Neuronal and brain morphological changes in animal models of schizophrenia

Accepted Manuscript Title: Neuronal and brain morphological changes in animal models of schizophrenia Author: Gonzalo Flores Julio C´esar Morales-Medina Alfonso Diaz PII: DOI: Reference:

S0166-4328(15)30343-0 http://dx.doi.org/doi:10.1016/j.bbr.2015.12.034 BBR 9963

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Behavioural Brain Research

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12-10-2015 8-12-2015 24-12-2015

Please cite this article as: Flores Gonzalo, Morales-Medina Julio C´esar, Diaz Alfonso.Neuronal and brain morphological changes in animal models of schizophrenia.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2015.12.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Neuronal and brain morphological changes in animal models of schizophrenia

Gonzalo Floresa, Julio César Morales-Medinab and Alfonso Diaz c

a

Laboratorio de Neuropsiquiatría, Instituto de Fisiología, Universidad Autónoma de

Puebla. 14 Sur 6301, Puebla, México, 72570 b

Centro de Investigación en Reproducción Animal, CINVESTAV- Universidad

Autónoma de Tlaxcala, Mexico AP 62, 90000 c

Departamento de Farmacia, Facultad de Ciencias Químicas, Benemérita

Universidad Autónoma de Puebla. Puebla, México, 72570

*Corresponding author: Gonzalo Flores, MD, PhD. Laboratorio de Neuropsiquiatría. Instituto de Fisiología, Universidad Autónoma de Puebla. 14 Sur 6301, CP. 72570, Puebla, México Tel No.: (522) 2295500 ext. 7322 Email Address: [email protected] or [email protected]

Running title: Animal models in schizophrenia

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GRAPHICAL ABSTRACT

Highlights    

Schizophrenia produces neural remodeling in the prefrontal cortex in humans Changes in the shape of dendritic arbor result of either gain or loss of connectivity Animal models are useful tools to understand Schizophrenia Schizophrenia animal models present dendritic and spine density alterations

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Abstract Schizophrenia, a severe and debilitating disorder with a high social burden, affects 1% of the adult world population. Available therapies are unable to treat all the symptoms, and result in strong side effects. For this reason, numerous animal models have been generated to elucidate the pathophysiology of this disorder. All these models present neuronal remodeling and abnormalities in spine stability.

It

is well known that the complexity in dendritic arborization determines the number of receptive synaptic contacts. Also the loss of dendritic spines and arbor stability are strongly associated with schizophrenia. This review evaluates changes in spine density and dendritic arborization in animal models of schizophrenia. By understanding these changes, pharmacological treatments can be designed to target specific neural systems to attenuate neuronal remodeling and associated behavioral deficits.

Abreviations Ace; centromedial amygdala BDNF; brain-derived neurotrophic factor BLA; basolateral amygdala CNS; central nervous system DA; dopamine DAD2R; DA D2 receptor DAT; dopamine transporter

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DG; dentate gyrus DH; dorsal hippocampus GD; gestational day HPA; hypothalamic–pituitary–adrenal axis IH; intermediate hippocampus IL-1 β; interleukin 1β KO; knock-out LPS; lipopolysaccharide mPFC; middle PFC   N-LNNA; N-Omega-Nitro-L-Arginine NAcc; nucleus accumbens   NO; nitric oxide nPFCL; neonatal prefrontal cortex lesion PCP; phencyclidine PD7; postnatal day 7 PFC; prefrontal cortex poly I:C; polyriboinosinic-polyribocydylic acid   PTSD; posttraumatic stress disorder PWSI; post weaning social isolation SNc; substantia nigra pars compacta TDL; total dendritic length TNF-α; tumor necrosis factor alpha VH; ventral hippocampus VTA; ventral tegmental area  

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Keywords: animal model, hippocampus, neurodevelopment, morphology, prefrontal cortex, Schizoprenia,

1. Schizophrenia (word count 249) Schizophrenia is a devastating disorder not only for the patient but also for the family. Indeed, this disorder alters the relation between the patient and the family and could induce a family breakdown modifying the prognostic of the schizophrenic patient. This complex disorder affects 1% of the world’s population. Interestingly, this mental disorder starts in early adulthood during the time when synapses are pruned [1,2] with a particular combination of positive, negative, affective symptoms as well as cognitive deficits. The severity of these symptoms can change over time depending on the disease stage [1,2]. Positive symptoms include hallucinations, delusions and thought disorders; negative symptoms comprise flat emotional expression, poor quality of speech, inability to derive pleasure from activities previously enjoyable and inability to initiate and persist in goal-directed activities. Finally, cognitive symptoms include deficits in executive functioning, attention and working memory. The neural mechanisms of schizophrenia are not yet fully known, and available pharmacological treatments are often ineffective to bring back the schizophrenic patient to normal functionality. In recent years, dendritic morphological studies using the Golgi and Golgi-Cox procedures (Fig. 1) have demonstrated changes in arborization and dendritic spine density in limbic regions such as prefrontal cortex (PFC), hippocampus and amygdala in postmortem tissue from schizophrenic 5   

patients and in animal models of schizophrenia [1,3,4]. These studies suggest that schizophrenia may involve dendritic spine abnormalities. In addition, recently a structural and biochemical study reported synaptic pathology in postmortem PFC tissue of schizophrenic patients using multiple label fluorescence confocal microscopy [5].

2. Neuronal staining as a tool to evaluate neuronal morphology The so-called Golgi–Cox method is a histological technique widely used as a tool to study neurons in the central nervous system (CNS). This staining procedure together with the Sholl analysis for light microscopy provides information about morphology, distribution, location, and intrinsic connections of neurons. Although this method does not reveal details of the internal structure of nerve cells, it does provide a unique view of the entire neurons and the relation of dendrites and axons to the neuron body, associated with their functional role in the normal CNS, for full details see the following book chapter [6]. The chromate precipitate staining was discovered by Camilo Golgi, when he observed that the whole neuron looked black and entitled this procedure as “black reaction”. There are two major groups of techniques using chromate precipitations known respectively as the Golgi (silver chromate) and Golgi-Cox (mercury chromate). Strangely, the Golgi-Cox staining is a better method than the Golgi procedure, especially to demonstrate the dendritic architecture of neurons in the mammalian brain [7].

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3. Relevance of neuronal morphology in schizophrenia Neuronal rearrangement and alterations in dendritic spines are observed in postmortem brains of patients with schizophrenia and numerous animal models of schizophrenia-like behavior [4] however, their causes have yet to be established [6,7]. For example, a reduced dendritic spine number has been reported in the layer 3 of the PFC in schizophrenia [10,11,12] and this region is a major site for corticocortico and thalamo-cortico integration [4]. The shape of dendritic arbor of a neuron determines the number and distribution of receptive synaptic contacts [13]. Moreover, dendritic arbors are dynamic during development, extend and retract branches as they mature [14, 15]. When the neurons reach adulthood, their structural plasticity is reduced and connections are stabilized. This process is not static, and while more than 80% of F actin in spines turns over every minute, 75% of the microtubules in dendrites turn over within tens of minutes [13]. In addition, dendritic spines are the main sites of excitatory input and thus the alterations in spine density are the result of either gain or loss of connectivity [16]. Therefore, modifications in spine density or dendritic arbor are associated with modifications in synaptic contacts. Alterations in both processes present an added effect in disrupting neuronal stability. The stability of dendrites and spines during development as well as during adulthood is intimately related to numerous molecules particularly those that can modulate actin. Indeed, neurotrophins have the capacity to regulate the stabilization and maturation of existing spines and the generation of new spines, for review see [17]. Brain-derived neurotrophic factor (BDNF), a member of neurotrophin family, 7   

regulate synaptic efficiency and plasticity as well as the survival and growth of neurons [18]. Interestingly, BDNF levels are lower in the PFC of schizophrenic patients [19]. Recently Nikonenko et al. [20] suggested that nitric oxide (NO), a gaseous neurotransmitter also plays a critical role in regulating the development of excitatory synapse by local mechanism that promotes dendritic spine growth. Finally, DCC organizes PFC wiring specifically during adolescence [21]. Therefore, DCC, BDNF and NO contribute to plasticity at the synapse along different molecules at different ages of development. Altogether, we believe that these three endogenous molecules contribute to the plastic changes observed in schizophrenia at different ages.

4. Neurotransmitter hypothesis of the disease Several reports suggest that the etiology of schizophrenia may be related to dopamine (DA) overactivity in the mesolimbic system [22]. Indeed, chronic administration of d-amphetamine, an indirect DA-agonist, produces schizophrenialike behaviors in normal human beings [22]. In schizophrenic patients, administration of DA agonists exacerbates the symptoms of schizophrenia. On the contrary, antipsychotic drugs such as haloperidol, a DA D2 receptor (DAD2R) antagonist is effective in ameliorating the core symptoms of schizophrenia [23]. Based on known anatomical and physiological data, there are reports suggesting that the alterations in DA activity in PFC could alter the activity of DA mesolimbic neurons [24, 25]. PFC receives glutamatergic projections from the CA1 subregion of the hippocampus and

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basolateral amygdala (BLA) and all of these three regions send glutamatergic projections to the nucleus accumbens (NAcc) (Fig. 2). Therefore the activation of these glutamatergic pathways stimulates GABAergic neurons of the NAcc, which inhibit neural activity of the ventral pallidum and this in turn promotes activity in the dorsomedial thalamus [26]. Thus, this circuit activates the limbic cortex such as PFC (Fig. 2). However, several researchers have suggested that the etiology of schizophrenia is not directly related to mesolimbic DA overactivity. Indeed glutamate [particularly N-methyl-D-Aspartate (NMDA) receptors] and GABA are emerging as key contributors of schizoprenia. The participation of their systems is particularly observed in varios animal models of Schizophrenia including the phencyclidine model discussed later on this review. Interestingly, the NAcc also receives a dense DA innervation from the ventral tegmental area (VTA) and plays a key role in selection of action, integration of cognitive and affective information processed by PFC and temporal lobe [26]. Dysregulation of DA activity in the NAcc could have significant behavioral and cognitive consequences. The NAcc is also involved in attending and orienting to relevant stimuli, which are significantly impaired in schizophrenia. The PFC participates in the regulation of attention, inhibition, cognitive control, motivation, and emotion. A fundamental component of schizophrenia is that patients exhibit profound deficits in PFC functions [27]. Therefore, evidence suggest that PFC is a primary site of dysfunction in schizophrenia, for review see Flores and Atzori, [1]. In the following sections we will describe in detail the four brain regions (BLA, hippocampus, NAcc and PFC) most affected in schizophrenia.

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5. Cerebral key regions involved in schizophrenia Numerous studies have documented the presence of structural changes in schizophrenic patients’ brain, including loss of cortical volume (white and gray matter), ventricular dilatation, myelinated fibers damage and glial cell abnormalities. Altogether structural and neuronal alterations provoke a damaged communication among different brain regions [28]. The volumetric changes are detected principally in prefrontal cortical areas, hippocampus and amygdala, engaged in disrupted cognitive functions in schizophrenia. Also many researchers have found alterations in the temporary top circumvolution, parietal lobe, cerebral subcortical regions including basal ganglia, corpus callosum and thalamus [29, 30]. Neuroimaging studies have also showed a reduction in the BLA of schizophrenic patients [31, 32] and decrease in size of striatum-limbic area and thalamus. In addition, post-mortem studies have shown a decrease in the number of basal dendrites and spines in pyramidal neurons of the layers III and V of PFC [10, 33]. These cytoarchitectural abnormalities are accompanied with changes in the molecular architecture of schizophrenic patients’ brain

and

are

related

to

neurotransmitters

systems

(dopaminergic

and

glutamatergic) [34, 35, 36]. Due to multiple affected regions and the diversity of symptoms found in schizophrenic patients, the existence of aberrant neural connectivity in the limbic system has been proposed, which participates in emotion, learning, memory, attention, and executive functions [37, 38]. All these processes are altered in schizophrenia [39]. In this review, we will only discuss about brain regions primarily 10   

affected in this psychiatric disorder such as the PFC, hippocampus, NAcc and amygdala.

5.1 The prefrontal cortex The PFC is a limbic system key component with a complex functional organization, and participates in numerous functions of superior order including selective attention, visceromotor control, decision making, directed behaviors toward a goal [38], working memory, attention, cognition, emotion and executive functions [40, 41]. Several anatomical studies have shown that the PFC is constituted by threeregions: superior PFC, medial PFC (mPFC) and lower PFC. However, for study it has been divided in the cingulate area, prelimbic area and infralimbic area. In the PFC, there is an extensive dopaminergic nerve input from the VTA [42]. The cerebral cortex is organized in 6 layers, numbered from the outer layer of the cortex to white matter. Layer I (molecular layer); layer II (external granular layer); layer III (external pyramidal layer or neuronal layer); layer IV (grained inner layer); layer V (inner pyramidal layer); and layer VI (polymorph layer or multiform layer) [43]. The PFC is one of the most top cortical regions and occupies nearly a third of the neocortex. This region was later developed during the evolutionary course, achieving its maximum development in human brain [44, 45, 46]. It is known that the mPFC exerts control over subcortical dopaminergic system; this cortical dysfunction produces various cognitive, emotional and motor deficits, and morphological alterations in certain psychiatric disorders [47, 48] including schizophrenia and 11   

attention deficit [49, 50, 51]. These findings are extremely relevant to the mPFC dopaminergic activity, involving cognition and motor functions [ 52, 53, 54].

5.2 Hippocampus Hippocampus is a key component of neural circuits and has connections with anterior cingulate cortex, NAcc, amygdala and midbrain raphe nuclei. It’s most important intrinsic pathways use glutamate as a neurotransmitter [55]. The hippocampus is a cognitive structure involved particularly in memory and emotional processes. This brain region is divided in dorsal (DH), intermediate (IH) and ventral (VH) hippocampus. While the DH is involved in cognitive functions and their dysfunction leads to amnesia, the IH only separates both regions [56, 57, 58]. The VH is responsible for emotional control such as frustrations [59, 60] and regulates the hypothalamic–pituitary–adrenal axis (HPA) activity through glucocorticoids negative feedback [61, 62, 63] and could cause a dysfunction in the hippocampus [56]. Dysfunction and decrease in the hippocampus volume are associated with different disorders such as posttraumatic stress disorder (PTSD), depression and schizophrenia [64]. Efferent connectivity indicates that the VH can modulate the circuit of reward and emotional behavior through projections to NAcc, PFC, and amygdala as well as the stress response by regulation of the HPA axis [57, 58]. Therefore, the participation of the hippocampus is significant in schizophrenia since its afferent and efferent pathways play an important role in the processes related to

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learning and memory, as well as stress, addiction, wakefulness state and synaptic plasticity [43].

5.3 The nucleus Accumbens The NAcc is part of the ventral striatum [65] and is involved in the integration between motivation and motor action known as limbic-motor interface [38].The NAcc is populated by spiny neurons and these neurons have a medium-sized cell soma, approximately 10-15 µm in diameter, dendritic spines and long axons that establish connections outside of nucleus. While GABAergic neurons comprise 95% of the total population of neurons in the NAcc [66], the rest of the population is comprised of GABAergic and cholinergic interneurons. Cholinergic neurons are typically multipolar, possess soma with fusiform or oval morphology and are neither bigger than 40 μm nor smaller than 20 μm. Cholinergic neurons constitute small portions of different regions of striatum, and represent 1% of NAcc. Dopaminergic projections maintain different topographies into dorsal striatum subdivisions and two divisions of the NAcc, the shell and the core [67]. These subregions present anatomical and biochemical differences. While the shell primarily receives glutamatergic limbic input from hippocampus and the amygdala, and important dopaminergic input from VTA, the core receives glutamatergic inputs from PFC and dopaminergic inputs from the substantia nigra pars compacta (SNc) [65]. The convergence between dopaminergic terminals and mPFC on common targets in NAcc indicates that the dopaminergic afferents can modulate the cortico13   

accumbens transmission [50, 52, 68, 69]. Therefore a possible loss of dopaminergic synaptic inputs to cortico-accumbens neurons can produce secondary changes in dopaminergic transmission into other mesolimbic structures [52, 68, 70]. Numerous reports indicate that schizophrenic patients present dopaminergic transmission abnormalities. However, cumulative evidence suggest that a cholinergic hyperactivity state in PFC is responsible for negative symptoms, while the cholinergic hypoactivity present in NAcc is considered to be a physiopathological part of positive symptoms inducing dopaminergic activity predominantly above cholinergic activity [71].

5.4 The amygdala Amygdala is an essential component of neural circuits that mediate stress and fear response, behavioral and endocrine autonomic functions and hypothalamic stimulation [60]. It is also considered a key structure in memory processing [72, 73]. The amygdaline complex is divided into the centromedial amygdala (Ace) which regulates the autonomic motor flow and BLA which is involved in cognitive and motivational functions such as behavior, emotions and exploratory activity. In addition, the amygdala also regulates the stress response. Interestingly, the amygdala only receives direct input from the VH [57]. The amygdala is a complex sub-internuclear group which is associated with neocortex sensors, frontal lobes, ventral striatum, PFC and hypothalamus [74].

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6. Animal models 6.1 Animal models of schizophrenia-like behavior Animal models are critical in research to further understand the disease mechanisms and design new treatments [75]. Animal models are valuable preclinical tools which investigates the neurobiological basis of schizophrenia. They offer a platform to quickly monitor numerous elements of the disease progression. Furthermore, they give the opportunity to analyze structural and molecular changes that occur in response to therapeutic agents [76]. Recently, it has been estimated, that more than twenty different animal models of schizophrenia have been developed [77], although each model has its own limitations. [78]. The majority of rodent models of schizophrenia replicate aspects of positive symptoms of schizophrenia such as hyperactivity. This abnormal behavior probably reflects an increase in the mesolimbic DA function. Among the models: lesions in the limbic system, social isolation from weaning and chronic administration of phencyclidine (PCP) and amphetamine have provided the most information about schizophrenia [79, 80]. These models show cortical dopaminergic dysfunction and sensorimotor system deficits, which can result from altered development of frontal cortical-limbic circuits [80]. In particular, lesion models have contributed to improve the understanding of the physiopathology and neurodevelopmental functions of several brain regions in relation to schizophrenia. Lesion models in limbic system have provided a greater evidence about the changes or alterations in dopaminergic mesolimbic functions [81,

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82, 83, 84, 85, 86]. Lesions with kainic and ibotenic acid in the hippocampus [87], PFC [81, 86], dorsolateral cortex and BLA [88, 89, 90, 91] develop psychosis structural models in animals. These acids kill vulnerable neurons, causing abnormalities in the cytoarchitecture of the hippocampus, amygdala and PFC, which modify the dopaminergic activity and produce hyperactivity and cognitive damage in animals [82, 92, 93] In the following sections, we will discuss in detail the neuronal modifications in various animal models of schizophrenia related behavior. 6.1.1 neonatal ventral hippocampal lesion (nVHL) Numerous studies have shown that a bilateral excitotoxic lesion of the VH at postnatal day 7 (PD7) in rats produces behavioral changes that appear only after puberty [1, 84, 85]. Lesioned rats are apparently normal at prepubertal age; but after puberty they exhibit locomotion hyper-responsiveness to novel environment, psychostimulants and stress with deficits in social interaction, sensorimotor gating, spatial learning and working memory, as well as reward and enhanced sensitivity to NMDA antagonists [82, 94, 95, 96, 97, 98, 99, 100]. At biochemical level, nVHL animals show changes such as a decrease in BDNF, nerve-growth factor-inducible B, glutamic acid decarboxylase-67, AMPA GluR3 mRNAs, and alterations in alpha1 adrenergic among others [101, 102, 103, 104, 105, 106]. This lesion reshapes the dendritic arbor in several brain regions (Table 1, Fig. 3). Indeed, our data demonstrated that nVHL produces a significant decrease in the dendritic length and number of dendritic spines on layer-3 and layer-5 of the pyramidal neurons of the PFC and pyramidal neurons of the BLA and medium spines neurons of the NAcc [93, 107, 108, 109]. These changes suggest a delayed neuronal

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reorganization particularly in the PFC, BLA and NAcc.

In addition, in vivo

microdialysis and autoradiography data demonstrated alterations in the release of acetylcholine, and the presence of muscarinic M1 receptors in the PFC and NAcc, respectively. [103]. VTA stimulation in animals with nVHL results in a higher excitability of PFC pyramidal neurons [110] and increased levels of the cytochrome oxidase I in PFC [111] with an altered excitability of NAcc neurons [52]. Interestingly, our recent report showed reduced number of cells in bilateral regions of the auditory temporal cortex with lower amplitude of the N40 auditory evoked potentials in adult animals with nVHL suggesting that nVHL leads to an inappropriate innervation in thalamic-cortical pathways, which results into altered function of cortical networks involved in processing of primary auditory information [112]. The nVHL is one of the most studied animal models of schizophrenia-related behavior and induces neuroanatomical rearrangement in the NAcc, BLA and PFC. The neuronal hypotrophy has been associated with the lack of input from the VH (Fig 3). Therefore, the constellation of behavioral deficits suggest that this neonatal VH manipulation is a key to further understand schizophrenia

6.1.2 Neonatal prefrontal cortex lesion (nPFCL) The nPFCL through ibotenic, aspartic or kainic acid injection has been used to create structural models of psychosis in animals [40, 82, 88, 113, 114]. In these models, lesions are created in populations of vulnerable neurons, causing abnormalities in cytoarchitecture of the PFC, which produces hyperactivity and cognitive damage. Ibotenic acid presents agonistic activity against NMDA receptors and metabolotropic quisqualate receptors and It is know that the overactivity of the NMDA receptors is 17   

a primary reason for neuronal death following cerebral ischemia [115]. An important and peculiar feature of schizophrenia is that the symptoms occur during or after puberty. An nPFCL induces morphological changes in some brain regions into adulthood (Table 1). Behavioral studies in rodents have shown that PFC lesions cause cognitive [116, 117, 118, 119], locomotion [83, 120, 121], social interaction and attention [48, 119] disorders, since it not only affects the injured structure, but also behavioral functions mediated by connected areas [122]. Lesioned animals early in development present physiological and neuroanatomical alterations including developmental abnormalities of cortex that alter the projections to many subcortical regions [123, 124]. It has been reported that a neonatal excitotoxic lesion in the PFC induces an increase in the expression of DA2R in the NAcc shell region, and potentiates sensitivity to stimulant locomotion effect of dopaminergic agonists after puberty [125, 126]. There has also been a dopaminergic activity change in both PFC and hippocampus of adult rats which were lesioned at neonatal stage [83, 84, 116, 127]. Ibotenic acid lesion of PFC at PD7 caused an increase in the locomotive activity in a new environment [83, 88, 116, 128]. Our group evaluated the effect of nPFCL on neuronal morphology at postpubertal age [88] and the results obtained were very intriguing. Indeed, nPFCL reduced the total dendritic length (TDL) as well as the spine density in medium spine neurons of NAcc (Table 1). While this lesion induced an increase in spine density in pyramidal neurons of the BLA, this lesion also decreased the spine density in neurons of the CA1 ventral hippocampus (Table 1). If the lesion in the PFC is performed in adult animals, the behavioral consequences are minimal. The neuronal alterations 18   

produced by nPFCL can be explained in terms that the brain continues to develop until puberty and connections are still being formed. Bayer et al. [129] also showed a decrease in glucose metabolism and synaptic density, as well as processes related with the neural system repair that occurs during puberty [130]. Altogether, these results suggest that nPFCL induces behavioral and neurochemical alterations in adult animals. A critical stage of neurodevelopment is the first postnatal week, and since alterations in the early development are contributing factor for schizophrenia, consequently the nPFCL may be related to these neural alterations

6.1.3 Administration of N-Omega-Nitro-L-Arginine on early postnatal days NO plays a key role in synaptogenesis and synaptic plasticity which may produce functional modifications in the brain circuits [131, 132]. Particularly, high levels of NO are observed in hippocampus, frontal cortex and striatum, after labor and these levels decrease after a couple of weeks post birth [133, 134]. Therefore NO may be important in the establishment of neuronal networks in the rat. Our group hypothesized that the postnatal blockade of NO levels may induce abnormal behaviors and neuronal rearrangement in the adult rat. Indeed, Mejorada et al., [135] observed that neonatal administration of the non-selective NO synthase inhibitor NOmega-Nitro-L-Arginine (N-LNNA) during PD4-6 induced hyperlocomotion in novel environment. Afterward, Morales-Medina et al., [136] observed amphetamineinduced hyperlocomotion in animals treated with N-LNNA at PD4-6 along with

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disruptions in NO levels in several brain regions and permanent changes in DAD2R in the caudate putamen in adulthood. Later on, N-LNNA was administered at PD-1-3, PD4-6 and PD7-9 and the animals were sacrificed at PD60 (postpubertal age) to evaluate the possible effects of this treatment on dendritic morphology and spine density in pyramidal neurons of the PFC layer 5 and CA1 hippocampus. N-LNNA administered at PD4-6 induced a decrease in TDL, a reduced number of intersections in the Sholl analysis and a decrease in the number of spines (Table 1, Fig. 3). In contrast, this treatment at PD13 only modified the TDL in the CA1 hippocampus (Table 1) [137]. The hyperlocomotion to novel environment, the hypersensitivity to amphetamine and the neuronal hypotrophy in the CA1 hippocampus are traits observed in other models of schizophrenia-related behavior (Table 1). Thus the constellation of behavioral and neuronal changes in animals with postnatal blockade of NO could be considered as a potential animal model of schizophrenia-related behavior.

6.1.4 Phencyclidine model PCP, a non-competitive NMDA receptor antagonist, induces schizophrenia-related behavior in normal subjects as well as rodents [138, 139]. Indeed, PCP induces behavioral deficits including positive, negative and cognitive symptoms similar to those observed in schizophrenic patients, [139]. When PCP is administered in acute, repeated or perinatal form, it induces hyperlocomotion in novel environment,

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sensorimotor gating deficits, and memory impairments in the passive avoidance test and reduced social contacts, for review see Mouri et al., [139]. Two research groups evaluated the effects PCP in neuronal morphology and spine density with fundamental differences in the experimental design. First, Hajszan et al., [138] administered PCP for one week and discontinued the treatment for one week before the neuronal study and showed a decrease in the spine density in neurons in the PFC. Later on, Flores et al., [140] administered PCP for one month and allowed the animals to rest for one month prior to neuronal analysis. PCP treatment for one month induced an increase in spine density in the PFC (layer 3 and 5, Fig. 3) and the NAcc with no changes in neuronal arborization in the PFC or NAcc (Table 1) [140]. The authors evaluate intermittent administration of PCP as it is often the case in human users. In addition, spiny neurons from the NAcc showed a strong trend towards a decrease in the TDL (*p=0.07). These two studies could show two stages of neuronal adaptation to the blockade of NMDA receptors. While Hajszan et al., [138] show changes resembling sub chronic exposure, Flores et al., [140]

present results after a chronic administration to PCP. Perhaps the data

collected by Flores et al., [140] show compensatory spinogenesis since PCP was not administered one month prior sacrifice. Moreover, the second study resembles more closely humans use and indicates that PCP produces long lasting effects in neuronal morphology since the changes in neuronal morphology are observed even one month after the termination of PCP treatment. Future studies should present whether acute or long term exposure to PCP produce neuronal remodeling or

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alterations in spine density if the animals are sacrified the day following the last injection.

6.1.5 Post weaning social isolation Social dysfunction is certainly implicated in the development and course of Schizophrenia [141]. Prior to the appearance of full range of schizophrenic symptoms, patients tend to isolate and when the full range of symptoms emerge, the social isolation is exacerbated. In rats, post weaning social isolation (PWSI) produces numerous abnormal behaviors, such as hyperactivity in novel environment as well as after administration of DA or glutamate antagonists [142, 143]. Early on, our group investigated the neuronal consequences of PWSI in the PFC and CA1 hippocampus [98]. Although the TDL was normal in animals that underwent social isolation for eight weeks, a rearrangement in the arborization of these neurons. PWSI increased the number of third order branches and decreased the number of fourth order branches toguether with a reduction in spine density in the PFC(Table 1, Fig. 3). Neuronal hypotrophy and reduction in spine density was present in CA1 hippocampus neurons (Table 1). This study demonstrated that PWSI produces robust neuronal changes in the brain and exacerbated sensibility to DA and glutamatergic agents.

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6.2 Genetic models Cumulative evidence suggest that schizophrenia has a strong genetic component with an estimated 80% of heritability [144, 145]. Despite this, it is important to indicate that a single genetic alteration cannot explain this complex psychiatric disorder [146, 147]. Indeed, a large number of studies have focused to investigate genes associated with schizophrenia, and observed that many of these genes act synergistically. Besides the genetic component, epigenetic processes and environmental factors certainly contribute to trigger schizophrenia [148, 149, 150]. Several genetic models have contributed significantly to the understanding of aberrant role of dopaminergic and glutamatergic function and are discussed briefly in this document.

6.2.1 NR1 receptor The NMDA receptor plays a key role in the aetiology of schizophrenia [151, 152]. Moreover, several studies report postmortem brains of patients with this condition present abnormal low levels of NR1 subunit expression, causing a lower density of NMDA receptors [153, 154]. NR1 knock out (KO) animals observed motor changes as hyperlocomotion, stereotyped movements, decreased social interaction and lack of motivation [155, 156]. Drastic changes in brain morphology mainly in the PFC and hippocampus were observed in NR1 receptor KO animals, demonstrating that this subunit promote the formation or maintenance of dendritic spines, and the regulation of the actin cytoskeleton in these brain regions [156, 157, 155]. All these 23   

features are similar to those observed in animal models and patients with schizophrenia [82, 83, 109, 159]. In addition, pharmacological treatment with antipsychotics improved moderately these anomalies [160, 161]. These results show that dysfunction of NMDA receptor for the suppression of the NR1 subunit in neurons in animal’s corticolimbic system reproduced symptoms similar to schizophrenia.

6.2.2 Dysbindin Genetic studies in Irish families suggests that dysbindin (dystrobrevin Binding Protein 1: DTNBP1) is involved in schizophrenic patients [162] as dysbindin is a synaptic protein that regulate exocytosis and vesicle formation to neurotransmitter release [163]. Postmortem studies show a reduced expression of dysbindin in the prefrontal cortex and hippocampus of patients with schizophrenia. Genetic studies indicate a clear relationship between dysbindin expression and negative symptoms of schizophrenia [164, 165]. Dysbindin KO mice showed an increase in locomotor activity, reduced social contact [166], dysfunction of reference memory [167, 168] and cognitive impairments [169]. To induce neuronal remodelling in limbic regions, it suggested that dysbindin participates in the dynamic dendritic morphogenesis protrusions and stabilizing dendritic protrusions during neuronal development [170, 171].

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6.2.3 DISC1 The gene DISC1 identified in Scottish pedigree was linked to schizophrenia as DISC1 is a synaptic protein expressed in early development and plays a key role in neurogenesis, neuronal migration and synaptic plasticity [172]. Reports suggest that DISC1 is involved particularly in the onset of schizophrenia [173]. Mice with DISC1 mutation showed spontaneous hyperlocomotion in new environment, decrease in social interaction and motivation, followed by a decline in working memory [174, 175]. In contrast, spatial object recognition memory is not affected in these animals [167, 175, 176]. Moreover, the mice showing knockdown of DISC1 in granule cells present soma hypertrophy, ectopic dendrites and incorrect placement of new granule cells due to overextended migration modifying neuronal morphology [177].

6.2.4 Reeler Finally, the mice "reeler" is a spontaneous mutant heterozygous of reelin gene. Reelin is an extracellular matrix glycoprotein and regulates the processes of neuronal migration in the developing brain and is involved in synaptic plasticity in the adult brain [178, 179]. Studies indicate that Reelin mRNA decreased dramatically in the hippocampus and PFC of patients with schizophrenia causing a long-term decrease of glutamic acid decarboxylase synthase GABA [180, 181]. In the genetic model, where Reelin expression is suppressed, the expression of GAD67 is decreased and the dendritic spine morphology is altered, parallel deterioration in hippocampal synaptic function and associative learning and memory observed. Also 25   

these mice exhibited reduced social interaction and decreased motor activity, and altered spatial learning. Clearly, all these genetic studies are insufficient to understand the origin of schizophrenia. Since schizophrenia is a heterogeneous disorder (with genetic and environmental contributing factors), it makes impossible for a single model to mimic the complete pathology. Although genetic models have improved our understanding of behavioral and morphological characteristics of this pathology, further studies are certainly warranted.

6.3 Animal models of risk factors Prenatal manipulations and obstetric complications during labor induce behavioral deficits resembling some schizophrenia-related behaviors in adulthood [182, 183]. This hypothesis is associated with changes occurring in the immune system of the mother and the maturation of the central nervous system (CNS) in the offspring in this particular time window. Indeed, the immune system and inflammatory responses are dramatically affected during pregnancy [184]. In addition, maternally generated cytokines can cross the placenta and modulate the development of the fetus [185, 186]. The placenta synthetizes interleukin 1β (IL-1 β), IL-6 and tumor necrosis factor alpha (TNF-α) and may act as another source of cytokines to the fetus [185]. Finally, after infection in the mother, the fetus can mount an inflammatory response through cytokines particularly with high levels of IL-6 [186, 187]. In addition to all the sources of cytokines that can affect the brain, and since the blood-brain barrier is incomplete 26   

in the fetus, the inflammation-induced cytokines can likely enter in the fetal brain [188]. In vitro IL-1 β, IL-6 and TNF-α can reduce the TDL of neurons as well as the neuronal survival. Thus the inflammatory response has an enormous effect on the formation of neuronal networks in the fetal CNS. Apparently, another factor important to take in consideration is the time of the insult performed. In this regard, gestational day (GD) 15 in rats correspond to the late first trimester of human pregnancy and GD18 is parallel to the early second trimester [189, 190]. The prenatal immune challenge at different prenatal times can differentially compromise the integrity of neuronal circuitry in adulthood. In addition, early postnatally the neuronal networks are still being actively formed as well as pruning of synapses takes place in rodents. Therefore manipulations during pregnancy and early postnatally are considered as models of risk factors for schizophrenia-related behavior.

6.3.1 Perinatal anoxia Epidemiological evidence correlates obstetric complications during labor and delivery as a risk factor for schizophrenia [6, 7, 182]. Two models have been used to measure perinatal anoxia: cesarean at birth and cesarean plus anoxia. Cesarean per se produces some level of hypoxia [191]. Also, since rats are born at an earlier ontogenic stage compared to humans, these animal models more closely mimic birth anoxia in premature infants [192].

27   

Adult rats that were delivered by cesarean, present stress-induced hypolocomotion and normal locomotion after amphetamine or apomorphine administration Meanwhile, cesarean plus anoxia induced several behavioral abnormalities observable after puberty, for example, amphetamine- and stress-induced hyperlocomotion [193, 194]. Both processes produced differential behavioral alterations in the later life of the offspring. Cesarean at birth induced a complex neuronal rearrangement in the PFC, hippocampus and NAcc (Table 1). At PD21, neurons in the PFC present hypertrophy, however neurons tend to become normal at later stage. At PD35, these neurons present a transient decrease in spine density. Hippocampal CA1 pyramidal neurons present a decrease in spine density only at PD35 and PD60. In the NAcc, medium spiny neurons showed neuronal hypertrophy at PD35 (Table 1). Animals with cesarean plus anoxia presented neuronal hypotrophy in the PFC at PD21 and reduction in spine density at PD21 and PD35 (Fig. 3). An increase in spine density was observed in the CA1 hippocampus (PD21 and PD35) and NAcc (PD35) (Table 1). Consequently, both interventions produced completely different maladaptive neuronal patterns including neuronal hypertrophy (cesarean) or neuronal hypotrophy (cesarean plus anoxia) in the PFC.

6.3.2 Prenatal immune challenge with lipopolysaccharide Maternal infections have been associated with the development of Schizophrenia in the adult offspring as observed in large epidemiological studies in humans [183,

28   

195]. Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative bacteria and is recognized by the toll-like receptor 2 and 4. Thus administration of LPS mimics a bacterial infection and exacerbates the immune response. Maternal exposure to LPS at GD15 and 16 reduced the number of DAD2R expressing cells in the PFC at PD60 and reduced dopamine transporter (DAT) binding in the NAcc at PD35. Moreover LPS administration at GD17 resulted in an anxiogenic phenotype in the adult offspring, suggest that prenatal infection produces altered dopaminergic activity and hyper anxiety in the offspring, traits observed in animal models of schizophrenia-related behavior. The possible changes in neuronal arborization and spine density in the PFC layer 3 and 5 as well as the CA1 hippocampus were evaluated at three critical ages of neurodevelopment (Table 1, Fig. 3): PD10, PD35 and PD60 [196]. A dramatic change in the number of intersections and reduced TDL at PD10 was observed in the PFC layer 3 [196]. A similar effect was observed at PD35 but the neurons presented no significant rearrangement at PD60 in this brain region. No change in spine density in the PFC layer 3 was observed at three ages evaluated. Moreover, pyramidal neurons of the PFC layer 5 presented normal arborization but decreased spine density at PD60 [196]. In contrast to the results obtained in the PFC, while the offspring of pregnant dams treated with LPS showed hypotrophy in the CA1 hippocampus neurons at PD60, these neurons presented no changes at PD10 or PD35 [196]. The spine density remained unchanged in these animals at three ages evaluated. However, a further

29   

study that dissected the surface, length and volume of spines showed a consistent decrease in all the spine parameters measured in LPS-treated animals. LPS-treated animals during pregnancy present abnormal dopaminergic activity, anxiety-related behavior and neuronal rearrangement in the PFC and CA1 hippocampus. Further studies are required to fully characterize this model to observe if these animals present hyperlocomotion in novel environment or hypersensitivity to amphetamine.

6.3.3 Prenatal immune challenge with polyriboinosinic-polyribocydylic acid Polyriboinosinic-polyribocydylic acid (poly I:C) mimic a viral infection. The administration of this agent was performed in pregnant mice at gestational day 9.5 and a series of behavioral and neuronal modifications using the Golgi-Cox staining was performed at postpubertal age [197]. Prenatal administration of poly I:C induced hypolocomotion in the open field test and impaired object recognition memory [197]. poly I:C administration during pregnancy induced neuronal rearrangement in the PFC and granule cells of the dentate gyrus (DG) of the hippocampus [197]. In particular, poly I:C treatment reduced the TDL, the number of branch order as well as the spine density in adult animals (Table 1, Fig. 3). In contrast to other models of schizophrenia-related behavior, poly I:C increased the TDL of pyramidal neurons in the PFC. This treatment also reduced the spine density in the PFC (Table 1). These results suggest that poly I:C or LPS administered during pregnancy alter the phenotype inducing schizophrenia-related behaviors. Several researchers suggest 30   

that the inflammatory response is responsible for the neuronal changes and not the agent per se [188] and these results confirm this hypothesis.

7. Pharmacological treatment reshaping the neurons in animal models of schizophrenia The rat model of nVHL has proven a highly valuable tool for the analysis of numerous behavioral deficits and alterations in the central nervous system including neuronal hypotrophy in several brain regions. Pioneer work from our group recently showed that Clozapine reshape neurons in animals with nVHL [109]. Indeed, Clozapine increased the TDL in neurons from the PFC, NAcc and BLA. In addition to the neuronal remodeling, this treatment dramatically decreased the hyperlocomotion in novel environment. Therefore these results may suggest that Clozapine reverses the behavioral deficits by modulating neurons. Further research is needed to find correlations between behavior and neuronal remodeling and more importantly whether traditional antipsychotics such as Haloperidol or novel treatments modify neuronal hypotrophy in different animal models.

31   

8. Concluding remarks (word count 396) Schizophrenia symptoms appear in early adulthood during the time when synapses are pruned [2]. While all animal models of schizophrenia present behavioral deficits and neuronal remodeling, studies presenting data from prepubertal animals is scarce. Thus, the search for neuronal remodeling in animal models at prepubertal age is certainly warranted to know the degree of validity of the model. As reviewed above, animal models of schizophrenia have different origins and target various neurotransmitter systems. Our group combined the nVHL with the pwSI in an attempt to know if those models of different origin will lead to the same behavioral and neuronal deficits. Surprisingly, pwSI increased the hyperlocomotion to novel environment to nVHL animals [107]. Moreover, pwSI further decreased the TDL in PFC and NAcc in nVHL animals. Therefore the increased locomotion is associated the neuronal alterations in PCF and NAcc. Schizophrenic patients respond differentially to antipsychotic treatments, these results are particularly important as they suggest that different origins of Schizophrenia would negatively impact different behaviors.

In addition, these results show that while antipsychotic treatment

rescued, another experimental manipulation further increased the behavioral de deficit. In both cases neuronal reorganization, at least partially, is required to modify the behavior. Moreover, these results suggest that the PFC, NAcc and hippocampus present a high degree of synaptic plasticity. All the animal models evaluated in this review, prenatal, perinatal or postpubertal manipulations lead to alterations in neuronal morphology that differs in a spatiotemporal way. Among all the models, the PFC consistently presents neuronal 32   

alterations. Indeed the PFC is one of the brain regions with the most plastic changes due to experience, stimulants, lesions or learning task [198]. These alterations are not limited to changes in spine density but in neuronal rearrangement as well. These changes suggest influence on neuronal networks in the brain. We must emphasize that the neuronal alterations are not uniform (for complete comparison see Fig 3). For example, in the PFC while cesarean at birth produces neuronal hypertrophy, cesarean plus anoxia induces neuronal hypotrophy. Surprisingly, animals with cesarean or cesarean plus anoxia present amphetamineinduced hyperlocomotion. Since the output is similar for both, we suggest that the behavioral outcome is the net result of a change in a neuronal circuit rather than a specific brain region. The brain regions that seem to have a key involvement in animal models of schizophrenia are the PFC, BLA, NAcc and CA1 hippocampus.

9.0 Acknowledgements Funding for this study was provided by project 129303 from CONACYT, Mexico (GF). CONACyT has no role in the writing or discussion of the present review. GF, JCMM and ADDF acknowledge the National Research System (CONACYT) for membership. We thank Mira Thakur for editing and proofreading the manuscript.

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Figure legends

Figure 1: This illustrates the Golgi-Cox method as a tool to characterize the cytoarchitecture of dendrites, fibers and neuronal somata in the central nervous system of a rat, as an animal model of schizophrenia. Left, all layers of the prefrontal cortex (PFC) were observed at lower magnification (6X). Right, the pyramidal neurons in the PFC were observed at medium magnification (40X).

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Figure 2: Diagram shows the connections of limbic regions affected in schizophrenia. The limbic pathways involved monosynaptic and multisynaptic connections. Red lines indicate glutamatergic projection. Blue lines show gabaergic projections. PFC: prefrontal cortex.

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Figure 3: Alterations in neuronal arborization and spine density in the prefrontal cortex in various animal models of schizophrenia-related behavior at post-pubertal age. The schematic diagrams show how diverse manipulations lead to changes in dendritic arborization or spine density. In red are depicted neurons and spines of animal models of schizophrenia and in blue are shown neurons and spines of animal models of risk factors.

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Table legends

Table 1: Data from neuroanatomical studies Table 1: Neuroanatomical characteristics on the dendrite arbor and spines, using the Golgi-Cox procedure and Sholl analysis in animal models of schizophrenia related behavior. Abbreviations:

=, no effect; ↑, increase; ↓, decrease;

BLA,

basolateral amygdala; DG, dentate gyrus; L-NNA, N-Omega-Nitro-L-Arginine; NA, not available; LPS, lipopolysaccharide; NAcc, nucleus Accumbens; PCP, phencyclidine; PD, postnatal day; PFC, prefrontal cortex; Poly I:C, polyriboinosinicpolyribocydylic acid; TDL, total dendritic length; Treatment

Brain region

TDL

Branch order

L-NNA administration at PD1-3 L-NNA administration at PD4-6 L-NNA administration at PD7-9 Neonatal ventral hippocampus lesion (nVHL)

Hippocampus CA1 PFC layer 5 Hippocampus CA1 PFC layer 5 Hippocampus CA1 PFC layer 5 PFC layer 3

=

NA

Spine density ↓

= ↓

NA NA

= ↓

= =

NA NA

= =

NA =

= ↓

=

=

Neonatal ventral hippocampus lesion + Social Isolation Neonatal ventral hippocampus lesion + Clozapine

PFC layer 3 NAcc

= ↓ compared to nVHL ↓ compared to nVHL ↓ ↓

= =

↑ compared to nVHL ↑ compared to nVHL ↑ compared to nVHL ↓ ↓

NAcc

PFC layer 3 NAcc BLA PFC layer 3

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Age

Reference

PD60

MoralesMedina et al., 2007

PD60

Flores et al., 2005

= =

PD80

Alquicer et al., 2008

=

=

PD80

Bringas et al., 2012

=

=

=

=

NA

=

PD10

NA

=

PD35

Baharnoori et al., 2009

LPS administration at GD 15-16

poly I:C administration at GD 9.5 PCP administration for 1 week PCP administration for 1 month Social isolation

Neonatal prefrontal cortex lesion Cesarean at birth

PFC layer 5 Hippocampus CA1 PFC layer 3 Hippocampus DG PFC PFC layer 3 PFC layer 5 NAcc PFC layer 3 Hippocampus CA1 Hippocampus CA1 Ventral BLA NAcc PFC layer 3

Hippocampus CA1 NAcc

Cesarean at birth plus anoxia

PFC layer 3

Hippocampus CA1 NAcc

= = = = ↓ ↑

= ↓ = = = ↓

↓

NA NA NA NA NA ↑ in intermodal/↓ in terminal ↓

NA

NA

↓

Adulth

Hajszan et al., 2006

= = *p=0.07 = ↓

= = = Order 3,4 4

↑ ↑ ↑ ↓ ↓

Adulth

Flores et al., 2007

PD80

=

NA

↓

PD60

SilvaGomez et al., 2003 Lazcano et al., 2015

= ↓ ↑ = = = = = NA ↑ = ↓ = = = = = NA = =

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA

↑ ↓ = ↓ = ↓ ↓ = = = = ↓ ↓ = ↑ ↑ = NA ↑ =

PD21 PD35 PD60 PD21 PD35 PD60 PD21 PD35 PD60 PD21 PD35 PD60 PD21 PD35 PD60 PD21 PD35 PD60

 

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PD60 PD60 PD10 PD35 PD60 PD80

Li et al., 2014

↓

Juarez et al., 2008

Juarez et al., 2008