25% sucrose overnight at room temperature with rotation. Sections were cut at 40 lm on a freezing microtome in the coronal plane. A one-in-three series of sections was mounted onto

5 POSTCOMMISSURAL FORNIX LESIONS AND MEMORY 949 gelatine-coated slides and stained with cresyl violet, a Nissl stain. For Experiments 2 and 3, additional series (one-in-three) were collected and processed for acetylcholinesterase staining. To verify the disconnection of the mammillary bodies in Experiment 2, eight of the dpfx2 lesions animals had 0.06 ll Fluoro-Gold (Fluorochrome, LLC, 4% solution in distilled water) injected into the mammillary bodies bilaterally (Supporting Information Fig. 1). The surgical procedures used were the same as those previously described except the coordinate placements were, relative to bregma: AP, 22.9 mm; LM, mm; and a depth of 9.1 mm from the top of cortex. Three days after the additional surgery, the animals were perfused and brain tissue cut and processed as above except the brains, and one series of sections, were light-protected throughout. This series was then cover-slipped and assessed under a fluorescent microscope. In addition, to confirm that the lesions disconnected the mammillary bodies from their subicular complex inputs but spared the hippocampal formation projections to the anterior thalamic nuclei, a bilateral postcommissural fornix lesion was made in an additional animal in the same way as previously described. Three weeks postsurgery this animal received an injection of 0.06 ll Fluoro-Gold in the mammillary bodies and 0.06 ll Fast Blue (Polysciences Inc, 3% solution in PBS) was injected into the anterior thalamic nuclei (coordinates relative to bregma AP, 20.4 mm; LM, 6 1.3; depth of 5.5 mm below top of cortex). A control animal had the same retrograde tracer injections but without previously receiving the postcommissural fornix lesion. All retrograde tracer injections were made using a 1-ll Hamilton syringe (Bonaduz, Switzerland) and the tracer was infused gradually over a 5-min period. The needle was left in situ for a further 5 min before being withdrawn. Acetylcholinesterase (AChE) Staining- (dpfx2, Fx3) AChE levels were visualized using a modified Koelle method (Koelle and Friedenwald, 1949). Sections were mounted onto gelatin-coated glass slides and dried overnight in a slide oven. Slides were then immersed in incubation medium (copper sulfate g/l, glycine 0.75 g/l, sodium acetate 2.88 g/l dissolved in distilled water and adjusted to ph5, acetylthiocholine iodide 1.15 g/l and ethopropazine 0.05 g/l) and left overnight. Slides were next washed four times in distilled water and then placed in sulfide solution (10 g/l dissolved in distilled water and adjusted to ph 7.5). Once sufficient color had developed, the sections were washed a further four times in distilled water. Sections were dried and then dehydrated in ascending alcohols and xylene before being cover-slipped using DPX mounting medium. Image Capturing and Assessment Images were captured using a Q Imaging MicroPublisher 3.3 RTV camera attached to a Zeiss Axiostar Plus microscope. Intensity measures for the AChE-stained sections were acquired on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at rsb.info.nih.gov/nih-image/). To assess the integrity of the septo-hippocampal cholinergic efferents, intensity measures were taken for the rostral (septal) hippocampus (Supporting Information Fig. 2) from between 23.0 to 23.5 mm behind bregma. Although the density measures were just taken from this level, they were representative of the septo-temporal extent of the hippocampus. Density measures were taken from a 1 mm 2 area that included the dentate gyrus and CA3. Density measures were taken for the hippocampus across four sections (i.e., eight total measures from both hemispheres) and for the overlying corpus callosum. The white matter (corpus callosum) value was then subtracted from the hippocampal value for each section separately, and the mean values calculated. Four lesions and four sham animals were assessed from each of Experiments 2 and 3. To indicate the extent of any mammillary body atrophy following postcommissural fornix and complete fornix lesions, mammillary body area measurements were estimated for both lesion and shams animals from Experiments 2 and 3. The dpfx2 rats (Experiment 2) were chosen as the same group received injections of the retrograde tracer Fluoro-Gold to test the completeness of the surgeries. Measurements were made using the NIH Image program, and were taken from a minimum of two sections per case where both medial and lateral mammillary nuclei were present. The total area (mm 2 ) of the mammillary bodies on each coronal section was measured. The mean mammillary body area and the maximum mammillary body area (across all sections) were determined for each animal. Statistical Analyses Group comparisons typically used parametric tests (t-tests and ANOVA). When significant interactions were found, the simple effects for each group were analyzed as recommended by Winer (1971) using the pooled error term. Throughout, the probability level of <0.05 was treated as significant. When tests involved several sessions, i.e., repeated measure analyses, corrections for any violations of sphericity were made using the Greenhouse-Geisser adjustment (SPSS Inc., Chicago, IL). In these instances, pooled error terms were not used for any subsequent simple effect analyses. On those instances where the training and testing procedures were the same in both Experiments 1 and 2, the data from both experiments were analyzed together with replication as an additional between-subject factor. RESULTS Where more than one experiment used the same behavioral task, the data are presented together. For this reason, the behavioral results are present by task and not by experiment.

6 950 VANN ET AL. perifornical nucleus and the tuber cinereum area. Final numbers for Experiment 2 were dpfx2, n 5 9 and Sham2, n FIGURE 1. Lesion photomicrographs. Coronal photomicrographs of descending postcommissural fornix lesions from Experiment 1 (B) and Experiment 2 (D, E, F) and corresponding control animals (A, C). MTT, mammillothalamic tract; dpf, descending postcommissural fornix. A, B, C, and D are taken 2.5 mm behind bregma; E and F are 1.4 mm behind bregma. Scale bars, 0.5 mm. Histological Analysis Experiment 1 Following histological analyses, five dpfx1 animals were excluded due to incomplete tract lesions. The remaining nine dpfx1 rats all appeared to have complete bilateral lesions of the descending bundle of the postcommissural fornix that were largely confined to the target area (Fig. 1). There were eight control (Sham1) rats. Experiment 2 Five dpfx2 animals were excluded due to partial sparing of the postcommissural fornix. The remaining animals all appeared to have complete, bilateral tract disconnections (Fig. 1). In both Experiments 1 and 2, the tract surgeries led to visible atrophy of the mammillary bodies (Supporting Information Fig. 3). Mammillary body area was significantly reduced in the dpfx2 group compared to the Sham2 group using measures of both mean area [t(17) , P ; mean 6 standard error of the mean (S.E.M.), dpfx mm ; Sham mm ] and maximum area [t(17) , P ; dpfx mm ; Sham mm ]. At the same time, the postcommissural fornix lesions in both experiments appeared very discrete. The mammillothalamic tract was intact in all cases across both experiments (Fig. 1). Any additional damage in Experiments 1 and 2 was restricted to very limited parts of the Experiment 3 The fornix lesions (Fx3) were consistently large and, as a consequence, completely severed the tract in both hemispheres in all cases (Fig. 1). The lesions extended rostrally to include the dorsal part of the caudal septum. In addition, the lesions reached the most dorsal part of the anterodorsal thalamic nucleus and anteroventral thalamic nucleus, along with the most rostral part of the (septal) hippocampus. In all cases, there was damage to the corpus callosum at the level of the lesion. One animal also had additional damage to the cingulum bundle and so was removed from all analyses. There was visible mammillary body atrophy following the complete fornix lesions, and this change was significant using measures of both mean area [t(15) , P < 0.001; Fx mm ; Sham mm ] and maximum area [t(15) , P < 0.001; Fx mm ; Sham mm ]. Furthermore, there were no differences between the mammillary body measurements following postcommissural fornix or complete fornix lesion (mean area and maximum area, both t < 1). The final numbers for this cohort were Fx3, n 5 9 and Sham3, n 5 8. Acetylcholinesterase Assessment Optical density measures were taken of the hippocampus to assess indirectly the extent of septohippocampal disconnection (Supporting Information Fig. 2). For each section, the density measure for the overlying corpus callosum was subtracted from the density measure for the hippocampal section. A mean density was then obtained for the eight measurements from each animal. There was no difference between the density measures for the postcommissural fornix lesions and their respective controls (t < 1; mean 6 S.E.M. dpfx ; Sham ). There was, however, a very striking difference between the density measures obtained for the fornix lesion group and their controls [t(6) , P < 0.001; Fx ; Sham ; Supporting Information Fig. 2]. Retrograde Tract Tracing Direct evidence that the postcommissural fornix lesions disconnected the mammillary bodies from their subicular complex inputs came from the lack of Fluoro-Gold label in the pyramidal cells in the dorsal subiculum following tract sectioning (Fig. 2; Supporting Information Fig. 1). In some cases, with seemingly complete lesions, a couple of labeled cells (usually two or three) were still present in the pyramidal layer II of the subiculum in occasional sections, but such tiny numbers represent a huge reduction compared with the dense label found in intact animals (Fig. 2; Supporting Information Fig. 1). The additional case, with paired tracer injections into the mammillary bodies and anterior thalamus, still contained Fast Blue label (thalamic injection) in the deep layers of the subiculum (Fig. 2), so confirming that as intended the subicular complex projection

7 POSTCOMMISSURAL FORNIX LESIONS AND MEMORY 951 FIGURE 2. Extent of disconnection following descending postcommissural fornix (dpf) lesion. Left panel, schematic of retrograde tracing procedure for surgical control (A) and descending postcommissural fornix lesion (B). Right panel, the descending postcommissural fornix lesion produced an almost complete disconnection of subicular complex-mammillary body projections (MB) shown by loss of yellow Fluoro-Gold label (B) compared to the anterior thalamic nuclei remained intact. Finally, it is noteworthy that the presence or absence of subiculum label in the eight dpcfx2 rats closely matched the reconstructions based on cresyl-stained sections used to discriminate between cases with complete lesions or only partial tract lesions. Delayed-Matching-to-Place in the Water-Maze (dpfx1, dpfx2, Fx3) Both path length and latency data were recorded and analyzed. Analyses are presented for all trials (1 4). Descending postcommissural fornix lesions (Exp. 1 and Exp. 2) An overall analysis was carried out using the combined latency data from Experiments 1 and 2 as the training procedures were identical (Fig. 3a). There was no effect of replication (F < 1) and with a control (A) in coronal sections of the dorsal subiculum (taken 5.5 mm behind bregma). In contrast, blue (Fast-Blue) label is still present in the deep layers of the dorsal subiculum demonstrating the presence of the subiculum-anterior thalamus projection. MTT, mammillothalamic tract. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.] no overall group difference was found between the dpf lesions and their surgical controls (F (1,32) , P ; Fig. 3a). There was, however, a significant effect of day (F (8.6,275.1) , P < 0.001) and a highly significant trial effect (F (2.3,74.2) , P < 0.001) showing that the animals were learning the platform position within the session; this trial effect was apparent for both groups (dpfx, P ; Sham, P ). There was a borderline group x trial interaction (F (2.3,74.2) 5 3.0, P ) as the combined lesion group were slightly faster than their shams at locating the platform on trial 4 (P ), but there were no group differences for any of the other trials (all P > 0.2). Analysis of path-length data gave the same pattern of results (Fig. 3b). Again, there was no effect of replication (F < 1) and no overall group difference (F (1,32) , P 5 0.1). There were significant effects of day (F (8.6,276.5) , P < 0.001) and trial (F (2.6,83.8) , P < 0.001) with both groups showing significant effects of trial (dpfx, P ; Sham,

8 952 VANN ET AL. P ). Again, there was a significant group x trial interaction (F (2.6,83.7) , P ) as there was, once again, a significant group difference at trial 4 (P ). Complete fornix lesions (Exp. 3) Analysis of latency data across the 12 sessions of testing revealed longer escape latencies following complete fornix lesions (main effect of group, F (1,15) , P ; Fig. 3c). While there was no overall effect of session (F (11,165) , P ) there was a significant group x session interaction (F (11,165) , P ) reflecting differential degrees of improvement on the task. Overall, performance improved across trials within a session (F (3,45) , P < ) and there was a borderline group x trial interaction (F (3,45) , P ) as the Fx3 group was significantly slower at finding the platform on trial 2 (P ). Path length analyses for the 12 sessions of testing were less clear-cut as there was no overall lesion effect (group F < 1; Fig. 3d). There was a significant improvement across sessions (F (11,165) , P ) but no group x session interaction (F (11,165) , P ). There was a highly significant effect of trial (F (3,45) , P < ) due to the animals finding the platform with shorter path lengths within a session. There was, however, a borderline group x trial interaction (F (3,45) , P ) brought about by evidence of a possible group difference at trial 2 (P ). As Trials 1 and 2 provide the most direct measure of one-trial learning, analyses were performed on just these trials. While there was an overall lesion effect on escape latency (group F (1,15) 55.99, P ) there was no overall lesion effect for path length (group F < 1). More importantly, both measures gave significant group x trial interactions (Path length, F (1,15) 56.40, P ; Latency F (1,15) 56.66, P ) due to the poorer Fx3 performance on trial 2 (Path length, P ; Latency, P ) but not on trial 1 (Path length, P ; Latency, F < 1). These analyses highlight the reduced rate of one-trial learning following complete fornix lesions. Reinforced Spatial Alternation in the T-Maze Due to the severity of the impairment following complete fornix lesions (Fx3), this group was not tested beyond Stage 1. Stage 1: Acquisition (Six Spaced Trials Per Session) Descending postcommissural fornix lesion (Exp. 1 and Exp. 2) There was no effect of replication (i.e., experiment 1 vs. Experiment 2; F (1,32) , P ). The combined dpfx lesion animals made significantly more errors than the sham animals (Fig. 4a; F (1,32) , P < 0.001). There was a significant effect of session (F (7,224) , P < 0.001) but no group x session interaction (F < 1). FIGURE 3. Delayed-matching-to-place in the water-maze. Mean escape latency (6S.E.M.) to find the hidden platform across the four trials of a session for Experiments 1 and 2 combined (A) and Experiment 3 (C). Path lengths to find the hidden platform (6 S.E.M.) are also presented for Experiments 1 and 2 combined (B) and Experiment 3 (D). Abbreviations: dpfx(1 and 2) (n 5 18), descending postcommissural fornix lesion animals from Experiments 1 and 2, combined; Fx3 (n 5 9), complete fornix lesion animals from Experiment 3; Sham(1 and 2) (n 5 18), surgical controls from Experiments 1 and 2, combined; Sham3 (n 5 8), surgical controls from Experiment 3; ***indicates an overall group difference (P < 0.005).

9 POSTCOMMISSURAL FORNIX LESIONS AND MEMORY 953 Stage 2: Continuous Alternation (10 Massed Trials Per Session) Descending postcommissural fornix lesions The next six sessions were designed to increase proactive interference. There was no significant effect of replication (F (1,32) , P ). Again, the dpf lesion animals made significantly more errors than their sham controls (F (1,32) , P < 0.001; Fig. 4a) but there was no effect of session (F (5,160) , P ) and no group x session interaction (F < 1). FIGURE 4. T-maze alternation for Experiments 1 and 2 combined (A) and Experiment 3 (B). Percentage of correct arm choices (6S.E.M.). For Experiments 1 and 2: Stage 1 (blocks 1 4), standard spaced trials (six trials per session); Stage 2 (blocks 5 7), continuous alternation (10 massed trials per session), Stage 3 (blocks 8 10), continuous alternation (16 massed trials per session); Stage 4 (blocks 11 12), continuous alternation (16 massed trials per session) with maze rotation; Stage 5 (block 13), continuous alternation (repeat of Stage 3). Animals in Experiment 2 were tested for two blocks in Stage 5; the means (6S.E.M.) for this additional block were PCFx , Sham Animals in Experiment 3 were only tested on standard spaced trials (Stage 1; blocks 1 4). dpfx(1 and 2) (n 5 18), descending postcommissural fornix lesion animals from Experiments 1 and 2, combined; Fx3 (n 5 9), complete fornix lesion animals from Experiment 3; Sham(1 and 2) (n 5 18), surgical controls from Experiments 1 and 2 combined; Sham3 (n 5 8), surgical controls from Experiments 3. Asterisks indicate a group difference for that training stage; * P < 0.05, ** P < 0.01, *** P < Stage 3: Continuous Alternation (16 Massed Trials Per Session) Descending postcommissural fornix lesions The combined analyses revealed a significant lesion effect (F (1,32) , P < 0.001) but no session effect (F < 1) or session x lesion interaction (F (5,160) , P ). However, there was a significant effect of replication (F (1,32) , P ) so the analyses for each experiment are presented separately. Over the six sessions of testing the postcommissural fornix lesion animals were significantly impaired in both experiments (Experiment 1: F (1,15) , P ; Experiment 2: F (1,17) , P ). In neither experiment was there an effect of session (Experiment 1: F < 1; Experiment 2: F (5,85) , P > 0.2) or a group x session interaction (Experiment 1: F < 1; Experiment 2: F (5,85) , P > 0.2; Fig. 4a). Stage 4: Continuous Alternation (Maze Rotation, 16 Massed Trials Per Session) Descending postcommissural fornix lesions Animals were tested for four sessions with the maze rotated inbetween trials to discourage the use of intramaze cues. There was no difference between Experiments 1 and 2 (F (1,32) , P ). Again, the combined dpf lesion animals made more errors than the sham controls (F (1,32) , P ; Fig. 4a). There was no effect of session (F (3,96) , P ) and no group x session interaction (F (3,96) , P ). Complete fornix lesion (Exp. 3) The fornix lesion animals (Fx3) made many more errors than their controls during Stage 1 (group F (1,15) , P < ; Fig. 4b). There was also a significant effect of session (F (7,105) , P ) and a group x session interaction (F (7,105) , P ) as the Sham3 group, but not the Fx3 group, improved with training. Consequently, there was a significant group difference for all sessions except session 1 (session 2, P ; session 3, P ; sessions 4 8, all P < 0.001). As the Fx3 rats remained at, or close to, chance they did not progress to the later stages. Stage 5: Continuous Alternation (No Maze Rotation, 16 Massed Trials Per Session) Descending Postcommissural Fornix Lesions Animals were tested for either a further two sessions (Experiment 1), or four sessions (Experiment 2), with 16 trials per session but without maze rotation. There was no clear lesion effect in either experiment when animals were returned to this condition (Experiment 1: F (1,15) , P ; Experiment 2: F (1,17) , P ; Fig. 4a) and there was no group x session interaction for either experiment (both F < 1).

10 954 VANN ET AL. now made fewer correct entries in the first eight arm choices (F (1,15) , P ) and made significantly more errors (F (1,15) , P < 0.0,001; Fig. 5). There was no effect of session for either measure Entries, F (5,75) , P ; Errors, F < 1) and no group x session interaction (both F < 1). Comparing the last six days of standard testing with the six days of rotation, there was a significant effect of rotation (Entries, F (1,15) , P ; Errors, F (1,15) , P < 0.001), a significant effect of group (Entries, F (1,15) , P ; Errors, F (1,15) , P < ) and a significant group x condition interaction (Entries, F (1,15) , P ; Errors, F (1,15) , P ). This interaction reflected the finding that there was only a significant group difference when the maze was rotated mid-trial (Entries and Errors, both P < 0.001). The final three sessions (Stage 3) were a repeat of those in Stage 1. There was now no overall group difference in the number of errors made (F (1,15) , P ) although the dpfx1 group did make significantly fewer correct entries made in the first eight choices than the Sham1 group (F (1,15) , P ). There was no effect of session (Entries, F < 1; Errors, F (1,15) , P ) and no group x session interaction (Entries, F (2,30) , P ; Errors, F (2,30) , P ) using either measure (Fig. 5a,b). FIGURE 5. Radial-arm maze. Performance of postcommissural fornix lesion animals (dpfx1, n 5 9) and their controls (Sham1; n 5 8) from Experiment 1. Both total errors (A) and number of correct entries in first 8 arm choices (B) are presented. The first five blocks represent performance during task acquisition (Stage 1); during blocks 6 7 the maze is rotated mid-way through the trial to control for use of intramaze cues (Stage 2). For the final block (Stage 3) animals were retested on the standard task (without maze rotation). Data are presented as mean 6 SEM Asterisks indicate a group difference for that training stage; * P < 0.05, *** P < Radial-arm Maze Experiment 1 (Acquisition and Rotation; dpfx1) For the radial-arm maze, both the number of correct entries in the first eight choices and the total errors made were analyzed. During acquisition (Stage 1), both groups performance improved on the radial-arm maze task across the 15 training sessions (effect of session: Entries, F (14, 210) , P < 0.001; Errors, F (5.3,79.2) , P < 0.001), and there was no lesion effect (Entries, F < 1; Errors, F (1, 15) , P > 0.1) or group x session interaction (both F < 1; Fig. 5). An analysis of sequential choice responses showed no group differences (F (1,15) , P ; mean values 6SEM, dpfx ; Sham ), no overall effect of session (F (14,210) , P ) and no group x session interaction (F < 1). The next six sessions of testing (Stage 2) involved rotating the maze after the first four choices. This manipulation was primarily to tax the use of extra-maze cues. The dpfx1 group DISCUSSION This study used the morphology of the fornix to test, for the first time, the importance of one specific set of hippocampal formation connections for spatial learning and memory. The fornix divides into precommissural and postcommissural components, the latter occupying a crucial position within the extended hippocampal memory system as it connects the subicular complex to the mammillary bodies and anterior thalamic nuclei (Raisman et al., 1966; Poletti and Creswell, 1977; Aggleton et al., 1986). The importance of the fornical fibers that innervate the mammillary bodies can then be examined by selective disconnection of the descending postcommissural fornix as these fibers form a discrete bundle, distinct from the fibers innervating the anterior thalamic nuclei (Guillery, 1956). As the disconnection surgery was novel, its effectiveness and selectivity were carefully tested. Retrograde tracers placed in the mammillary bodies, after transecting the target region of the descending postcommissural fornix, confirmed the loss of hippocampal formation projections to the mammillary bodies. In contrast, the hippocampal formation projections to the anterior thalamic nuclei remained intact (as intended), even though these projections also rely on the postcommissural fornix. Furthermore, assays for hippocampal levels of AChsterase confirmed that septal fibers were spared. At the same time, there was comparable mammillary body atrophy following either complete fornix or descending postcommissural fornix lesions; an expected finding as the mammillary body atrophy principally reflects the loss of incoming fibers and not the loss of mammillary body neurons (Loftus et al., 2000). The surgery may also have disconnected some other poste-

11 POSTCOMMISSURAL FORNIX LESIONS AND MEMORY 955 rior hypothalamic nuclei as a small number of fornical fibers terminate in the perifornical area and supramammillary nuclei (Kishi et al., 2000), while a few additional fornical fibers may pass through the mammillary body to reach the caudal midbrain (Sprague and Meyer, 1950) and rostral central gray (Valenstein and Nauta, 1959). As the direct projections from the supramammillary nucleus to the hippocampus do not run in the postcommissural fornix (Pasquier and Reinoso-Suarez, 1978), these fibers would have been spared by the surgery. Two cohorts of rats with descending postcommissural fornix lesions (dpfx) were tested on spatial memory tasks sensitive to the loss of the hippocampus, fornix, anterior thalamic nuclei, and mammillary bodies (e.g., Warburton et al., 1997; Cassel et al., 1998; Bannerman et al., 2002; Vann and Aggleton, 2003). Taken together, the dpfx rats were unimpaired on the delayedmatching-to-place task in the water-maze. The dpfx rats were mildly impaired on initial acquisition of T-maze alternation and although increasing the amount of proactive interference impaired performance, there was no indication that the dpfx rats were especially sensitive to this manipulation. Likewise, the dpfx rats were unaffected on initial acquisition of the radial-arm maze task although deficits emerged when the maze was rotated midway through each trial. This impairment may well reflect decreased reliance on distal visual cues by the dpfx rats, although this manipulation also introduces a longer retention delay (but see Vann and Aggleton, 2003). These mild or null effects contrasted with the impact of complete fornix lesions, which produced substantial deficits, especially on the T-maze task. It is, perhaps, unsurprising that selective mammillary body disconnection had less impact than complete fornix transection as the former surgery spares many hippocampal connections, including those with other sites implicated in spatial memory, i.e., anterior thalamic nuclei, septum, nucleus accumbens, and prelimbic cortex. The spared thalamic connections are particularly relevant as direct comparisons have shown that anterior thalamic lesions are more disruptive than mammillary body lesions on some (Sutherland and Rodriguez, 1989; Aggleton et al., 1991; Aggleton et al., 1995), but not all (Aggleton et al., 1995; Gaffan et al., 2001), tests of spatial memory. In addition, anterior thalamic and fornix lesions can have comparable disruptive effects on spatial tasks (e.g., Sutherland and Rodriguez, 1989; Aggleton et al., 1991; Gaffan et al., 2001) and disconnection evidence shows that the anterior thalamic nuclei function conjointly with the hippocampus on the same spatial tasks used in this study (Warburton et al., 2001; Henry et al., 2004). Thus, one explanation for the greater impact of complete fornix lesions than descending postcommissural fornix lesions is that after the latter surgery spatial memory is still supported by the dense hippocampal formation (anterior thalamic projections. This explanation does not preclude a role for other fornical connections, e.g., precommissural fornix lesions can disrupt T-maze alternation (Henderson and Greene, 1977). Nevertheless, it is most unlikely that complete fornix lesion effects solely reflect the loss precommissural fibers given the mild effects of both prefrontal lesions (Mogensen et al., 2004) and the removal of septo-hippocampal cholinergic inputs (Winters and Dunnett, 2004). More unexpected was the repeated, mildness of the behavioral deficits following descending postcommissural fornix lesions. This pattern was most striking for delayed matching-to-place in the water-maze. This task is highly sensitive to both mammillary body and mammillothalamic tract lesions (Vann and Aggleton, 2003), yet there was no evidence of a descending postcommissural fornix lesion-induced impairment. Traditional theories of mammillary body function have often emphasized their primary role as a relay for indirect hippocampal formation inputs to the anterior thalamic nuclei, via the mammillothalamic tract (Papez, 1937; Delay and Brion, 1969; Gaffan, 1992; Aggleton and Brown, 1999). The apparent lack of interneurons in the mammillary bodies seems consistent with this relay role (Veazey et al., 1982). The above theories would, therefore, predict that disconnecting the descending postcommissural fornix should have an effect comparable to lesions of the mammillary bodies or the mammillothalamic tract. In fact, as noted above, other studies have consistently found clear spatial memory deficits after mammillary body or mammillothalamic tract lesions on working memory tasks in the water-maze (Vann and Aggleton, 2003) and radial-arm maze (Sziklas and Petrides, 1993; Vann and Aggleton, 2003) tasks either unimpaired or only mildly affected by the descending postcommissural fornix lesions in the present study. The present findings are, therefore, problematic for memory models that emphasize the importance of sequential hippocampal formationmammillary body-anterior thalamic projections (e.g., Delay and Brion, 1969; Gaffan, 1992; Aggleton and Brown, 1999). There are several reasons why hippocampal formation-mammillary body disconnection need not be as disruptive as removal of the mammillary bodies themselves. The first is that indirect projections from the hippocampal formation to the mammillary bodies via some other structure remain intact and, hence, might still support performance. While there are potential routes via the prefrontal cortex or septum to the mammillary bodies, the lack of consistent evidence that these relay sites are vital for the present tests of spatial working memory (e.g., Dias and Aggleton, 2000; Kirby and Rawlins, 2003; Vuckovich et al., 2004) makes this explanation less attractive. A more obvious possibility is that the direct projections from the hippocampal formation to the thalamus compensate for removal of the mammillary body relay. However, this account cannot explain how both mammillary body and mammillothalamic tract lesions disrupt tasks seemingly unaffected by descending postcommissural fornix lesions. A more likely explanation is that spatial learning in rats with descending postcommissural fornix lesions is still supported by nonhippocampal inputs to the mammillary bodies, including those from the tegmental nuclei of Gudden. The dorsal and ventral tegmental nuclei of Gudden are reciprocally connected with the lateral and medial mammillary nuclei, respectively, though both may contribute memory in qualitatively different ways (Vann and Aggleton, 2004). The dorsal nucleus of Gudden is thought to be necessary for generating the head-direction signal (e.g., Bassett et al., 2007) and, hence, is required for tasks that require path integration (Frohardt et al., 2006). Despite the consensus that head-direction information is integral for effective navigation and the potential