Deficits in parvalbumin and calbindin immunoreactive cells in the hippocampus of isolation reared rats

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1 J Neural Transm (2007) 114: DOI /s Printed in The Netherlands Deficits in parvalbumin and calbindin immunoreactive cells in the hippocampus of isolation reared rats M. K. Harte 1, S. B. Powell 2, N. R. Swerdlow 2, M. A. Geyer 2, G. P. Reynolds 1 1 Division of Psychiatry and Neuroscience, Whitla Medical Building, Queen s University, Belfast, U.K. 2 Department of Psychiatry, University of California, San Diego, La Jolla, CA, U.S.A. Received: August 8, 2006 = Accepted: January 11, 2007 = Published online: February 15, 2007 # Springer-Verlag 2007 Summary Post-mortem studies have provided evidence for abnormalities of the g-aminobutyric acid (GABA)-ergic system in schizophrenia. The calcium-binding proteins (CBPs), parvalbumin (PV), calbindin (CB) and calretinin (CR) can be used as markers for specific subpopulations of GABAergic neurons in the brain. Isolation rearing of rats is a non-pharmacological, non-lesion manipulation that leads to deficits in prepulse inhibition of the startle reflex (PPI) and other behavioural and neurochemical alterations reminiscent of schizophrenia. Female rats were reared in social housing (groups of three) or singly for 11 weeks post weaning and PPI was measured. Brains were removed and hippocampal CBP containing neurons determined following immunocytochemical staining. Compared to socially housed rats, isolated rats exhibited PPI deficits and reductions in PV and CB-immunoreactive cells in the hippocampus, with no significant change in CR. These findings demonstrate selective abnormalities of sub-populations of GABAergic interneurons in the hippocampus of isolation reared rats, which resemble the neuronal deficits seen in this region in schizophrenia. Keywords: Schizophrenia, isolation rearing, hippocampus, GABA, calcium binding proteins Introduction Schizophrenia is a complex disorder resulting from a combination of genetic and environmental factors, which increase the risk of the disorder. Animal models of schizophrenia are inevitably limited in validity but allow us to study certain features of this disorder and investigate how these features respond to known or potential antipsychotic drugs. Such models may also contribute to our understanding of Correspondence: Michael K. Harte, Division of Psychiatry and Neuroscience, Whitla Medical Building, Queen s University, Belfast, 97 Lisburn Road, BT9 7BL, Northern Ireland M.K.Harte@qub.ac.uk the relationships between brain pathology and symptoms of schizophrenia. Rearing of rats in social isolation from weaning is a non-pharmacological, non-lesion manipulation that leads to a number of long-lasting behavioural and neurochemical abnormalities, including deficits in sensorimotor gating mechanisms, neophobia, increased anxiety, deficits in cognitive function, and increased locomotor activity in a novel environment (Geyer et al., 1993; Hall et al., 1998; Jones et al., 1992; Sahakian et al., 1977; Weiss and Feldon, 2001). As some of these changes show similarity to clinical aspects of schizophrenia, isolation rearing may provide a useful animal model for studying the neurobiological basis of psychiatric disorders such as schizophrenia. Recent research has defined further the subtle neuronal abnormalities seen in schizophrenia. Deficits in g-aminobutyric acid (GABA)-containing neurons are reported in the frontal cortex (Beasley and Reynolds, 1997; Beasley et al., 2002) and particularly in the hippocampus. Several studies have provided evidence for dysfunction of GABAergic interneurons in the hippocampal formation in schizophrenia (Benes et al., 1998; Zhang and Reynolds, 2002). Benes et al. (1998) reported significantly reduced density of interneurons in CA2 and CA3 of brains of schizophrenia patients, including drug-free patients, suggesting that deficits in interneurons may play a contributory role in the pathophysiology of schizophrenia. Consistent with this interpretation, neurochemical studies have demonstrated a deficit of GABAergic uptake sites in the hippocampus (Reynolds et al., 1990), while other reports showed a widespread compensatory up regulation of post-synaptic specific GABA A

2 894 M. K. Harte et al. receptor binding activity throughout most sub-fields of the hippocampal formation of schizophrenia patients (Benes et al., 1996). Therefore, there is substantial evidence for a functional impairment of GABAergic inhibitory interneurons in the hippocampus in schizophrenia (Benes, 1999). However, our understanding of the GABA system in schizophrenia must take into account the fact that there are several different types of non-pyramidal neurons that utilize GABA as a neurotransmitter, and that each subset of neurons may show a unique pattern of change. GABAergic neurons can be defined further by the presence of one of three calcium binding proteins (CBPs): parvalbumin, calbindin or calretinin. Calcium binding proteins represent a family of small, acidic proteins that accept Ca 2þ with high selectivity. It is now generally accepted that the major neuronal role of these CBPs is the buffering and transport of calcium ions and the regulation of various enzyme systems. As cellular degeneration is often accompanied by impaired calcium homeostasis, a protective role for CBPs has been postulated (reviewed in Heizmann, 1992). For the purpose of the present study we are using CBPs as markers for different subtypes of GABAergic interneurons in the hippocampus of the rat brain. The present study was designed to determine the effect of isolation-rearing on inducing regionally specific schizophrenia-like neuropathological patterns. In particular we investigated the effect of isolation rearing on neuronal expression of the CBPs, parvalbumin, calbindin and calretinin, in the hippocampus. After a pre-determined period of isolation, rats were characterised for deficits in prepulse inhibition (PPI) of the startle reflex, before determination of parvalbumin, calbindin and calretinin immunoreactivity in sub-fields of the hippocampal formation. Material and methods Animals A total of 18 female Sprague-Dawley rats were used in this experiment. Female rats were used because of recent evidence that the isolation-rearing induced deficits in PPI may be more robust in female rather than male rats (Powell et al., 2002). At the time of weaning (postnatal day 23), rats were assigned to either social housing (n ¼ 9) or isolation housing (n ¼ 9). Social housing consisted of 3 rats and isolation housing consisted of one rat per standard polycarbonate cage ( cm). All rats were kept on a 12-hour reverse light-dark cycle (lights off from 0700 to 1900) in the same colony room with temperature and humidity remaining constant. Thus, rats could see, hear, and smell the other rats in the room. All behavioral testing occurred during the dark phase between 0900 and 1700, 11 weeks after isolation. Food (Harlan Teklad; Madison, WI) and water were available ad libitum except during behavioral testing. Experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No ) revised All efforts were made to minimize the number of animals used and the suffering of animals in these experiments. Startle testing The startle testing employed in this study is described in detail elsewhere (Geyer and Swerdlow, 1998; Mansbach et al., 1988; Powell et al., 2003). Briefly Rats were tested in the acoustic startle=ppi paradigm 11 weeks postweaning. The acoustic startle session consisted of a 5-min acclimation period in the chamber with a constant background noise (65 db), followed by 52 presentations of acoustic stimuli to measure acoustic startle. The 52 acoustic trials consisted of: twenty-two 40-ms presentations of a 120-dB broadband pulse, ten 20-ms presentations of each prepulse intensity (68, 71, 77 db) 100 ms prior to a 40-ms presentation of a 120-dB broadband pulse, and NOSTIM trials in which no acoustic pulse was delivered in order to assess general motor activation in the rats. Five of the 120-dB trials were presented at the beginning of the session and five at the end of the session, with the remaining twelve 120-dB trials presented in the middle of the session. Startle magnitude was quantified from the PULSE ALONE (120 db) trials and the PREPULSE þ PULSE trials. Percent PPI (% PPI) for the three prepulse intensities was calculated according to the formula: % PPI ¼ 100 {[(startle response for PREPULSE þ PULSE trial)=(startle response for PULSE ALONE trial)]100}. An average % PPI value was obtained for each animal by combining the data from the three prepulse intensities. Immunohistochemistry Immediately following the behavioural testing, rats were sacrificed by deep anaesthesia with pentobarbital followed by transcardial perfusion with 10% formalin=saline. Brains were removed and fixed in 10% phosphate buffered formalin for 3 days before being embedded in wax. Three sections (10 mm) from the hippocampus (bregma 3.3 mm), determined using a rat brain atlas (Paxinos and Watson, 1998) were mounted onto slides coated with 3-aminopropyltriethoxysilane (APES) (Sigma) and stained for parvalbumin, calbindin and calretinin. immunoreactivity (Beasley et al., 1997). Briefly, following clearing in xylene and rehydrated in graded alcohol, sections were incubated in hydrogen peroxide solution to inhibit endogenous peroxidase activity and a microwave treatment protocol was employed to aid antigen retrieval. Sections were then blocked with 5% normal horse (for parvalbumin and calbindin) or 5% goat serum (for calretinin), diluted in 0.1 M phosphate buffered saline (PBS) containing 0.1% triton X-100. After incubation at 4 C, with either a mouse monoclonal antibody against parvalbumin at a dilution of 1:10,000 in PBS for 36 h, rabbit anti-calretinin antibody or mouse anti-calbindin D-28K antibody diluted 1:7500 for 24 h (Swant, Bellinzona, Switzerland), biotinylated secondary antibody was added. Sections were processed by the avidin-biotin method using a Vectastain ABC kit (Vector Laboratories) and peroxidase was visualised using 3 0,3 0 - diaminobenzidine (DAB) intensified with nickel chloride. No immunoreactivity could be detected in control sections, in which the primary antibody was omitted from the staining protocol. All slides were coded and analyzed blind to housing conditions. Hippocampal sections (bregma 3.3 mm) were scanned at 4 magnification using an Olympus microscope interfaced to an Image ProPlus analysis system via a JVC 3-CCD video camera. Estimations of neuronal density (cells=mm 2 ) were carried out, in three horizontal sections (30 mm apart), using composite images of total left and right hippocampus. Immunostained neurons were counted at a higher magnification in defined complete subfields of each region according to the Atlas of Paxinos and Watson (1998) (Fig. 1). Neuronal body size (area) for each animal was obtained from mean values calculated from the threshold images of 20 (per section) individual arbitrarily chosen PV, CB and CR-containing positive neurons. Within the hippocampal formation, three regions were examined, the dentate gyrus (DG), CA2 plus CA3 and CA1 plus subiculum.

3 Deficits in parvalbumin and calbindin immunoreactive cells 895 of PPI revealed a significant main effect of housing on PPI [F(1,16) ¼ 6.27, p< 0.05], with isolated rats showing reduced average %PPI compared to socially housed rats after 11 weeks of isolation. Immunohistochemical analyses revealed deficits in parvalbumin immunoreactive cell density in the hippocampus of rats reared in isolation. ANOVA of parvalbumin immunoreactive cell density showed a significant effect of housing for two of the sub-fields examined. Dentate Gyrus [F(1,16) ¼ 4.67, p<0.05]; CA2=3 [F(1,16) ¼ 4.84, p<0.05] (Fig. 1A). There were also deficits in calbindin immunoreactive cell density in the hippocampus of rats reared in isolation. ANOVA of calbindin immunoreactive cell density showed a significant effect of housing for each sub-field examined. Dentate Gyrus [F(1,16) ¼ 16.84, p< 0.001]; CA2=3 [F(1,16) ¼ 8.65, p<0.01]; CA1 [F(1,16) ¼ 7.41, p<0.01] (Fig. 1B). Total calretinin immunoreactive cell density did not differ between housing groups in any region investigated (Fig. 1C). ANOVA of hippocampal area showed no effect of housing conditions for any single sub-field or for the hippocampus as a whole. Furthermore ANOVA of cell size showed no effect of housing conditions for any of the CBP-IR neurons (results not shown). There were significant positive correlations between average %PPI and both parvalbumin (Pearson correlation ¼ 0.493, p ¼ 0.038) and calbindin (Pearson correlation ¼ 0.551, p ¼ 0.018) specific to the dentate gyrus. Significant correlations of behaviour and neurochemical measures were not found in other sub-fields examined or in the hippocampus as a whole. Discussion Fig. 1. Relative density (cells=mm 2 ) of parvalbumin (PV), calbindin (CB) and calretinin (CR) immunoreactive neurons in the hippocampal sub-fields of rats reared in isolation and in their socially housed controls. Data are expressed as mean S.E.M, n ¼ 9 per group. p < 0.001, p < 0.01, p <0.05 vs. control Statistical comparisons of behavioural and morphological data in socials and isolates were performed using ANOVA and correlations examined using the Pearson correlation; a p-value of <0.05 was considered to be significant. All Statistical analysis was undertaken using SPSS v13. Results There was no difference in startle magnitude between socially housed and isolation reared rats. However ANOVA Here we demonstrate that social isolation induces a behavioural deficit (reduction in PPI) and is associated with selective deficits in the relative density of subsets (defined by calcium binding protein expression) of GABAergic interneurons in the hippocampus. Prepulse inhibition of the startle reflex (PPI) is an operational measure of sensorimotor gating (Geyer et al., 1993; Graham, 1975); deficits in PPI have been reported in several neuropsychiatric conditions including schizophrenia (Braff et al., 1978, 1992, 2001). Similarly, deficits in PPI have been reported by numerous investigators in animals reared in isolation (Cilia et al., 2001; Geyer et al., 1993; Varty et al., 1999; Wilkinson et al., 1994). The disruption of PPI in isolation-reared rats can be reversed by both

4 896 M. K. Harte et al. typical (Geyer et al., 1993; Varty and Higgins, 1995) and atypical antipsychotics (Bakshi et al., 1998; Varty and Higgins, 1995) suggesting that this model may also be useful for identifying therapeutic agents for the treatment of schizophrenia. Deficient PPI is a common finding associated with isolation rearing (Bakshi et al., 1998; Cilia et al., 2001; Geyer et al., 1993; Varty et al., 1999). As reviewed elsewhere, however, deficits in PPI are not always observed in studies of isolation-reared rats (Powell et al., 2002; Weiss and Feldon, 2001). That the animals studied here demonstrated a deficit in PPI indicates that early, postweaning social isolation resulted in a functional neural deficit. Consistent with previous reports, parvalbumin, calbindin and calretinin immunoreactivity was present in intensely labelled non-pyramidal neurons in the rat hippocampus (Cahir et al., 2005; Greene et al., 2001; Solbach and Celio, 1991). Although calbindin is present in some pyramidal cells, initial studies in our laboratory (unpublished results) have demonstrated that the anti-calbindin D-28K antibody causes darker staining in a subset of neurons in the hippocampus (presumably GABAergic interneurons) with lighter staining in the majority of hippocampal cells (presumably pyramidal cells). Parvalbumin, calbindin and calretinin have been shown to act as markers of separate populations of local circuit neurons in the cortex, indicating that they may be useful tools in unravelling the intrinsic inhibitory circuitry of the brain (Conde et al., 1994). In the present study we found deficits in parvalbumin and calbindin immunoreactive neurons in the hippocampus of animals reared in isolation compared to their group housed controls. GABA-containing interneurons in the hippocampus can control large populations of principal cells through their extensive axonal arborizations. Parvalbumin immunoreactive neurons in the hippocampus have been shown to make connections with the somata of pyramidal cells (Ribak et al., 1990), and thus may play an important role in controlling the output from these principal neurons. Parvalbumin is interesting in that it shows a late onset of expression, occurring after GABAergic neurons have been formed (Grateron et al., 2003; Solbach and Celio, 1991), imparting a period during which these cells may be particularly susceptible to neurotoxic insult. We have previously reported deficits of parvalbumin immunoreactive cells both in the frontal cortex (Beasley and Reynolds, 1997; Beasley et al., 2002), and more profoundly in the hippocampus in schizophrenia (Zhang and Reynolds, 2002). These GABAergic deficits in schizophrenia could well be an initial deficit, perhaps of neurodevelopmental origin, that subsequently results in further progressive neuronal dysfunction and the development of schizophrenia in the second or third decade of life. These GABAergic deficits are unrelated to antipsychotic drug treatment, age or duration of illness. In animals long-term repeated administration of the NMDA receptor antagonist phencyclidine (PCP), has been shown to mimic certain aspects of the disorder. Utilizing a sub-chronic or chronic intermittent treatment regime, our laboratory and others have found that compared to vehicle treated controls, animals receiving PCP exhibit reductions in PV-immunoreactive cells in the frontal cortex (Cochran et al., 2003) and hippocampus (Abdul-Monim et al., 2006). Here we show a similar structural abnormality in animals that were reared in isolation, providing further evidence that early environment plays an important role in normal neuronal development. Calbindin immunoreactivity is widely distributed within the hippocampus, and is contained in about 10 12% of GABAergic interneurons (Freund and Buzsaki, 1996). These calbindin interneurons receive input from afferents to the hippocampus, including serotonergic fibres from the raphe (Halasy et al., 1992). Pyramidal cell dendrites are the primary targets of calbindin immunoreactive neurons in the hippocampus (Seress et al., 1993) placing them in a position to exercise global control over hippocampal activity. Interestingly deficits in calbindin have also been reported in schizophrenia (Beasley et al., 2002; Cotter et al., 2002; Iritani et al., 1999). Calbindin immunoreactive cell bodies and fibres were also found to be disordered in their arrangement in the hippocampus compared to normal control brains (Iritani et al., 1999). A reduction in calbindin neurons in the isolates could have important consequences in terms of neuronal connectivity and integration in the hippocampus. The decrease in density of parvalbumin and calbindin may represent a loss of these types of local circuit neurons. A decrease in the expression of parvalbumin or calbindin below a detection threshold, however, can not be ruled out. It is not yet possible to determine which of these explanations is correct. If the latter is true, it is still likely that there would be important consequences in terms of neuronal function because of the protective role played by calcium binding proteins. In the present study, no changes were found in the density of calretinin immunoreactive neurons. This is in contrast to a similar immunohistochemical study in which an increase in calretinin was observed in the dentate gyrus, with no change in other sub-fields, in animals reared in isolation (Greene et al., 2001). Some discrepancies between this work and the previous study exist in that they looked at a shorter isolation period in a different strain and

5 Deficits in parvalbumin and calbindin immunoreactive cells 897 sex (male wister rats), in a smaller group and only in the right hemisphere. The counting method was similar but they used thicker sections at different levels throughout the hippocampus. The method we use here is very similar to that used in our previous studies in our human postmortem tissue of schizophrenia (Reynolds and Beasley, 2001; Zhang and Reynolds, 2002). Indeed the majority of studies using human post-mortem tissue have reported that calretinin immunoreactive neurons are unaffected in schizophrenia (Cotter et al., 2002; Daviss and Lewis, 1995; Porebski et al., 1999; Reynolds and Beasley, 2001; Zhang and Reynolds, 2002). This study used a non-stereological two dimensional quantification of neuronal density. The two major sources of error in this approach lie in possible group differences in hippocampal volume and cell size. A previous study in isolates showed no effect of isolation rearing on hippocampal volume (Greene et al., 2001). The present study also compared areas of the hippocampal regions investigated. No significant differences in the mean area of the hippocampal sub-fields were detected between housing conditions. There was also no significant difference in mean cell size of parvalbumin, calbindin or calretinin containing neurons in the hippocampal sub-fields between housing conditions, indicating that differences in hippocampal volume, hippocampal area and=or cell size did not substantially confound the comparisons of neuronal density. In addition, Benes and Lange (2001) has discussed the unbiased measurement of neuronal density, suggesting that a two dimensional approach is appropriate for the determination of non-pyramidal cells in the hippocampus due to the relatively low density of interneurons in this structure. The hippocampus has long been known to regulate PPI (Caine et al., 1991, 1992; Koch, 1999; Swerdlow et al., 1992, 2001). The present study demonstrated behavioural changes and hippocampal pathology in the same animals, and further demonstrated that these changes were related to each other: there was a significant positive correlation between PPI and both parvalbumin and calbindin in isolationreared rats. This effect was only found in the dentate gyrus. These findings should be viewed as preliminary, based on the small sample of isolation-reared rats. Further work is required to determine the association between abnormal functioning of inhibitory interneurons in the dentate gyrus and PPI in these animals. Nonetheless, the present results underscore the usefulness of the social isolation model in mimicking both behavioural (PPI deficits) and neuropathological (deficit in parvalbumin and calbindin immunoreactive neurons) phenomena characteristic of schizophrenia. Acknowledgements Michael K. Harte is supported by a MSD Pharmacology Fellowship. Mark A. Geyer, Neal R. Swerdlow and Susan B. Powell are supported by grants from the National Institute of Mental Health (MH52885, MH01436). Statement of interest Mark Geyer holds an equity interest in San Diego Instruments, Inc. References Abdul-Monim Z, Neill JC, Reynolds GP (2006) Sub-chronic psychotomimetic phencyclidine induces deficits in reversal learning and alterations in parvalbumin-immunoreactive expression in the rat. J Psychopharmacol [Epub ahead of print] Bakshi VP, Swerdlow NR, Braff DL, Geyer MA (1998) Reversal of isolation rearing-induced deficits in prepulse inhibition by Seroquel and olanzapine. Biol Psychiatry 43: Beasley CL, Reynolds GP (1997) Parvalbumin-immunoreactive neurons are reduced in the prefrontal cortex of schizophrenics. 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