An environment-dependent modulation of cortical neural response by forebrain cholinergic neurons in awake rat

brain research 1513 (2013) 72–84

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Research Report

An environment-dependent modulation of cortical neural response by forebrain cholinergic neurons in awake rat Mohammed Zacky Ariffina,1, Lai Seong Changa,1, Han Chow Koha,1, Chian-Ming Lowb,c,d, Sanjay Khannaa,b,n a

Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore Neurobiology Program, Life Sciences Institute, National University of Singapore, Singapore 117597, Singapore c Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore d Department of Anesthesia, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore b

ar t ic l e in f o

abs tra ct

Article history:

The forebrain cholinergic neurons project to cortex, including the hippocampus and the

Accepted 23 March 2013

cingulate cortex (Cg). However, the relative influence of these neurons on behavior-linked

Available online 4 April 2013

neural processing in the two cortical areas remains unclear. We have now examined the

Keywords:

effect of destruction of the cholinergic neurons with microinjection of the immunotoxin

Medial septum

192 IgG-saporin into the medial septum on the induction of c-Fos protein, an index of

192 IgG-saporin

neuronal synaptic excitation, in the two forebrain areas to varied episodic experiences.

C-Fos

Separate groups of rats were (a) re-exposed to the laboratory where they had previously

Cingulate cortex

undergone a surgery for intraseptal microinjection or (b) exposed to a novel environment.

Hippocampus

Re-exposure evoked a differential increase in the number of c-Fos positive neurons in

Novelty

dorsal CA1 compared to novelty, while a robust increase was observed in the Cg selectively in the novel environment. Both the differential and the selective increases were strongly attenuated by the cholinergic destruction with intraseptal-immunotoxin. These findings suggest that the cholinergic modulation of the neural processing in the two forebrain areas varies partly in an environment-dependent fashion affecting CA1 neural activation on repeat exposure to an environment where they had a relatively complex aversive experience while favoring Cg neural activation more during novelty. & 2013 Elsevier B.V. All rights reserved.

1.

Introduction

The cholinergic nuclei of the basal forebrain, including the medial septal region, are implicated in behavioral activation and, indeed, they are suggested to play a role in attention, associative learning and memory, sensory–motor integration, affect-motivation and cognition (Horita and Carino, 1988; n

Baxter et al., 1997; Ma et al., 2002; McNaughton and Corr, 2004; Sarter et al., 2005; Dwyer et al., 2007; Bland, 2009; Easton et al., 2011; Fuller et al., 2011; Lee et al., 2011). The medial septal region, consisting of the medial septum, the vertical and the horizontal limbs of the diagonal band of Broca, is anatomically and functionally linked to cortex, especially to the allocortex hippocampus (Kiss et al., 1990). Stimuli that

Corresponding author at: Department of Physiology (MD9), Yong Loo Lin School of Medicine, National University of Singapore, 2 Medical Drive, Singapore 117597, Singapore. Fax: +65 6778 8161. E-mail addresses: [email protected], [email protected] (S. Khanna). 1 Contributed equally to the work performed. SK contributed to the writing of the manuscript together with C-ML and MZA. 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.03.046

brain research 1513 (2013) 72–84

affect septal neurons, including the cholinergic neurons also potently excite neurons in the hippocampus. In this regard, the septo-hippocampal cholinergic neurons are activated in rats on exposure of the animals to behaviorally arousing stimuli of varied hue including exposure to open field (Dudar et al., 1979; Imperato et al., 1991; Inglis and Fibiger, 1995; Thiel et al., 1998; Ceccarelli et al., 1999; Giovannini et al., 2001). Likewise, arousing stimuli, especially animal exposure to novel open field, elicit the expression of c-fos and the corresponding protein, c-Fos, in the neurons of the hippocampus, c-fos being an immediate early gene (IEG) that is expressed in neurons on synaptic excitation (Emmert and Herman, 1999; Pace et al., 2005; Sheth et al., 2008; VanElzakker et al., 2008). The effect is marked and consistent in the hippocampal field CA1. Additionally, both the septal cholinergic neurons and acetylcholine exert a strong modulatory influence on hippocampal neural activity (Zheng and Khanna, 2001; Leung et al., 2003). Exposure to arousing stimulus, such as novelty also evokes an activation of other structures including the amygdala and the cingulate cortex (Cg; Nagahara and Handa, 1997; Salome et al., 2004; Hale et al., 2008). Moreover, the medial septum, including the septal cholinergic neurons and the hippocampus projects to these regions, while the amygdala and the Cg project, directly and/or indirectly, back to the hippocampus (Stewart et al., 1985; Senut et al., 1989; Kiss et al., 1990; Petrovich et al., 2001; Risold, 2004; Cenquizca and Swanson, 2007; Jones and Witter, 2007). The interaction between these regions, for example between septal cholinergic neurons and Cg, may have bearing on aspects of animal behavior that is consistent with the idea that the above-mentioned regions are interrelated components of an affective-cognitive network (Marston et al., 1994; McNaughton and Corr, 2004). We have used c-Fos expression in the present study to examine the relative pattern of activation of the two cortical structures, namely hippocampus and the Cg, to two different environmental milieus and their modulation by the cholinergic

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neurons. The influence of septal cholinergic neurons was examined by destroying these with the immunotoxin, 192 IgG-saporin. The immunotoxin-induced septal lesion have a profound effect on cholinergic markers in both the hippocampus and the Cg suggesting that such lesions affect key cholinergic afferents to the two cortical regions.

2.

Results

2.1. Effect of 192 IgG saporin on ChAT positive neurons in the medial septal region The average number of ChAT positive neurons in the medial septal region of the representative control, namely the IS veh nov group, were 1538.007156.60 neurons (n¼7; range 919–2176; Fig. 1). On the other hand, robust destruction was observed following IgG pretreatment (e.g. Fig. 1). Thus, the total number of ChAT positive neurons in the two test groups, namely the IS IgG Nov and the IS IgG reex were 39.00713.07 (n¼ 8) and 71.00713.33 (n¼6), respectively. The counts in the two test groups were significantly different from the control but were not different from each other (Groups, F2, 18 ¼ 88.78, Po0.0001, 1way ANOVA followed by Newman–Keuls test). In all cases the IgG-induced loss was 490% when compared to the average number of ChAT positive neurons (1538.00) in the control group. Despite the near absence of ChAT positive neurons, robust parvalbumin staining was still observed suggesting a relatively selective effect of the neurotoxin on septal cholinergic neurons (Fig. 1).

2.2. Effect of environmental exposure on the number of c-Fos like immunoreactive (FLI) neurons in whole hippocampus and Cg Compared to the control groups (IS veh basal), the number of FLI positive neurons were increased in the whole CA1 subfield

Fig. 1 – Intra-septal immunotoxin 192 IgG-saporin (0.8 μl of 0.42 μg/μl) selectively destroyed choline acetyl transferase (ChAT, A) positive cholinergic neurons while largely sparing parvalbumin (PV, B) positive GABAergic septohippocampal neurons. The panels are digitized images (600 dpi) through the medial septum of animals treated with intra-septal vehicle (Ai and Biii) or the immunotoxin (Aii and Biv). Cells stained for ChAT or PV stand out as darkly stained relative to the background. Scale bar represents 100 lm.

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Fig. 2 – (A) Digital images illustrating the c-Fos-like immunoreactive (FLI) cells in sections taken through the dorsal part of the hippocampus field CA1 (Dorsal CA1; left panels) and cingulate cortex (Cg; right panels) in animals (a) habituated in home cage following microinjection of vehicle (IS veh basal), (b) re-exposed to the laboratory where the animals had previously undergone a day-surgery for intraseptal microinjection (i.e. IS veh reex) and (c) exposed to novel environment (i.e. IS veh nov). The FLI cells stand out as darkly stained relative to the background. Notice the increased number of FLI cells in the pyramidal cell layer (dark gray band relative to background) of CA1 in sections representing the IS veh reex and IS veh nov groups relative to the sections of the IS veh basal group. The number of FLI cells were most marked in the section representing the IS veh reex group. The number of FLI cells in the Cg was most marked in the section representing the IS veh nov group vis-a`-vis the sections for the other groups. Scale bar on the CA1 and Cg images represents 20 lm and 100 lm, respectively. (B) Histograms illustrating the environment-dependent expression of FLI in the CA1 pyramidal cell layer of whole hippocampus (total CA1, left) and cingulate cortex (Cg, right). The number of c-Fos/section represents the bilateral mean7S.E.M. of FLI cells per section of the selected region averaged for the group. Significant difference: Po0.05, * vs. ‘IS veh basal’ groups, \widehat vs. ‘IS veh nov’ groups, # vs. ‘IS veh reex’ groups (one way ANOVA followed by Newman–Keul’s test).

of the hippocampus in the two test groups, i.e. IS veh reex and IS veh nov (Groups, F2, 14 ¼ 21.13, Po0.0001, Fig. 2). Interestingly, the increase in CA1 was much more during the repeat

exposure (IS veh reex) as compared to that observed with exposure to the novel environment (IS veh nov; Fig. 2). An increase in FLI positive neurons was also observed in the

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whole CA3 subfield of the two test groups (Groups, F2, 14 ¼ 13.25, Po0.0007, data not shown). However, the numbers of c-Fos positive neurons in CA3 were similar under the two test conditions. Unlike CA1, the number of FLI positive neurons in Cg was significantly higher in the IS veh nov group as compared to both the control and re-exposure groups (Groups, F2, 14 ¼ 15.35, Po0.0005; Fig. 2). The numbers of FLI positive neurons in the control and re-exposure groups were not different from each other, although the count for the latter tended to be higher than control (Fig. 2).

2.3. Effect of environmental exposure on the number of FLI neurons in amygdala and medial septal region Compared to control, the numbers of c-Fos positive neurons in the three subcortical regions, namely the BLA, the Me and the medial septum region, were significantly increased on repeated exposure to the laboratory and with exposure to novel room (Groups, F2, 14 ¼45 and Po0.02 for BLA and Me; Groups, F2, 12 ¼8.91 and Po0.005 for the medial septal region; Fig. 3). Due to an oversight we did not process the medial septal region for c-Fos in two animals of the IS veh reex group. Nonetheless, the increase in c-Fos in the remaining animals was clear and marked (Fig. 3).

2.4. Effect of 192 IgG saporin pretreatment on c-Fos induction in whole hippocampus and Cg In context of Cg, IgG pretreatment reduced the number of c-Fos positive neurons observed on exposure of the animals to the novel room compared to the vehicle pretreated control. Indeed, the number of neurons in Cg was nearly halved by IgG pretreatment compared to vehicle pretreated animals and was not different from the levels observed in IS veh reex and IS IgG reex groups (Groups, F3, 23 ¼10.56, Po0.001; Figs. 4 and 5). IgG pretreatment reduced the number of c-Fos positive neurons in the whole CA1 of animals re-exposed to the laboratory as compared to the levels in the corresponding vehicle pretreated control animals, though such pretreatment did not affect the number of c-Fos positive neurons in CA1 of animals exposed to the novel room (Groups, F3, 23 ¼6.03, Po0.004; Figs. 4 and 5). Overall, the number of c-Fos positive neurons seen in IS veh nov, IS IgG nov and IS IgG reex groups were not significantly different from each other (Fig. 5). Unlike CA1, pretreatment with the immunotoxin did not significantly affect the number of c-Fos positive neurons observed in the field CA3 of the hippocampus on reexposure to the laboratory or on exposure to the novel room when compared to the corresponding vehicle pretreated control animals (Groups, F3, 23 ¼ 1.19, P40.3; Fig. 5).

2.5. Effect of 192 IgG saporin pretreatment on c-Fos induction in dorsal and ventral CA1 The c-Fos count for the whole of CA1 was further sub-divided into its dorsal and ventral components to identify their respective contributions to the change seen in the whole

Fig. 3 – Re-exposure to the laboratory (IS veh reex group) and exposure to a novel environment (IS veh nov group) increased the number of FLI cells in the medial septal region (A), the basolateral amygdala (BLA, B), and the medial amygdala (Me, C). The plot is built as in Fig. 2. Data are mean7SEM. Significant difference: Po0.05; * vs. ‘IS veh basal’ groups (one way ANOVA followed by Newman– Keul’s test).

CA1. It turned out that the pattern of change seen with the whole CA1 was mimicked by the counts from the dorsal, but not the ventral regions of CA1. Thus, IgG pretreatment reduced the number of c-Fos positive neurons in the dorsal CA1 of animals re-exposed to the laboratory as compared to the levels in the corresponding vehicle pretreated control animals (Groups, F3, 23 ¼ 6.43, Po0.003, Fig. 6). Indeed, the number of neurons in dorsal CA1 was nearly halved by IgG pretreatment compared to vehicle pretreated animals (Fig. 6). On the other hand, IgG pretreatment did not significantly affect the number of c-Fos positive neurons in animals

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Fig. 4 – Digital images illustrating the FLI cells in sections taken through the dorsal part of the hippocampus field CA1 (Dorsal CA1; left panels) and the cingulate cortex (Cg; right panels) in animal microinjected with the immunotoxin 192 IgG-saporin (the IS IgG groups). The animals from the corresponding control groups were microinjected with vehicle (the IS veh groups). Notice the comparatively low number of FLI cells in dorsal CA1 and Cg upon re-exposure to the laboratory (A) and exposure to the novel room (B), respectively, following 192 IgG-saporin pretreatments as compared to the corresponding controls. Scale bars on the CA1 and Cg images represent 20 lm and 100 lm, respectively.

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Fig. 6 – Intraseptal pretreatment with the immunotoxin 192 IgG-saporin attenuated the number of FLI cells seen on reexposure in the pyramidal cell layer of dorsal (A), but not ventral CA1 (B). The plot is built as in Fig. 2. Data are mean7S. E.M. Significant difference: Po0.05; * vs. ‘IS veh reex’ group (one way ANOVA followed by Newman–Keul’s test). Although not depicted statistically on the figure, the ‘IS veh nov’ and the ‘IS IgG nov’ group cell counts, while similar, were lower than ‘IS veh reex’ group cell count in the dorsal CA1.

Fig. 5 – Environment- and region-specific effect of intraseptal immunotoxin, 192 IgG-saporin, on the number of FLI cells in the cortex. The plot is built as in Fig. 2. Note that the immunotoxin pretreatment reduced the number of FLI cell seen on exposure to novel environment in the cingulate cortex (Cg, A) but not the whole CA1 (B) while reducing the re-exposure-evoked FLI cell count in the whole CA1 (B), but without affecting FLI in Cg (A). The numbers of FLI cells were invariant across the different groups in the hippocampal field CA3 (C). Data are mean7S.E.M. Significant difference: Po0.05; * vs. ‘IS veh nov’ group, # vs. ‘IS veh reex’ group (one way ANOVA followed by Newman–Keul’s test). Although not depicted statistically on the figure, the ‘IS veh nov’ and the ‘IS IgG nov’ group cell counts, while similar, were lower than ‘IS veh reex’ group cell count in whole CA1. Whereas, the ‘IS veh reex’ and ‘IS IgG reex’ group cell counts, while similar, were lower than the ‘IS veh nov’ group cell count in the Cg (A).

exposed to the novel room vis-à-vis the corresponding vehicle pretreated control animals, though the average count tended to be lower than the control (Fig. 6). Unlike dorsal CA1, no significant difference in c-Fos count was observed in ventral CA1 between the different groups (Groups, F3, 23 ¼ 2.44, P40.08; Fig. 6).

2.6. Effect of 192 IgG saporin pretreatment on c-Fos induction in amygdala IgG pretreatment did not significantly affect the counts of c-Fos positive neurons in the BLA or the Me of the different groups of animals (Groups, F3, 23 ¼o1.40, P40.1). The counts of c-Fos positive neurons in BLA per section of the 4 groups were as follows: IS veh reex and IS veh nov vs. IS IgG reex and IS IgG nov being 93.89714.81 (n ¼6) and 74.78710.82 (n¼ 7) vs. 87.84713.27 (n ¼6) and 58.47715.62 (n ¼8). The counts of c-Fos positive neurons in Me per section of the 4 groups were as follows: IS veh reex and IS veh nov vs. IS IgG reex and IS IgG

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nov being 275.1725.47 (n¼ 6) and 245.9739.64 (n¼7) vs. 240.7733.45 (n¼ 6) and 184.6719.55 (n ¼8).

an initial high exploratory activity that decreased toward a low by the 30th min of exposure.

2.7. Effect of 192 IgG saporin pretreatment on animal's exploratory behavior

3.

The re-exposure-induced animal horizontal locomotion was unaffected with intraseptal pretreatment with IgG vis-à-vis the corresponding vehicle pretreated control animals (Treatment, F1, 50 ¼ 0.48, P40.5, 2-way ANOVA; Fig. 7). The animal exploration was measured by a number of quadrant crossed every 15 min over a 90 min of observation. Similarly, the immunotoxin treatment did not affect the ambulatory distance covered by animals on exposure to the novel room vs. the corresponding control (Treatment, F1, 65 ¼1.37, P40.2, 2-way ANOVA; Fig. 7). In all groups, the animals displayed

Fig. 7 – The lack of effect of intraseptal pretreatment with 192 IgG-saporin on animal horizontal locomotion (A) and ambulation (B) on re-exposure to the laboratory and exposure to the novel room, respectively. Horizontal locomotion on re-exposure was assessed as the number of quadrants crossed by the animal when it moved from one quadrant to the next with all four paws in the observation chamber. The animal horizontal locomotion in the observation chamber placed in the novel room was scored as the ambulatory distance covered during ambulatory episodes. An ambulatory episode was signaled when the rat moved beyond 4 inbuilt infra-red beams (i.e. box size) in 500 ms. In both cases, the data were analyzed in blocks of 15 min. Data are mean7S.E.M. Statistical comparison was made using two-way ANOVA.

Discussion

3.1. Cholinergic neurons facilitate environment- and region-dependent expression of FLI The present findings indicate that the forebrain cholinergic neurons strongly facilitate the environment- and regiondependent neural processing in the dorsal hippocampus CA1 and the Cg. In this context, re-exposure of the animal to laboratory evoked a differential increase in the numbers of dorsal hippocampal CA1 neurons expressing FLI, a marker of neural synaptic excitation. The increase was nearly 50% more as compared to the levels of c-Fos induced on first exposure to the novel environment. The differential expression of cFos in dorsal CA1 on re-exposure was strongly attenuated by the intraseptal immunotoxin-induced destruction of the cholinergic neurons which, however, had only a weak and statistically non-significant effect on the novelty-induced expression of c-Fos in the region. The former observation is novel while the latter observation reinforces the findings that septal cholinergic neurons are not crucial to the noveltyinduced excitation of dorsal CA1 neurons (Ikonen et al., 2002; Fletcher et al., 2007). Rather, the novelty-induced induction of c-Fos in hippocampus is strongly attenuated by the inactivation of the BLA region (Sheth et al., 2008). Exposing the animal to the novel room evoked a marked and selective increase in the number of FLI neurons in Cg. In this context, the number of FLI neurons in Cg nearly doubled with exposure to the novel room as compared to the levels seen on re-exposure to the laboratory environment. The selective induction of c-Fos in the Cg in the novel room was strongly attenuated by cholinergic destruction with the intraseptal microinjection of the immunotoxin that pared the levels of c-Fos in Cg to that seen on re-exposure. This novel observation suggests that the septal cholinergic neurons modulate Cg molecular plasticity linked to processing of novel information. Previously, the septal cholinergic neurons were linked with modulation of theta wave-like neural activity in Cg of the freely moving rat (Borst et al., 1987). The environment-specific effects of the cholinergic lesion on CA1 and Cg in the present study were, however, not due to an inability of lesioned animals to respond to the specific environments. Thus, despite the lesion, the pattern of c-Fos expression in regions outside of dorsal CA1 and Cg was unaffected. Furthermore, the environment- and region-specific effects of cholinergic lesion indicates that the change in FLI in dorsal CA1 and Cg was not due to: (a) a systemic methodological anomaly that may have led to under-staining and, therefore, undercounting of FLI cells in dorsal CA1 and Cg, and (b) lesioninduced non-specific change such as non-specific decrease in animal arousal and suppression of synaptic processing in the network. In this context, the cholinergic lesion also did not attenuate the animal exploratory drive. Additionally, cholinergic lesion also did not preclude the induction of c-Fos per se since the lesions did not attenuate the induction of c-Fos in CA1 with novelty or the high levels of neural c-Fos seen in Cg in the home cage and on re-exposure.

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3.2. Cholinergic mechanisms modulate CA1 neural processing to re-exposure The pattern of change in FLI with cholinergic lesion suggests that both cholinergic-dependent and -independent mechanisms affect dorsal CA1 neural activation on re-exposure to the laboratory. The cholinergic-independent component of expression of FLI is indistinguishable in strength from that seen with novelty, which is also relatively insensitive to cholinergic destruction. The cholinergic-sensitive differential increase likely reflects a response to some aspect of the familiar context in the laboratory, despite the long gap of about 14 days before re-exposure. This is strengthened by the evidence that a mismatch, induced by exposing the animals to the adjacent novel room, did not elicit differential increase in dorsal CA1 but evoked a novelty response. Conversely, the Cg was only mildly activated in the laboratory which is in contrast to the robust increase in FLI in the region on animal exposure to the novel room. This contrasting responses of Cg further reinforces the idea that re-exposure to the laboratory was not novel. Interestingly, a similar increase in FLI positive cells was observed in amygdala and medial septum on re-exposure to the laboratory vs. exposure to novel environment, suggesting that the affective valence of the re-exposure and exposure was broadly similar. Thus, the differential increase in CA1 on re-exposure is unlikely to reflect generalized affective representation in CA1 on re-exposure. The present evidence, that septal cholinergic neurons facilitate behavior-dependent molecular plasticity, i.e. induction of c-Fos in CA1 neurons, strengthens the idea that the septal cholinergic neurons facilitate neural plasticity in CA1. For example, septal cholinergic neurons facilitated the afferent stimulation-induced long-term potentiation of excitatory synaptic transmission (LTP) in dorsal CA1 during behavioral activation (Leung et al., 2003; Leung and Péloquin, 2010), LTP being a cellular mnemonic mechanism. Although the implication of c-Fos induction in the present study remains unclear, it is notable that c-fos facilitates the hippocampal LTP and the consolidation of long-term memory in behavioral experiments. For example, deletion of the IEG in mice attenuates the strength of hippocampal LTP (Fleischmann et al., 2003). Further, such deletion or the intrahippocampal administration of antisense oligonucleotide that knocked down the local c-fos also attenuated the hippocampal based learning and memory (Guzowski, 2002; Fleischmann et al., 2003). Furthermore, the destruction of septal cholinergic neurons attenuates aspects of episodic experiences (Easton et al., 2011; Cai et al., 2012; Lecourtier et al., 2011).

3.3. A diverse imprint of cholinergic neurons in the registration of novelty The effect of the intraseptal immunotoxin-induced cholinergic lesion on the induction of FLI cells in the Cg in the current study, when seen in conjunction with previous evidence from the hippocampus, suggests a relatively wide and diverse imprint of these neurons in registration of novelty. In context of the hippocampus, the septal cholinergic lesions prevent the re-mapping of novel environment by hippocampal CA1 pyramidal cells (Ikonen et al., 2002).

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The effect of cholinergic lesion in attenuating the noveltyinduced c-Fos in Cg is reminiscent of the proposed role of the basal forebrain cholinergic neurons. These neurons are suggested to detect saliency of an event and, in turn, interact with the prefrontal cortex to bring out attributes such as novelty or affective significance (Sarter et al., 2005). Moreover, the current findings, seen in juxtaposition with published evidence, suggest that the cholinergic neurons participate in a continuum of effects to novelty complementing other modulatory system(s). For example, lesion of the noradrenergic neurons attenuate induction of c-Fos in the Cg on sustained, but not relatively brief behavioral activation to complex social and object novelty (Gompf et al., 2010). Whereas, in the current study the novelty of the exposure lasted for a relatively brief period. Thus, the animals displayed a gradual decrease in exploratory activity, from a high seen on exposure to the novel environment to a low within 30 min of exposure. This indicates that the cholinergic innervations, including the septo-cingulate cholinergic neurons play a prominent role during relatively brief environmental novelty.

3.4. An enlarged perspective on the neural effects of cholinergic neurons In conclusion, the current findings enlarge the perspective on the neural effects of the cholinergic neurons. They suggest that the modulatory effect of the forebrain cholinergic neurons, especially the septal cholinergic neuron, on CA1 and Cg varies partly in an environment-dependent fashion and potentiates c-Fos induction in dorsal CA1 on re-exposing the animals to the familiar context where they had a relatively complex aversive experience while favoring c-Fos induction in Cg during exposure to spatial and object novelty. Such environment- and region-specific effects emphasize that diverse functional connectivity of the cholinergic neurons in the medial septum may underpin their varied effects on affect-cognition.

4.

Experimental procedure

4.1.

Animals, anesthesia and test environments

Adult male Sprague-Dawley rats were used in these experiments (260–300 g, including at time of surgery). Animal had access to water and food ad libitum. The local IACUC and the local animal committee of the National Medical Research Council, Singapore, approved the experimental procedures. Intracerebral microinjections were performed under pentobarbital anesthesia (60 mg/kg, i.p., Sigma, USA; see Section 4.3) so as to microinject the immunotoxin 192 IgG saporin or the corresponding vehicle into the septal region. Post-surgery, all the animals were allowed to recover in the laboratory before being taken back to the Animal Holding Unit. At the end of an experiment, the animal was deeply anaesthetized with urethane (1.5 g/kg, i.p.; Sigma, USA) to facilitate perfusion through the heart. The brain of the perfused animals was taken for later histological analyses of c-Fos expression in the following regions: hippocampus

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(fields CA1 and CA3), Cg, amygdala and the medial septal region. Alternate sections taken through the medial septum were also stained for choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. In some separate experiments the relative selectivity of the immunotoxin was assessed by qualitatively comparing the effect of intraseptal immunotoxin on medial septal ChAT vs. parvalbumin expression, parvalbumin being a marker for septohippocampal GABAergic neurons. The animal's neural response (i.e. induction of c-Fos) was compared across the following test environments, namely (a) re-exposure to the laboratory where the animals had previously undergone surgery for microinjection and (b) exposure of animal to a novel environment. In context of (a), the animals were re-exposed to the laboratory a couple of weeks after surgery. The animals were exposed to the laboratory for the first time on the day of surgery. The surgery comprised part of the multidimensional experience in the laboratory that ranged over few hours and encompassed exposing the animal for about 1.5 h to the laboratory prior to surgery, anesthesia and post-surgical recovery over 3–4 h in the laboratory. The animals exhibited purposeful movement and exploratory behaviors at the time they were returned to the animal holding unit after recovery. The surgical experience, thus, mimicked somewhat the human experience during day-surgery and presumably resulted in a more complex experimental set than the more established fear-conditioning paradigm where the animals are also re-exposed to the aversive context to test their recall of the previous experience. Generally, a relatively discrete conditioning stimulus and a discrete unconditioned stimulus are used during fear conditioning (e.g. Tronson et al., 2009). However, unlike fear-conditioning paradigm, the exact nature, i.e. aversive or neutral, of the surgical experience in the laboratory is unknown since this was not tested during the experiments. In context of (b), the exposure to novel environment consisted of exposing the animals to a ‘novel room’ a couple of weeks after surgery (or novelty). The novel room was distinct from the laboratory in that it contained a set of objects separate from the laboratory and was unoccupied. No personnel stayed back to observe after the animal was placed in the novel room.

4.2.

Experimental groups

The following groups of surgically manipulated animals were analyzed for changes in c-Fos: i. Intraseptal-vehicle basal (IS veh basal): these animals were habituated for 10–15 days in Animal Holding Unit after receiving intraseptal microinjection of the vehicle in the laboratory. They were deeply anaesthetized in the home cage and perfused in the laboratory. ii. Intraseptal-vehicle re-exposure (IS veh reex): these animals were habituated for 10–15 days in an animal holding unit after undergoing surgery for intraseptal microinjection of the vehicle in the laboratory. On the test day they were reexposed to the laboratory to determine the effect of reexposure on c-Fos expression. The laboratory setting was

similar on re-exposure as during the surgery. During the re-exposure the animals were kept for 2 h in a clear plastic observation chamber (47.5 cm  26.5 cm  20.5 cm, L  W  H) that also served as the observation chamber. Animal horizontal locomotion was monitored during the period of observation (see Section 4.4 below). iii. Intraseptal-192 IgG saporin re-exposure (IS IgG reex): these animals received intraseptal microinjection of 192 IgG saporin. Subsequently, they were handled as in (ii). iv. Intraseptal-vehicle novelty (IS veh nov): these animals were habituated for 10–15 days in an animal holding unit after receiving intraseptal microinjection of the vehicle in the laboratory. On the test day they were exposed to the ‘novel room’ to determine the effect of novelty on c-Fos expression in the regions of interest. The ‘novel room’ was one door removed from the laboratory and consisted of an open field apparatus (Med Associates Inc.; the open field being a clear transparent box with following dimensions: 43.2 cm  43.2 cm  30.5 cm, L  W  H) that was positioned near one wall while a variety of other external cues were spread out around the room. For the purpose of the experiment the open field apparatus was subdivided into two halves by placing an insert in the chamber. The size of each half was 43.2 cm  21 cm  30.5 cm (L  W  H). On the day of the experiment, the animal was brought up from the Animal Holding Unit and immediately introduced into one half of the open field and allowed to remain there for the next 2 h. v. Intraseptal-192 IgG saporin novelty (IS IgG nov): these animals received intraseptal microinjection of 192 IgG saporin. Subsequently, they were handled as in (iv).

4.3. Surgical manipulations and microinjection of 192 IgG saporin Animals were surgically manipulated so as to microinject the immunotoxin 192 IgG saporin (0.42 μg/μl, Chemicon International, USA) into the medial septal region and, thus, destroy the cholinergic neurons in the region. Control animals received intraseptal injection of the vehicle (0.5% w/v alcian blue dye in saline). The animals were pretreated with atropine (1 mg/kg, i.p., Sigma, USA), deeply anaesthetized with pentobarbital (60 mg/kg, i.p., Sigma, USA) and mounted on a stereotaxic frame. Surgical procedure and intraseptal microinjections of the immunotoxin 192 IgG saporin were carried out under aseptic conditions (Zheng and Khanna, 2001). Briefly, a small burr hole was made on the skull overlying the medial septum (anterior 0.7 mm from Bregma, lateral 70.2 mm; Paxinos and Watson, 2007) to expose the underlying dura mater that was punctured using a fine needle (33G) attached to an Exmire microsyringe (Ito Corporation, Japan). Subsequently, either vehicle or the immunotoxin in 0.5% w/v alcian blue saline solution was microinjected bilaterally at V 6.0 mm from the dura mater. At each injection point, 0.4 μl of the solution was injected over a period of 1 min. To minimize the backdiffusion of fluid along the needle track and facilitate diffusion into the tissue the needle was left undisturbed at the microinjection site for an additional 10 min following each microinjection before retracting.

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The volume of injection and the concentration of 192 IgGsaporin were selected based on previous evidences, including from the laboratory that such treatment evoked a selective loss of cholinergic neurons in the medial septum and attenuated the power of hippocampal theta (e.g. Lee et al., 1994; Zheng and Khanna, 2001). Such a lesion also evokes a robust loss of cholinergic markers in the Cg indicating that the immunotoxin-induced lesions in the region disrupt the key cholinergic afferents to both the hippocampus and the Cg (Amaral and Kurz, 1985; Heckers et al., 1994; Torres et al., 1994; Walsh et al., 1996; Pizzo et al., 1999). Here it is notable that the immunotoxin 192 IgG saporin binds selectively to the nerve growth factor receptor on the cholinergic neurons in the medial septum. This triggers endocytosis of the immunotoxin of which the saporin component, which is a ribosome-inactivating immunotoxin, causes cell death on entering the neuron (Schweitzer, 1989; Wiley et al., 1991).

4.4.

Behavioral observations

The floor of the observation chamber in the laboratory was subdivided into 4 similar quadrants and animal horizontal locomotion was monitored up to 2 h. Horizontal locomotion (or ambulation) was assessed as the number of quadrants crossed by the animal when it moved from one quadrant to the next with all four paws. The animal movements in the observation chamber in the novel room animal were monitored by means of infrared sensors in-built into the open field apparatus. The following parameter was derived using the infrared sensors: Ambulatory distance: the horizontal exploration was scored as the ambulatory distance. An ambulatory episode was signaled when the rat moved beyond 4 inbuilt infra-red beams (i.e. box size) in 500 ms.

4.5.

Immunocytochemistry

The animals were deeply anaesthetized in the laboratory following 2 h of observation and perfused through the heart. Deep anesthesia was induced with urethane (1.5 g/kg, i.p; Sigma, USA), while the perfusion was performed using 1% sodium nitrite (Merck, Germany) in 0.5 M sodium phosphate buffer solution followed by 4% paraformaldehyde (Merck, Germany) solution in 0.1 M sodium phosphate buffer. The brain was removed and preserved in 4% paraformaldehyde. Following 24 h of preservation the brain was sectioned into 60 μm sections using a vibratome (Campden Instruments, USA) for immunohistochemistry. Alternate coronal sections were collected in 0.05 M Tris-buffered saline (Fisher Scientific, USA). Previously described methods (Zheng and Khanna, 2001; Khanna et al., 2004; Lee et al., 2008) were adapted for c-Fos immunocytochemistry and ChAT labeling, while modified version of the method described by Kiss et al. (1990) was adapted for parvalbumin labeling. The ChAT and parvalbumin label was used to identify cholinergic and GABAergic neurons, respectively, in the medial septal region while c-Fos was used as an index of synaptic activation in regions of interest. Briefly, the tissue sections were rinsed with 0.3% hydrogen peroxide (Merck, Germany) and then incubated for 2 h at room temperature in the blocking solution of 3% bovine

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serum albumin (BSA; Sigma, USA) in 0.05 M TBS with 0.3% Tritons X-100 (Bio-Rad Labs, USA). Subsequently, the tissues were incubated for 70 h with the primary antibody followed by 24 h incubation with the secondary antibody. Finally, the sections were next treated with the avidin–biotin–peroxidase complex (Vectastain Elite ABC Kit, Vector Labs, USA) for 3 h at room temperature followed by diaminobenzidine treatment (Sigma, USA). Once the brown immunolabel had developed, the reaction was stopped by rinsing the sections in TBS in a Petri dish and mounted on chrome alum gelatin-coated slides, air dried, dehydrated via ethanol (BDH Lab Supplies, UK) and ethanol– xylene (J.T. Baker, UK) and cover-slipped with DePeX (BDH Lab Supplies, UK). The immunoreactivity was visualized as brown reaction product. Omission of the primary antibody abolished the labeling. The primary antibodies used were rabbit anti-Fos polyclonal antibody (1:2000, Ab-5, Calbiochem, USA), rabbit antiChAT polyclonal antibody (1:800, Calbiochem, USA) and mouse anti-parvalbumin monoclonal antibody (1:5000, Sigma, USA). Biotinylated goat anti-rabbit (Calbiochem, USA) and goat antimouse (Sigma, USA) antibodies were used as secondary antibody at concentration of 1:1000.

4.6.

Data analysis

The destructive effect of the intraseptal immunotoxin on cholinergic neurons was determined by counting the number of remaining neurons labeled for the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), in alternate sections taken through medial septum region from anterior 1.20 mm to posterior 0.26 mm (Fig. 8; average number of sections¼ 9; Paxinos and Watson, 2007). As a measure of the number of cholinergic neurons in non-lesioned animals, ChAT positive neurons in the medial septum region of the IS veh nov group were counted. The medial septal region comprised of the medial septum, the vertical and the horizontal limb of the diagonal band of Broca. In the hippocampal formation, neurons positive for c-Foslike immunoreactivity (FLI) were counted in the pyramidal cell layer of field CA1 and CA3 from posterior 1.80 mm to posterior 6.04 mm (Fig. 8; average number of section for whole CA1 and CA3 being 21 and 23, respectively). Contours of these cell layers stood out in the brain sections, due to dense packing of the principal neurons as compared to the surround. Likewise, FLI-positive cells were also counted for amygdaloid subdivisions, corresponding to the basolateral and the medial amygdala (BLA and Me, respectively) from posterior 1.80 mm to 5.04 mm, and the Cg corresponding to Cg1 and Cg2 from anterior 4.70 mm to posterior 1.30 mm (Fig. 8; average number of sections for amygdala and Cg being 23 and 16, respectively; Paxinos and Watson, 2007). The total count for each of the region was averaged for the sections of each animal, and then for the experimental group. In case of the hippocampus such averages were also calculated for anterior segments (posterior 2.3 mm–4.52 mm), and posterior segments (posterior P 4.8 mm–6.04 mm) of field CA1. The posterior region was demarcated from the anterior by the C-shaped organization of the hippocampal formation in coronal sections. In the frontal sections, posterior field CA1 is demarcated into dorsal and ventral divisions based on the intervening field CA2.

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that might arise otherwise. In addition, the counts were made from alternate sections covering the length of the regions of interest. This was done so as to minimize the bias that might arise with selective sampling of sections. c-Fos-positive cells were identified by brown nucleus that was distinct from the background at 40  and 100  . For the purpose of illustration brain sections were digitized using Nikon Eclipse E400 and JVC color video camera KY-F55B. The software used to build the digitized section was Montage (Synoptics). The diagrams illustrated are 600 dpi.

4.7.

Statistical analysis

The results are expressed as mean7S.E.M. The c-Fos data were commonly analyzed using 1-way ANOVA. Since the groups were of unequal sizes, the data were first validated for homogeneity of variance using Bartlett's test. In instances, where the Bartlett's test showed unequal variance, the data was normalized by log transform followed by ANOVA. In all instances the post hoc comparison after ANOVA were performed using the Newman–Keuls test for multiple comparisons. The time course of behavioral change among different groups was compared using 2-way ANOVA followed by Bonferroni multiple comparison. Statistical significance was accepted at Po0.05.

Acknowledgment This work was supported by the National Medical Research Council.

r e f e r e n c e s

Fig. 8 – Diagrams of the coronal sections of the rat brain adapted from Paxinos and Watson (2007). The open symbols on the diagrams identify the different regions of interest from where c-Fos-like immunoreactive (FLI) cells were counted. These regions include the hippocampus (A; star and diamond), cingulate cortex (B; circles and triangles), medial septum (C; inverted triangle) and the amygdala (D; oval for basolateral amygdala, and pentagon for medial amygdala). An anterior, middle and a posterior section of each region are represented. The star and the diamond in A mark the fields CA1 and CA3 of the hippocampus, respectively. Within each hippocampal field, the FLI cells were counted from the pyramidal cell layer given by the broken line in the respective field. The numerical values below each diagram represent the anterior–posterior coordinate with respect to the Bregma.

The immuno-positive cells were counted using an Olympus microscope (Khanna et al., 2004). All counts were performed by one individual to minimize individual variations

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