Post-weaning social isolation rearing influences the expression of molecules related to inhibitory neurotransmission and structural plasticity in the amygdala of adult rats

doi:10.1016/j.brainres.2012.01.073

Brain Research

Volume 1448, 11 April 2012, Pages 129-136

https://doi.org/10.1016/j.brainres.2012.01.073 Get rights and content

Abstract

Several lines of evidence indicate that alterations in the structure of neural circuits and inhibitory neurotransmission underlie the physiopathogenesis of schizophrenia. Most of the studies on these parameters have been focused on cortical regions and, despite the crucial role of the amygdala in this psychiatric disorder, there is less information on this region. In order to expand this knowledge, we have studied the expression of molecules related to inhibitory neurotransmission and structural plasticity in rats subjected to post-weaning isolation rearing, an animal model that reproduces several core symptoms of schizophrenia. We have analyzed, using qRT-PCR and immunohistochemistry, the expression of synaptophysin, GAD65, GAD67, the neural cell adhesion molecule (NCAM), its polysialylated form (PSA-NCAM) and its synthesizing enzymes (St8siaII and St8SiaIV). Isolation-reared rats showed significant increases in the expression of GAD67 protein in the centromedial, medial and basolateral amygdaloid nuclei, but no significant changes in GAD65 or synaptophysin expression were found in these regions. The expression of PSA-NCAM and NCAM was significantly increased in the basolateral and medial nuclei respectively. Our results indicate that isolation-rearing influences positively inhibitory neurotransmission and neuronal structural plasticity in the amygdala, probably through PSA-NCAM. These findings are in contrast to reports describing decreased expression of molecules related to inhibitory neurotransmission in the amygdala of schizophrenic patients. Consequently, although the social isolation rearing model can reproduce some of the behavioral traits of schizophrenics it may fail to reproduce some of the neurobiological features of this disorder, particularly in the amygdala.

Highlights

► Alterations in neuronal structure and inhibitory circuits in schizophrenia. ► Study of post-weaning social isolation rearing (SIR) in rats, a schizophrenia model. ► Analysis (IHC and qRT-PCR) of synaptophysin, GAD65/67, NCAM, PSA-NCAM and ST8SiaII/IV. ► SIR increases GAD67, NCAM and PSA-NCAM expression in some amygdaloid nuclei.

Introduction

Schizophrenia is a complex disease, affecting approximately 1% of the population worldwide. The heritability of this disease is around 80% (Owen, 2005), highlighting the importance of altered genetic pathways in their development. Current pathophysiological theories of schizophrenia are pointing to the GABAergic system as responsible for some of the alterations in schizophrenic brains, because of its implication in the regulation of other neurotransmitter systems, such as dopaminergic or serotoninergic, and its tight relationship with the excitatory transmission (Benes and Berretta, 2001). GABA is the principal inhibitory neurotransmitter in the central nervous system (CNS) and genes implicated in its metabolism have been associated with schizophrenia in different human populations (Straub et al., 2007, Zai et al., 2009) and in animal models of this psychiatric disorder (Peleg-Raibstein et al., 2008). Almost all of these studies have found important alterations in the isoenzymes responsible for GABA synthesis (GAD65 or GAD67) and in their coding genes, showing that alterations in the synthesis of GABA may be one of the crucial facts in schizophrenia.

In addition to these changes in inhibitory neurotransmission, structural changes have also been found in the brains of schizophrenic patients, as well as in animal models of this disorder (Phillips et al., 2003). This structural remodeling involves changes in synaptic density, which are frequently estimated analyzing the expression of synaptophysin, a synaptic vesicle membrane protein, whose expression is linked to synaptic remodeling (Eastwood and Harrison, 2001, Greengard et al., 1993). One of the mechanisms involved in this structural plasticity is the addition of polysialic (PSA) acid to the Neural Cell Adhesion Molecule (NCAM): This process, mediated by the two polysialyltransferases (St8SiaII and St8SiaIV), facilitates the formation of new synapses, the remodeling of neurites (see Bonfanti, 2006, Rutishauser, 2008 for review), or the partial isolation of neuronal elements (Gomez-Climent et al., 2011), because, when polysialylated, NCAM becomes anti-adhesive. Both NCAM and ST8SIAII genes have been associated with schizophrenia and alterations in the expression of NCAM and PSA-NCAM have been found in postmortem brain studies of this disorder (Brennaman and Maness, 2010, Sullivan et al., 2007, Tao et al., 2007).

Most of these data on the expression of molecules related to inhibitory neurotransmission or structural plasticity have been obtained from the prefrontal cortex, the nucleus accumbens and the hippocampus, regions specially affected in schizophrenia. However, despite the fact that the amygdala is also severely affected in schizophrenic patients, this knowledge is still scarcer in this region. The amygdaloid complex plays a critical role in the recognition and the response to emotional stimuli, including fear and anxiety (Adolphs et al., 1995, Cahill and McGaugh, 1998, Davis et al., 1994, LeDoux, 2000), which are frequently abnormal in schizophrenic patients. Recent brain imaging studies have shown that abnormalities in limbic lobe regions including the amygdala may be responsible of the inadequate affective responses in schizophrenics (Phillips et al., 2003). Several studies have reported changes in the volume of the amygdala in psychiatric patients, which probably reflect structural changes in amygdaloid neurons (reviewed in Drevets et al., 2008, Sheline et al., 1998, Tebartz van Elst et al., 2000). In light of these set of evidence pointing to the amygdala, we have decided to study the expression of molecules related to structural plasticity and inhibitory neurotransmission using an animal model, which reproduces some of the core symptoms of schizophrenia.

The post-weaning social isolation-paradigm offers a well established and characterized animal model to study schizophrenic symptoms in a rodent. Some of the behavioral and neurochemical changes induced by the isolation-rearing are disrupted prepulse inhibition (Geyer et al., 1993), reduced expression of AMPA glutamate receptors in hippocampus (Sestito et al., 2011), impaired spatial cognition with affected prefrontal cortical synaptic plasticity (Quan et al., 2010), impaired novel object recognition (McLean et al., 2010), increased aggression (Ferdman et al., 2007) and reduced prefrontal cortex volume (Day-Wilson et al., 2006). Similar environmental interventions during early-life in humans may contribute to the development of common psychiatric disorders, such as depression and schizophrenia in genetically predisposed individuals (see Fone and Porkess, 2008 for review).

The main objective of this study is to determine specific changes in the amygdaloid expression of different molecules involved in structural plasticity and inhibitory neurotransmission. For this purpose we have analyzed the expression of GAD67, GAD65, synaptophysin, NCAM and PSA-NCAM by means of immunohistochemistry and optical densitometry in the centromedial (CeM), medial (Me) and basolateral (BLa) amygdala. We have also studied the expression of mRNAs for GAD67, GAD65, synaptophysin, NCAM and the polysialyltransferases (St8SiaII and St8SiaIV) using quantitative real-time PCR (qRT-PCR) of total amygdala.

Section snippets

Immunohistochemical analysis reveals that the expression of GAD67, but not of GAD65 or synaptophysin, is altered in the amygdala of isolation-reared rats

No changes were observed in the expression of GAD65 in any nucleus of amygdala when comparing social versus isolation-reared rats. By contrast, the expression of GAD67 was significantly increased in all the three amygdaloid nuclei of the isolation reared rats respect to the socially housed animals: Me, CeM and BLa (t₉ = − 2.822, p = 0.02; t₉ = − 3.572 p = 0.006 and t₉ = − 2.835 p = 0.0196 respectively; Fig. 1, Fig. 2). The expression of synaptophysin, which is a general marker of synapses (Greengard et al.,

Discussion

Early-life adverse events markedly influence the development of the nervous system and may facilitate, in genetically predisposed individuals, the development of psychiatric disorders such as schizophrenia or major depression. Exposing rodents to postweaning social isolation affects brain development and leads to behavioral, morphological and neurochemical alterations during adulthood, which resemble core symptoms of schizophrenic patients. The behavioral alterations include neophobia,

Animals

Eight pregnant Lister Hooded rats were purchased from Jackson laboratories (Bar Harbor, Maine, USA) and bred in our animal facility. Pregnant rats were housed individually in a controlled temperature room (25 ± 1 °C) and on a 12-h light/dark cycle with food and water available ad libitum. 27 male rats were born from the pregnant rats and were used for the experiments. All efforts were made to minimize the number and suffering of animals used. All animal experimentation was conducted in accordance

Acknowledgments

Spanish Ministry of Science and Innovation (MICINN-FEDER) BFU2009-12284/BFI, MICINN-PIM2010ERN-00577/NEUCONNECT in the frame of ERA-NET NEURON and the Stanley Medical Research Institute supported JN. Javier Gilabert-Juan has a FPU predoctoral fellowship from the Spanish Ministry of Education (AP2008-00937).

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Some years later, we became interested in the effects of stress during brain development and their link to neuropsychiatric disorders. We found that male rats reared in isolation (from P21 until adulthood) had a reduced expression of PSA-NCAM in the BLA (Gilabert-Juan et al., 2012a), but not in the mPFC or the hippocampus (Gilabert-Juan et al., 2013a). This same postweaning isolation stress also reduced PSA-NCAM expression in the whole amygdala in male mice (Castillo-Gómez et al., 2017).
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In addition, it is also worth noting that to the best of our knowledge SIR-induced PFC volume reductions have only been reported twice (Day-Wilson et al., 2006; Schubert et al., 2009), while SIR-induced HPC volume reduction is still controversial (Fabricius et al., 2010; Schubert et al., 2009). In any case, some of the above mentioned SIR-induced frontocortico-limbic neurochemical/molecular changes are reminiscent of neural sensitization phenomena, and it is interesting that sensitization processes of central dopaminergic (Cain et al., 2012; Gilabert-Juan et al., 2012; Marsden et al., 2011) and serotoninergic mechanisms have been described as a consequence of isolation rearing (Lukkes, 2009). It thus may be conceivable that some sensitization-like process could occur in the PFC during the present prolonged social isolation treatment (much longer than those used in the previous literature) which could lead to the observed volume increase, although this hypothesis deserves further research.
Social isolation rearing of rodents is an environmental manipulation known to induce or potentiate psychotic-like symptoms and attentional and cognitive impairments relevant for schizophrenia. When subjected to a 28-week isolation rearing treatment, the Roman high-avoidance (RHA-I) rats display the common behavioral social isolation syndrome, with prepulse inhibition (PPI) deficits, hyperactivity, increased anxiety responses and learning/memory impairments when compared to their low-avoidance (RLA-I) counterparts. These results add face validity to the RHA-I rats as an animal model for schizophrenia-relevant behavioral and cognitive profiles and confirm previous results. The aim here was to further investigate the neuroanatomical effects of the isolation rearing, estimated through volume differences in medial prefrontal cortex (mPFC), dorsal striatum (dSt) and hippocampus (HPC). Results showed a global increase in volume in the mPFC in the isolated rats of both strains, as well as strain effects (RLA > RHA) in the three brain regions. These unexpected but robust results, might have unveiled some kind of compensatory mechanisms due to the particularly long-lasting isolation rearing period, much longer than those commonly used in the literature (which usually range from 4 to 12 weeks).
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Research ReportPost-weaning social isolation rearing influences the expression of molecules related to inhibitory neurotransmission and structural plasticity in the amygdala of adult rats

Abstract

Highlights

Introduction

Section snippets

Immunohistochemical analysis reveals that the expression of GAD67, but not of GAD65 or synaptophysin, is altered in the amygdala of isolation-reared rats

Discussion

Animals

Acknowledgments

Neuropsychopharmacology

Prog. Neurobiol.

Trends Neurosci.

Exp. Neurol.

Trends Neurosci.

Neuroscience

Brain Res. Bull.

Behav. Brain Res.

Neurosci. Biobehav. Rev.

Biol. Psychiatry

Exp. Neurol.

Neuroscience

Brain Res.

Brain Res.

Clin. Ther.

Behav. Brain Res.

Biol. Psychiatry

Neuroscience

J. Neurol. Sci.

Biol. Psychiatry

Schizophr. Res.

Neurosci. Lett.

Neuroscience

J. Psychiatr. Res

Research Report
Post-weaning social isolation rearing influences the expression of molecules related to inhibitory neurotransmission and structural plasticity in the amygdala of adult rats