Interneuron heterotopia in the Lis1 mutant mouse cortex underlies a structural and functional schizophrenia-like phenotype

fcell-09-693919 July 8, 2021 Time: 17:35 # 1 ORIGINAL RESEARCH published: 13 July 2021 doi: 10.3389/fcell.2021.693919 Edited by: Tae Ho Lee, Fujian Medical University, China Reviewed by: Rita M. Cowell, Southern Research Institute, United States Javier Gilabert-Juan, Autonomous University of Madrid, Spain *Correspondence: Salvador Martinez [email protected] †These authors have contributed equally to this work and share first authorship Specialty section: This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology Received: 12 April 2021 Accepted: 16 June 2021 Published: 13 July 2021 Citation: Garcia-Lopez R, Pombero A, Estirado A, Geijo-Barrientos E and Martinez S (2021) Interneuron Heterotopia in the Lis1 Mutant Mouse Cortex Underlies a Structural and Functional Schizophrenia-Like Phenotype. Front. Cell Dev. Biol. 9:693919. doi: 10.3389/fcell.2021.693919 Interneuron Heterotopia in the Lis1 Mutant Mouse Cortex Underlies a Structural and Functional Schizophrenia-Like Phenotype Raquel Garcia-Lopez1†, Ana Pombero1†, Alicia Estirado1, Emilio Geijo-Barrientos1and Salvador Martinez1,2* 1Instituto de Neurociencias, UMH-CSIC, Alicante, Spain, 2Centro de Investigación Biomédica En Red en Salud Mental-CIBERSAM-ISCIII, Valencia, Spain LIS1 is one of the principal genes related to Type I lissencephaly, a severe human brain malformation characterized by an abnormal neuronal migration in the cortex during embryonic development. This is clinically associated with epilepsy and cerebral palsy in severe cases, as well as a predisposition to developing mental disorders, in cases with a mild phenotype. Although genetic variations in the LIS1 gene have been associated with the development of schizophrenia, little is known about the underlying neurobiological mechanisms. We have studied how the Lis1 gene might cause deficits associated with the pathophysiology of schizophrenia using the Lis1/sLis1 murine model, which involves the deletion of the first coding exon of the Lis1 gene. Homozygous mice are not viable, but heterozygous animals present abnormal neuronal morphology, cortical dysplasia, and enhanced cortical excitability. We have observed reduced number of cells expressing GABA-synthesizing enzyme glutamic acid decarboxylase 67 (GAD67) in the hippocampus and the anterior cingulate area, as well as fewer parvalbumin-expressing cells in the anterior cingulate cortex in Lis1/sLis1 mutants compared to control mice. The cFOS protein expression (indicative of neuronal activity) in Lis1/sLis1 mice was higher in the medial prefrontal (mPFC), perirhinal (PERI), entorhinal (ENT), ectorhinal (ECT) cortices, and hippocampus compared to control mice. Our results suggest that deleting the first coding exon of the Lis1 gene might cause cortical anomalies associated with the pathophysiology of schizophrenia. Keywords: LIS1 gene, schizophrenia, GABAergic system, interneurons, hippocampus, mPFC, c-fos INTRODUCTION Abnormal neuronal migration during brain development results in several brain organization alterations. One of the most significant cortical abnormalities is lissencephaly (Reiner et al., 1995;Kato and Dobyns, 2003;Barkovich et al., 2005;Wynshaw-Boris, 2007). This developmental alteration produces severe symptoms that include seizures, intellectual retardation, and a higher risk of developing psychotic disorders like schizophrenia (Dobyns et al., 1993;Tabarés-Seisdedos et al., 2008). Schizophrenia (SZ) is a chronic and serious psychiatric illness that occurs in 1% of Frontiers in Cell and Developmental Biology | www.frontiersin.org 1July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 2 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model the global population (Freedman, 2003) and which is caused by both genetic and environmental factors. The symptoms of SZ fall into three categories: (1) positive symptoms that include hallucinations, delusions, cognitive deficits, and movement disorders; (2) negative symptoms, such as the disruption of normal emotions and behaviors; and (3) cognitive symptoms: attention deficit and memory problems (Ross et al., 2006; Insel, 2010). The precise cause of SZ remains unclear, postmortem studies have shown a decrease of GABA-synthesizing enzyme (GAD67) together with a reduction in the number of parvalbumin (PV)+ interneurons in the medial prefrontal cortex (mPFC) (Hashimoto et al., 2003, 2008;Reynolds et al., 2004;Woo et al., 2008; Glausier et al., 2014;Hoftman et al., 2015). Other GABAergic subtypes of interneurons, such as cholecystokinin, vasoactive intestinal peptide, somatostatin, neuropeptide Y, and calbindin (CB) have also been reported to be less expressed in the mPFC of SZ patients (Hashimoto et al., 2003, 2008;Fung et al., 2010, 2014). These deficits in the GABAergic interneurons seem to be related to the alteration of PFC-dependent cognitive functions in SZ patients. Indeed, PFC is implicated in important cognitive processes such as attention, memory, and behavioral changes (Heidbreder and Groenewegen, 2003;Gabbott et al., 2005; Briand et al., 2007). Furthermore, functional failures of N-methyl-D-aspartate (NMDA) receptors, typically found in GABAergic neurons, affect the maturation of GABAergic interneurons (Thomases et al., 2013). Interestingly, previous studies provided evidence that NMDA receptor antagonists, such as ketamine, MK801, and phencyclidine (PCP), reproduce schizophrenia-like symptoms in mice. For this reason, blocking NMDA receptors is the approach most widely used to mimic SZ symptoms in animal models (Jentsch and Roth, 1999;Ozdemir et al., 2012;Gilabert-Juan et al., 2013;Kim et al., 2014;Li J.-T. et al., 2016;Castillo-Gómez et al., 2017). In mice, PCP treatments generate abnormal behaviors (Boyce et al., 1991), including hyperactivity, stereotypic behaviors, and motor dysfunction (Castellani and Adams, 1981;Nabeshima et al., 1987). Accordingly, PCP produces schizophrenia-like psychosis, and increases psychotic symptoms in SZ patients (Javitt and Zukin, 1991). Moreover, several studies have reported that PCP induces cFOS protein expression in pontine nuclei and the thalamus, as well as in cortical regions including the retrosplenial, pyriform, cingulate, and frontal cortices. These results suggest a potential relationship between increased cFOS and the psychotic effects of the NMDA receptor antagonists (Abdel-Naby Sayed et al., 2001). The LIS1 complex is a tetramer intracellular enzyme composed of two catalytic subunits, PAFAH-1B3 (ALPHA1) and PAFAH-1B2 (ALPHA2), and a regulatory dimer of PAFAH1B1 (LIS1) (Hanahan, 1986;Kornecki and Ehrlich, 1988;Koltai et al., 1991a,b;Chao and Olson, 1993;Stafforini et al., 2003;Bazan, 2005;Escamez et al., 2012). Lis1 is a gene strongly expressed in the cortical plate during embryogenesis and at postnatal stages in the hippocampus, neocortex and cerebellum (Reiner et al., 1995). Genetic variations in the lissencephalic critical region (17p13.3) around LIS1 gene, have been observed in schizophrenia patients. Previous studies of our group have shown that patients showed a greater tendency toward genetic variations of Lis1 markers (Tabarés-Seisdedos et al., 2006, 2008). Moreover, variations of LIS1 expression have been reported in the dorsolateral prefrontal cortex of patients with schizophrenia (Lipska et al., 2006) and functional alterations of LIS1 protein interactive complex in brain development are relevant to the pathogenesis of schizophrenia and related psychiatric illness (Bradshaw et al., 2011). In this study, we analyzed the effect that deleting the first coding exon of the Lis1 gene has on the distribution of GABAergic interneurons in the young-adult stages of Lis1/sLis1 mutant mice. We found a reduction of GABAergic interneurons in both the hippocampus and Anterior Cingulate area (ACA) in Lis1/sLis1 mutant mice compared to control animals. In addition, the immunohistochemistry of the cFOS protein was analyzed to evaluate neuronal activity in different brain areas. Furthermore, we explored the activation of GABAergic interneurons in Lis1/sLis1 mice. To do this, we analyzed the expression of cFOS in GABAergic interneurons in cortical regions and the hippocampus. Finally, behavioral studies demonstrate the dysfunction of mutant mice in terms of spontaneous locomotion and cognitive tests. Our results suggest that this mutation in the Lis1 gene could cause changes associated with some SZ endophenotypes. MATERIALS AND METHODS Animals All mutant Lis1/sLis1 mice had an ICR genetic background. The Lis1/sLis1 transgenic line has been described previously (Cahana et al., 2001). For genotyping, PCR was performed as described in Cahana et al. (2001), using one set of primers to detect the gene codifying the shorter protein (sLIS1): Mlis1 50-GGT GGC AGT GTT GAG ATG CCT AGC C-30and Mlis1 50-GCA TTC CTG TAA TCC AGT ACC TGG-30(Supplementary Figure 1). The 67-green fluorescent protein (Gad67-Gfp) line was described by Tamamaki et al., 2003. The Gad67-Gfp ×Lis1/sLis1 strain was generated by crossing heterozygous Lis1/sLis1 mice with Gad67-Gfp mice. The animal experiments complied with the Spanish and European Union laws on animal care in experimentation (Council Directive 86/609/EEC). To stage the animals, we took the vaginal plug formation date as embryonic day 0.5 (E0.5). To fix adult brains from postnatal day 30 (P30) or 60 (P60) mice, the animals were anesthetized with isoflurane (Zoetis, United Kingdom), perfused transcardially with phosphatebuffered saline (PBS, pH7.4), followed by 4% paraformaldehyde (PFA) in PBS. The brains were subsequently removed and postfixed overnight at 4◦C in the same fixative. Some animals were selected for P60 behavior tests. An equal number of males and females were used in each experiment. Immunohistochemical Staining The animals were processed for immunohistochemistry staining to quantify the three subtypes of GABAergic interneurons (PV+, CB+, and CR+cells), glutamic acid decarboxylase Frontiers in Cell and Developmental Biology | www.frontiersin.org 2July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 3 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model (GAD67) and cFOS. After perfusion, the brains were removed, postfixed in the same fixative overnight and embedded in 4% agarose in PBS, and transversely sectioned into 70 µmthick sections using a Vibratome (Leica). The brain slices were then rinsed with phosphate buffer solution containing 0.075% Triton X-100 (PBST) and processed for immunohistochemistry staining. For cFOS immunohistochemistry, the brains were soaked in 10, 20, and 30% sucrose solution at 4◦C in PBS, and coronal sections (40-µm thick) were cut using a cryostat (HM525, Microm/Thermo Fisher Scientific, Waltham, MA, United States). The vibratome sections were incubated with 0.09% hydrogen peroxide (H2O2) for 30 min, rinsed with phosphate buffer solution containing 0.075% Triton X-100 (PBST). Next, the tissue was incubated with Goat Serum (GS) (blocking solution) for 1 h to avoid any non-specific antigen binding. The sections were then incubated overnight at 4◦C with the proper primary antibodies diluted in EnVision FLEX Antibody Diluent (DAKO, Denmark). The antibodies used were: anti-green fluorescent protein (GFP) chicken polyclonal antibody (1:500; Aves Labs, Inc.,Tigard, Oregon); anti-calbindin D-28k (CB) rabbit polyclonal antibody (1:2000; Swant, Bellizona, Switzerland); anti-calretinin (CR) rabbit polyclonal antibody (1:2000; Swant, Bellizona, Switzerland); anti-parvalbumin (PV) polyclonal antibody (1:2000; Swant, Bellizona, Switzerland); and anti-cFOS rabbit polyclonal IgG (1:400; Calbiochem). The next day, the sections were rinsed three times at room temperature and incubated with the biotinylated secondary antibody for 1 h. Afterward, the sections were washed with PBS-T and incubated with Avidin–Biotin Complex for 1 h (1:300; ABC kit, Vector Laboratories CA-94010). For the colorimetric detection (black), the tissue was incubated with 1% 3,39Diaminobenzidine (DAB; Vector Laboratories SK-4100), 0.025% ammonium nickel sulfate hexahydrate, and 0.0018% H2O2 in PBS). In some cases, we performed double immunohistochemistry for anti-PV and anti-vesicular glutamate transporter 1 (VGLUT1) polyclonal guinea pig antibody (1:4000; Millipore, Temecula, CA, United States) or anti-cFOS rabbit and anti-GFP. For this, the samples were processed for immunofluorescence staining (free Triton X-100). For this immunostaining, the sections were incubated with the appropriate secondary antibody for 1 h, after being incubated overnight with either antiparvalbumin and anti-VGLUT1 or anti-cFOS and anti-GFP. The secondary antibodies used were: Alexa Fluor 488 donkey anti-rabbit IgG 1:500 (Molecular Probes/Invitrogen#A11055) and Alexa Fluor 488 goat anti-chicken IgG (A11039/Molecular Probes), and donkey anti-guinea pig IgG 1:200 (Biotium, Jackson Inmuno Research) or goat anti-rabbit IgG 1:300 (BA-9200/Vector) followed by Cy3-Streptavidin 1:500 (PA43001/Amersham). Next, the sections were washed with PBS-T RT and the nuclei were counterstained with 40,6-diamidino-2-phenylindole (DAPI, Molecular Probes/Invitrogen) diluted 1:1000 in PBS for 10 min and the sections were then washed in PBS. Finally, the sections were mounted on glass slides using 10:1 Mowiol (Calbiochem)- NPG (Sigma). Microscopy and Statistical Analysis Images were acquired using optical (Leica CTR6000) and confocal (Leica TCS SPE) microscopes, and the images were used to make immunopositive cell counts. To quantify the number of cells, we counted four sections per animal, in a parallel series of sections stained using different markers; we used ImageJ software (NIH, United States) for cell counting. The data used for statistical analysis was the mean of the four sections counted. To quantify VGLUT1+staining on PV-interneurons we selected two sections and five interneurons in each section (see Figure 3 below). On the GFP+channel (PV+neurons) we chose a segment of a dendrite in each selected neuron, and then we measured the area of that segment and the area of red boutons/clusters placed in contact with that dendrite segment. The total area of the dendritic segments selected was the same in WT and mutant sections and we measured the% of dendritic area covered by red stained boutons; these measurements were made with Image-J in a blind manner. We focused on three mPFC subregions: the ACA, the prelimbic cortex (PrL) and infralimbic cortex (IL); we studies also the hippocampal region and the perirhinal (PERI), entorhinal (ENT), ectorhinal (ECT) cortices in both the right and left hemispheres using at least four different sections for each brain area. To identify these regions, the Allen Brain Atlas1was used as a guide. The average of these determinations for each section was defined as the number of immunopositive cells within a specified area in the individual brain. The average of these values among different sections of an individual brain was used for statistical analysis. In all cases, the data are expressed as the mean ±standard error of the mean (SE). Statistical differences between Lis1/sLis1 mice and control littermates were determined using the Student’s t-test. The Mann–Whitney Utest was used to compare differences between two independent groups when the dependent variable was not normally distributed or when their variances were not equal. To perform statistical analysis Sigma Plot software was used. A value of p<0.05 was considered statistically significant. Significance levels were set to: ∗p<0.05, ∗∗p<0.01 and ∗∗∗p<0.001. Behavioral Tests Locomotor Activity For the Open Field Test, the spontaneous activity of the mice was assessed by monitoring their activity in an open field (SMART VIDEO TRACKING Software, Panlab, Barcelona, Spain). The apparatus consisted of a square plexiglass box (50 cm wide ×50 cm long ×40 cm high). The mice (P60) were placed, individually, in the center of the apparatus, and left undisturbed for 10 min. The cage was thoroughly cleaned with 75% ethanol between each test to eliminate any olfactory cues. The total ambulatory distance was analyzed. To record voluntary wheel-running activity, each cage was equipped with a running wheel (Med Associates Inc., Vermont, United States) at P60. The animals had constant voluntary access to the running wheel. The data was recorded with a standard computer using the Wireless Running Wheel Interface System 1http://atlas.brain-map.org/ Frontiers in Cell and Developmental Biology | www.frontiersin.org 3July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 4 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model (Med Associates Inc., Vermont, United States). The number of wheel revolutions each minute were recorded over a 72-h period. From the raw data, the total revolutions, day/night revolutions, and the 12-h revolution average were calculated using Excel 2016 (Microsoft Corporation, Washington, United States). Novel Object Recognition Test The Novel Object Recognition Test (NOR) (n= 12 for each group; P60) was used to assess non-spatial memory in a box (50 cm wide ×50 cm long ×40 cm high). After a 30 min habituation period the previous day, the behavior was recorded using a camera equipped with a computer-assisted data acquisition system (Smart, PANLAB, Spain), in both training and retention sessions. Two novel objects were symmetrically placed 15 cm from the walls during the training session. Each mouse explored for 5 min. Exploration of the objects was defined by the time a mouse spent with its head facing the objects within 1 cm of them, or if it was touching or sniffing them. The objects and box were cleaned with 75% ethanol after the training session. The retention tests were carried out 5 min after the training session, and one of the objects used during training was replaced with a novel object. Each mouse was then allowed to freely explore for 5 min. Exploratory preference was calculated by the ratio of the time spent exploring the novel object over the total time spent exploring the two objects. RESULTS GABAergic Signaling Data published to date has revealed the potential role of cortical GABAergic deficits in the symptoms and pathology observed in SZ patients (Lewis et al., 2012;Nakazawa et al., 2012;Stansfield et al., 2015;Li J.-T. et al., 2016). To study the GABAergic system in Lis1/sLis1 mutant mice, we quantified the number of PV, CR, CB and GAD67-positive neurons in the mPFC and hippocampus (Hi) of mice brains at 30 days (P30). Reduction of Number of Cells Expressing GAD67 Is Observed in the Anterior Cingulate Area and Hippocampus in Lis1/sLis1 Mutant Mice The number of cells expressing GABA-synthesizing enzyme glutamic acid decarboxylase 67 (GAD67), responsible for most GABA synthesis, was studied in Lis1/sLis1 mutant and control mice by using the mutant strain Gad67-Gfp ×Lis1/sLis1. GAD67positive cells were detected through GFP immunohistochemistry at P30. The distribution of GAD67+interneurons was studied in the hippocampus and mPFC (Figure 1). A decrease in the number of interneurons expressing this enzyme was observed in the hippocampus of the Lis1/sLis1 mutant mice (Figures 1A– E). GAD67 reactivity was noted in scattered cells of all the CA1–CA3 layers and, occasionally, in cells of the dentate gyrus (DG) in control mice (Figures 1A,C). The analysis of the Lis1/sLis1 mutant hippocampus showed that the number of cells expressing GAD67 was reduced in the entire hippocampal area (Figures 1B,D;n= 4; controls, 705 ±28; Lis1/sLis1, 623 ±26; p<0.05, Student’s t-test). These differences become more evident in the CA3 area, where a severe reduction of GAD67+cells was observed (Figure 1D). The mPFC is subdivided into three main parts: the anterior cingulate (ACA), prelimbic (PrL), and infralimbic (IL) areas (Tavares and Corrêa, 2006;Vertes, 2006). Although no differences were observed in the number of GAD67positive interneurons in the infralimbic (IL) and prelimbic (PrL) areas, a reduction of the number GAD67+interneurons was detected in the ACA (n= 4; Figures 1F–H;n= 4; controls, 235 ±50; Lis1/sLis1, 176 ±32; p<0.05, Student’s t-test). Fewer PV+Cells in the Anterior Cingulate Area of Lis1/sLis1 Mutant Mice Alterations of PV-expressing interneurons have been implicated in many neuro-psychiatric diseases, including schizophrenia (Flames et al., 2004;Fisahn et al., 2009;Fazzari et al., 2010; Ting et al., 2011;Huang et al., 2021). Accordingly, we analyzed the distribution of PV-positive interneurons in Lis1/sLis1 mutant and control mice in the PrL, IA, and ACA areas (Figure 2). No differences were observed in the number of PV-positive interneurons in the PrL and IL areas (n= 4, average number of PV-positive interneurons ±SE: controls, 63.9 ±7.54; Lis1/sLis1, 68.9 ±3.28). We detected a significant decrease of PV interneurons in the ACA of Lis1/sLis1 mutant mice compared to control animals (n= 4; Figures 2A,B;n= 4; average number of PV+neurons ±SE: controls, 103 ±3,30; Lis1/sLis1, 65 ±8,70; p<0.01, Student’s t-test). That reduction of PV+cells was prominent in layers 2/3 and 5, where they are most abundant, whereas differences in layer six were less marked (Figure 2B). The values shown correspond to the mean ±SE (n= 4; Figure 2E). No significant differences in PV interneurons were observed in the hippocampus (n= 4, average number of PVpositive interneurons ±SEM: controls, 104 ±6.77; Lis1/sLis1, 110 ±6.15). Decreased CR+Cells in the Anterior Cingulate Area of Lis1/sLis1 Mutant Mice As a continuation of the study of GABAergic populations of interneurons, we examined the distribution of CR and CB positive interneurons in the mPFC of control and Lis1/sLis1 mutant mice. In control mice, we observed CR+interneurons distributed throughout the ACA, most frequently being found in layer 2/3, whereas in layers 5 and 6 less CR-positive interneurons were marked (Figures 2C,D). In accordance with our previous results on PV interneurons, we observed no differences in the number of CR-positive interneurons in the PrL and IL areas of the mPFC (n= 4, average number of CR-positive interneurons ±SE: controls, 107 ±4.05; Lis1/sLis1, 98 ±5.46). Nevertheless, a decrease in CR interneurons was observed in ACA in Lis1/sLis1 mutant mice (Figures 2C,D). The values shown correspond to the mean ±SE (n= 4; Figure 2E;n= 4 average number of CR+ neurons ±SE: controls, 134 ±18; Lis1/sLis1, 84 ±4; p<0.01, Student’s t-test). No significant differences were observed for CBpositive interneurons in any of the mPFC regions (n= 4, average number of CR-positive interneurons ±SE: controls, 114 ±8.05; Lis1/sLis1, 112 ±9.70). The analysis of the hippocampus in Lis1/sLis1 mutant mice revealed no differences in CBor CRexpressing interneurons between mutant and control mice (n= 4, Frontiers in Cell and Developmental Biology | www.frontiersin.org 4July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 5 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model FIGURE 1 | Study of GABAergic interneurons in the hippocampus and anterior cingulate area in Gad67-Gfp ×Lis1/sLis1 and Lis1/sLis1 adult mice. Photomicrographs of coronal sections taken through the hippocampal area (A–D) and ACA (F,G), processed using immunoperoxidase staining for GAD67anti-green fluorescent protein (GAD67-GFP) chicken polyclonal antibody. (A–D) There were fewer GFP-positive cells in the hippocampus of Lis1/sLis1 mice compared with adult controls. (E) Histogram indicating the average number of GFP+ cells ±SE in the ACA, PrL/ILA and hippocampus. (F,G) The number of GFP-positive cells in the anterior cingulate area of Lis1/sLis1 mice was decreased compared to adult controls. For all panels scale bar is 200 µm. average number of CR-positive interneurons ±SE: controls, 109 ±8.50; Lis1/sLis1, 105 ±8.72; n= 4, average number of CB-positive interneurons ±SE: controls, 163 ±32.29; Lis1/sLis1, 169 ±17.60). Decreased VGLUT1+Terminals Contacting PV+ Interneurons in the Hippocampus of Lis1/sLis1 Mutant Mice Previous studies have reported decreased excitatory terminals in cortical interneurons in SZ patients (Chung et al., 2016) as well as in mouse models displaying a SZ-like phenotype (Del Pino et al., 2013). The reduced number of PV+interneurons and the lowered excitatory drive to PV+cells (Coyle et al., 2012; Gonzalez-Burgos et al., 2015;Chung et al., 2016) have been proposed as the neural substrate for cognitive dysfunction in SZ. Therefore, we first analyzed the distribution of PV-positive interneurons in the hippocampal areas of Lis1/sLis1 mutant and control mice. As we have described above, no differences were observed in the number of PV-positive interneurons in the hippocampus. Next, we quantified the excitatory synapses contacting PV+interneurons. To do this, we stained the vesicular glutamate transporter 1 (VGLUT1) by immunohistochemistry to detect excitatory presynaptic contacts with PV+interneurons in Lis1/sLis1 mutant mice and in their control littermates. We quantified the area of VGLUT1 boutons contacting PV+ interneurons in the hippocampal region and in the mPFC of Lis1/sLis1 mutant and control mice, using the Image J software. We observed a decrease in the area covered by VGLUT1 boutons in PV+cells in both the CA1-3 and DG regions of Lis1/sLis1 mutants compared to controls (Figures 3A–C;n= 4, average of% of PV+dendrite opposed to VGLUT1 ±SE: controls 21.3 ±2.1%; Lis1/sLis1, 14.6 ±2.6%, p<0.05, Student’s t-test). However, no significant differences were observed in the mPFC (n= 4, average of% of PV+dendrite opposed to VGLUT1 ±SE: controls 19.7% ±2.3%; Lis1/sLis1, 22.9% ±2.6%). These results demonstrate that hippocampal PV interneurons of CA13 and DG receive a reduced number of excitatory synapses in Lis1/sLis1 mutant mice. Frontiers in Cell and Developmental Biology | www.frontiersin.org 5July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 6 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model FIGURE 2 | Study of GABAergic interneurons in the anterior cingulate area in control and Lis1/sLis1 mutant mice in adults. (A–D) Photomicrographs of coronal sections taken through the anterior cingulate area and processed by immunoperoxidase staining for parvalbumin (PV) and calretinin (CR) using single immunostaining. There were fewer PV-positive cells in the anterior cingulate area of Lis1/sLis1 mice than the controls (A,B). A higher magnification of PV+ cells is inserted in the bottom right. CR immunostaining demonstrates a decrease in CR-positive neurons in Lis1/sLis1 mice versus adult control littermates (C,D). (E) Histogram indicating the average ±SE in WT and Lis1/sLis1 mutant mice of PV+ cells, CR+ cells and CB+ cells in the ACA and PrL/IL. For all panels the scale bar is 200 µm. For inserts in (A,B), scale bar is 50 µm. Increased Number of Positive Cells for cFOS in the mPFC, Hippocampus and Other Related Cortical Areas in Lis1/sLis1 Mutant Mice Proteins coded by immediate-early genes such as cFOS, are considered markers of neuronal activity in the central nervous system (Sagar et al., 1988;Herdegen et al., 1995;Kontkanen et al., 2002). Moreover, it has been established that the number of cFOSexpressing neurons increases in animal models of schizophrenia (Celada et al., 2013;Hervig et al., 2016;Calovi et al., 2020). Here, we analyzed the distribution pattern of cFOS immunopositive cells in the hippocampal areas, mPFC, PERI, ECT, and ENT of both control and Lis1/sLis1 mutant mice. To this end, we quantified the number of cells expressing cFOS. We counted cFOS positive cells at P60 in control and Lis1/sLis1 mutant mice that had been exposed to the same basal environmental stimuli in the animal facility (face-to-face cages in the same room). In the hippocampus of control young-adult mice most of the cFOS protein was expressed in the granular cell layer of the DG and the pyramidal cell layer of CA1–CA3. cFOS was also detected, but at lower densities, in the DG molecular layer (hilus), and in the stratum oriens of the CA1–CA3 (Figures 4A,C,E). We detected a significant increase in cFOS immunopositive cells in CA1–CA3 regions (Figures 4B,D), as well as in the dentate gyrus (Figure 4E) in the hippocampus of Lis1/sLis1 mutant mice compared to control mice (n= 4; average number of cFOS+cells in CA ±SE: controls, 359 ±20, n=4,Lis1/sLis1, 498 ±22, n=4;p<0.01, Student’s t-test; average number of cFOS+cells in DG ±SE: controls, 45 ±4, n=4,Lis1/sLis1, 148 ±9, n=4;p<0.001, Student’s t-test). Moreover, we analyzed cells positive for cFOS in both the suprapyramidal (DGsp) and infrapyramidal (DGip) blades of the DG. In control mice, cFOS positive cells were mainly observed in the DGsp, whereas fewer cFOS positive cells were seen in the DGip (Figure 4E), suggesting a different expression pattern of cFOS protein in the two DG blades. Strikingly, this difference in neuronal activity between DGsp and DGip was not observed in the DG of Lis1/sLis1 mice. The DGip of the DG in Lis1/sLis1 mutant mice there was a greater number of cells expressing cFOS compared to the control mice (Figures 4E,F,I). Subsequently, we analyzed cFOS-positive cells in the mPFC and observed that this area also presented more cFOS-positive cells in Lis1/sLis1 mice than the control animals (Figure 4G–I; Frontiers in Cell and Developmental Biology | www.frontiersin.org 6July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 7 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model FIGURE 3 | VGLUT1+ boutons next to PV+ interneurons in the hippocampal area (all CA1-3 and DG) in control and Lis1/sLis1 mutant mice. (A,B) PV+ interneurons displayed in green and VGLUT1 boutons in red in control (A) and Lis1/sLis1 mutant mice (B).(C) The histogram shows the percentage cover of hippocampal and prefrontal PV+ cells with VGLUT1 boutons in Lis1/sLis1 and control mice. The % of PV+ dendrite opposed to VGLUT1 in the hippocampus is reduced in Lis1/sLis1 mutant mice compared to controls. Scale bar 5 µmin(A,B). n= 4; average number of cFOS+cells in mPFC ±SE: controls, 322 ±36, n=4,Lis1/sLis1, 460 ±37, n=4;p<0.001, Student’s t-test). In addition, we analyzed the neuronal activity of GAD67+ interneurons. To this end, we quantified the number of cells coexpressing cFOS and GAD67 at P60 in control and Lis1/sLis1 mutant mice (Figure 5). We analyzed the number of GAD67+ interneurons that were immunopositive for cFOS in both the mPFC and hippocampal areas. We detected fewer cells coexpressing cFOS and GAD67 with respect to the total number of cFOS positive cells, in the mPFC area of Lis1/sLis1 mutant mice compared to control mice (Figures 5A–D;n= 4; average number of% cFOS-GAD67/cFOS cells ±SE: controls, 8.95 ±0.45; Lis1/sLis1, 3.48 ±1.28, p<0.001, Student’s t-test). No significant differences were observed in the number of cells co-expressing cFOS and GAD67 when the hippocampus was analyzed (n = 4, average number of% cFOS-GAD67/cFOS cells ±SE: controls, 5.56 ±1.79; Lis1/sLis1, 6.56 ±1.11). However, when the number of cells co-expressing cFOS and GAD67 with respect to the total number of GAD67 positive cells was studied in the mPFC area of Lis1/sLis1 mutant mice compared to control mice, no significant differences were detected suggesting that interneuron excitability remains intact. Hippocampal functions such as episodic memory and spatial orientation, are associated with other brain areas like the perirhinal (PERI), entorhinal (ENT), and temporal associative cortices, as well as the prefrontal cortex (Aggleton and Brown, 1999). Moreover, ENT axons are the most significant cortical input to the hippocampus (Warburton and Brown, 2010). We therefore decided to analyze whether the functional activity of these areas is affected in Lis1/sLis1 mutant mice. To this end, cellular activity in the PERI, ECT, and ENT Frontiers in Cell and Developmental Biology | www.frontiersin.org 7July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 8 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model FIGURE 4 | Cells expressing cFOS in the hippocampus and PFC in Lis1/sLis1 mutant mice. (A–H) Coronal sections are immunostained with anti-cFOS antibody in the hippocampus (A–F) and prefrontal cortex (G,H) at P60. There was an increase in cFOS+ cells in the CA (A–D) and DG (E,F) regions of the hippocampus in Lis1/sLis1 mutant mice. Moreover, Lis1/sLis1 PFC also showed an increased number of cells expressing cFOS (G,H).(I) The histogram represents the statistically significant increase in cFOS-positive cells in the areas analyzed (DG, CA, and PFC). For all panels scale bar is 200 µm. FIGURE 5 | Study of the number of co-expressing cFOS and GAD67 cells at P60 in the mPFC of control and Lis1/sLis1 mutant mice. (A,B) The number of cells co-expressing cFOS and GAD67 in the mPFC area of Lis1/sLis1 mutant mice was decreased compared to control mice. (C) The image was taken from the Allen Brain Atlasshowing the approximate area of panels (A,B), where the cells were counted. (D) Histogram indicating the average number of co-expressing cFOS and GAD67 cells ±SE in the young-adult mPFC and hippocampus. Scale bar is 200 µm. Frontiers in Cell and Developmental Biology | www.frontiersin.org 8July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 9 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model FIGURE 6 | Analysis of cells expressing cFOS in other hippocampus-related cortical regions. (A,B) The entorhinal, perirhinal, and ectorhinal cortices were assessed. The number of cells positive for cFOS was quantified in these areas and the perirhinal and entorhinal areas of the Lis1/sLis1 mutant mice presented a larger number of cells expressing cFOS (B) than the controls (A). No significant differences in the ectorhinal cortex were detected between the Lis1/sLis1 mutant mice and controls. (C) The image was taken from the Allen Brain Atlasshowing the approximate area of panels (A,B).(D) The histogram shows these results in the areas analyzed (ECT; PERI, and ENT). Scale bar is 200 µm. cortices was also analyzed through cFOS immunohistochemistry (Figure 6). We observed an increment of cFOS+cells in PERI and ENT in Lis1/sLis1 mutant mice compared to the controls (n= 4; Figures 6A–D average number of cFOS+ cells in the perirhinal area ±SE: controls, 108 ±8, n=4, Lis1/sLis1, 154 ±11, n=4;p<0.01, Student’s t-test; average number of cFOS+cells in the entorhinal area ±SE: controls, 91 ±10, n=4,Lis1/sLis1, 140 ±11, n=4;p<0.01, Student’s t-test). In contrast, no differences were observed when ECT was analyzed (Figures 6A–D;n= 4, average number of cFOS+cells in the ectorhinal area ±SE: controls, 129 ±11; Lis1/sLis1, 148 ±6). Our results show a significant increase in the number of cells expressing the cFOS protein in certain areas of the Lis1/sLis1 brain, which mimic those described in postmortem brains of SZ patients and schizophrenia-like phenotype mouse models (Dragunow and Faull, 1990;Abdel-Naby Sayed et al., 2001; Keilhoff et al., 2004). Behavioral Alterations in Lis1/sLis1 Mutant Mice To analyze the functional effects of the observed cortical alterations, behavioral analyses were performed. Firstly, ambulatory locomotor activity was assessed through the Open Field Test. The total distance traveled by the Lis1/sLis1 group was not significantly different to the control group (n= 12 for each group; Figure 7A). Sleep disorders and circadian rhythm alterations have been described in SZ patients (Wulff et al., 2011). Furthermore, studies in animal models have suggested that schizophrenia-like phenotype mouse models display circadian rhythm disruption (Tam et al., 2015). We therefore studied spontaneous locomotor activity by means of a Running Wheel Analysis over a 72h period. This test analyzes both circadian rhythms and locomotor activity. We observed decreased voluntary activity in the Lis1/sLis1 mutant mice compared to the controls. Mutant mice made significantly fewer rotations than controls when considering a 12 h average (Figure 7B;n= 12 for each group, average number of revolutions/12 h ±SE: controls, 8909 ±737, n=12,Lis1/sLis1, 6213 ±949; p<0.05, Student’s t-test). We then wanted to study whether these differences were due to a general decrease in activity and if there were differences between daytime and nighttime activity. Firstly, we analyzed the restactivity rhythms in the control and mutant mice, respectively. Our results showed that both the controls and mutants presented statistically significant differences between their night vs. day activity (n= 12; p<0.01 for control mice and p<0.001 for Lis1/sLis1 mutant mice). As expected, there was an increase in spontaneous locomotor activity at night in both control and mutant mice (Figure 7C). Next, we analyzed potential Frontiers in Cell and Developmental Biology | www.frontiersin.org 9July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 16 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model Heckers, S., and Konradi, C. (2002). Hippocampal neurons in schizophrenia. J. Neural Transm. 109, 891–905. doi: 10.1007/s007020200073 Heckers, S., Stone, D., Walsh, J., Shick, J., Koul, P., and Benes, F. M. (2002). Differential hippocampal expression of glutamic acid decarboxylase 65 and 67 messenger RNA in bipolar disorder and schizophrenia. Arch. Gen. Psychiatry 59, 521–529. doi: 10.1001/archpsyc.59.6.521 Heidbreder, C. A., and Groenewegen, H. J. (2003). The medial prefrontal cortex in the rat: evidence for a dorso-ventral distinction based upon functional and anatomical characteristics. Neurosci. Biobehav. Rev. 27, 555–579. doi: 10.1016/ j.neubiorev.2003.09.003 Herdegen, T., Kovary, K., Buhl, A., Bravo, R., Zimmermann, M., and Gass, P. (1995). Basal expression of the inducible transcription factors c-Jun, JunB, JunD, c-Fos, FosB, and Krox-24 in the adult rat brain. J. Comp. Neurol. 354, 39–56. doi: 10.1002/cne.903540105 Hervig, M. E., Thomsen, M. S., Kalló, I., and Mikkelsen, J. D. (2016). Acute phencyclidine administration induces c-Fos-immunoreactivity in interneurons in cortical and subcortical regions. Neuroscience 334, 13–25. doi: 10.1016/j. neuroscience.2016.07.028 Hoftman, G. D., and Lewis, D. A. (2011). Postnatal developmental trajectories of neural circuits in the primate prefrontal cortex: identifying sensitive periods for vulnerability to schizophrenia. Schizophr. Bull. 37, 493–503. doi: 10.1093/ schbul/sbr029 Hoftman, G. D., Volk, D. W., Bazmi, H. H., Li, S., Sampson, A. R., and Lewis, D. A. (2015). Altered cortical expression of GABA-related genes in schizophrenia: illness progression vs developmental disturbance. Schizophr. Bull. 41, 180–191. doi: 10.1093/schbul/sbt178 Huang, Y., Jiang, H., Zheng, Q., Fok, A. H. K., Li, X., Lau, C. G., et al. (2021). Environmental enrichment or selective activation of parvalbumin-expressing interneurons ameliorates synaptic and behavioral deficits in animal models with schizophrenia-like behaviors during adolescence. Mol. Psychiatry. doi: 10.1038/ s41380-020-01005-w [Epub ahead of print]. Insel, T. R. (2010). Rethinking schizophrenia. Nature 468, 187–193. doi: 10.1038/ nature09552 Javitt, D. C., and Zukin, S. R. (1991). Recent advances in the phencyclidine model of schizophrenia. Am. J. Psychiatry 148, 1301–1308. doi: 10.1176/ajp.148.10.1301 Jeans, A. F., Oliver, P. L., Johnson, R., Capogna, M., Vikman, J., Molnar, Z., et al. (2007). A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse. Proc. Natl. Acad. Sci. U.S.A. 104, 2431–2436. doi: 10.1073/pnas.0610222104 Jentsch, J. D., and Roth, R. H. (1999). The neuropsychopharmacology of phencyclidine: from NMDA receptor hypofunction to the dopamine hypothesis of schizophrenia. Neuropsychopharmacology 20, 201–225. doi: 10.1016/s0893133x(98)00060-8 Jinno, S. (2011). Topographic differences in adult neurogenesis in the mouse hippocampus: a stereology-based study using endogenous markers. Hippocampus 21, 467–480. doi: 10.1002/hipo.20762 Kargieman, L., Santana, N., Mengod, G., Celada, P., and Artigas, F. (2007). Antipsychotic drugs reverse the disruption in prefrontal cortex function produced by NMDA receptor blockade with phencyclidine. Proc. Natl. Acad. Sci. U.S.A. 104, 14843–14848. doi: 10.1073/pnas.0704848104 Kato, M., and Dobyns, W. B. (2003). Lissencephaly and the molecular basis of neuronal migration. Hum. Mol. Genet. 12 Spec No 1, R89–R96. Keilhoff, G., Becker, A., Grecksch, G., Wolf, G., and Bernstein, H.-G. (2004). Repeated application of ketamine to rats induces changes in the hippocampal expression of parvalbumin, neuronal nitric oxide synthase and cFOS similar to those found in human schizophrenia. Neuroscience 126, 591–598. doi: 10.1016/ j.neuroscience.2004.03.039 Kendler, K. S., MacLean, C. J., O’Neill, F. A., Burke, J., Murphy, B., Duke, F., et al. (1996). Evidence for a schizophrenia vulnerability locus on chromosome 8p in the Irish Study of High-Density Schizophrenia Families. Am. J. Psychiatry 153, 1534–1540. doi: 10.1176/ajp.153.12.1534 Kim, T.-W., Kang, H.-S., Park, J.-K., Lee, S.-J., Baek, S.-B., and Kim, C.-J. (2014). Voluntary wheel running ameliorates symptoms of MK-801-induced schizophrenia in mice. Mol. Med. Rep. 10, 2924–2930. doi: 10.3892/mmr.2014. 2644 Koltai, M., Hosford, D., Guinot, P., Esanu, A., and Braquet, P. (1991a). PAF. A review of its effects, antagonists and possible future clinical implications (Part II). Drugs 42, 174–204. doi: 10.2165/00003495-199142020-00002 Koltai, M., Hosford, D., Guinot, P., Esanu, A., and Braquet, P. (1991b). Platelet activating factor (PAF). A review of its effects, antagonists and possible future clinical implications (Part I). Drugs 42, 9–29. doi: 10.2165/00003495199142010-00002 Kontkanen, O., Lakso, M., Wong, G., and Castrén, E. (2002). Chronic antipsychotic drug treatment induces long-lasting expression of fos and jun family genes and activator protein 1 complex in the rat prefrontal cortex. Neuropsychopharmacology 27, 152–162. doi: 10.1016/s0893-133x(02)00289-0 Kornecki, E., and Ehrlich, Y. H. (1988). Neuroregulatory and neuropathological actions of the ether-phospholipid platelet-activating factor. Science 240, 1792– 1794. doi: 10.1126/science.3381103 Koyanagi, A., and Stickley, A. (2015). The association between sleep problems and psychotic symptoms in the general population: a global perspective. Sleep 38, 1875–1885. doi: 10.5665/sleep.5232 Lee, Y. J., Cho, S.-J., Cho, I. H., Jang, J. H., and Kim, S. J. (2012). The relationship between psychotic-like experiences and sleep disturbances in adolescents. Sleep Med. 13, 1021–1027. doi: 10.1016/j.sleep.2012.06.002 Lewis, D. A., Curley, A. A., Glausier, J. R., and Volk, D. W. (2012). Cortical parvalbumin interneurons and cognitive dysfunction in schizophrenia. Trends Neurosci. 35, 57–67. doi: 10.1016/j.tins.2011.10.004 Li, C., Tang, Y., Yang, J., Zhang, X., Liu, Y., and Tang, A. (2016). Sub-chronic Antipsychotic Drug Administration Reverses the Expression of Neuregulin 1 and ErbB4 in a Cultured MK801-Induced Mouse Primary Hippocampal Neuron or a Neurodevelopmental Schizophrenia Model. Neurochem. Res. 41, 2049–2064. doi: 10.1007/s11064-016-1917-x Li, J.-T., Su, Y.-A., Wang, H.-L., Zhao, Y.-Y., Liao, X.-M., Wang, X.-D., et al. (2016). Repeated Blockade of NMDA receptors during adolescence impairs reversal learning and disrupts GABAergic interneurons in rat medial prefrontal cortex. Front. Mol. Neurosci. 9:17. doi: 10.3389/fnmol.2016.00017 Lipska, B. K., Peters, T., Hyde, T. M., Halim, N., Horowitz, C., Mitkus, S., et al. (2006). Expression of DISC1 binding partners is reduced in schizophrenia and associated with DISC1 SNPs. Hum. Mol. Genet. 15, 1245–1258. doi: 10.1093/ hmg/ddl040 Lipska, B. K., and Weinberger, D. R. (2000). To model a psychiatric disorder in animals: schizophrenia as a reality test. Neuropsychopharmacology 23, 223–239. doi: 10.1016/s0893-133x(00)00137-8 Loring, J. F., Paszty, C., Rose, A., McIntosh, T. K., Murai, H., Pierce, J. E., et al. (1996). Rational design of an animal model for Alzheimer’s disease: introduction of multiple human genomic transgenes to reproduce AD pathology in a rodent. Neurobiol. Aging 17, 173–182. doi: 10.1016/01974580(95)02076-4 Lyon, L., Saksida, L. M., and Bussey, T. J. (2012). Spontaneous object recognition and its relevance to schizophrenia: a review of findings from pharmacological, genetic, lesion and developmental rodent models. Psychopharmacology 220, 647–672. doi: 10.1007/s00213-011-2536-5 Marín, O., and Rubenstein, J. L. R. (2003). Cell migration in the forebrain. Annu. Rev. Neurosci. 26, 441–483. Marrone, D. F., Ramirez-Amaya, V., and Barnes, C. A. (2012). Neurons generated in senescence maintain capacity for functional integration. Hippocampus 22, 1134–1142. doi: 10.1002/hipo.20959 Moghaddam, B., Adams, B., Verma, A., and Daly, D. (1997). Activation of glutamatergic neurotransmission by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex. J. Neurosci. 17, 2921–2927. doi: 10.1523/jneurosci. 17-08-02921.1997 Nabeshima, T., Fukaya, H., Yamaguchi, K., Ishikawa, K., Furukawa, H., and Kameyama, T. (1987). Development of tolerance and supersensitivity to phencyclidine in rats after repeated administration of phencyclidine. Eur. J. Pharmacol. 135, 23–33. doi: 10.1016/0014-2999(87)90753-9 Nakazawa, K., Zsiros, V., Jiang, Z., Nakao, K., Kolata, S., Zhang, S., et al. (2012). GABAergic interneuron origin of schizophrenia pathophysiology. Neuropharmacology 62, 1574–1583. doi: 10.1016/j.neuropharm.2011.01.022 Näkki, R., Sharp, F. R., Sagar, S. M., and Honkaniemi, J. (1996). Effects of phencyclidine on immediate early gene expression in the brain. J. Neurosci. Res. 45, 13–27. doi: 10.1002/(sici)1097-4547(19960701)45:1<13::aid-jnr2>3. 0.co;2-k Oh, H. Y., Singh, F., Koyanagi, A., Jameson, N., Schiffman, J., and DeVylder, J. (2016). Sleep disturbances are associated with psychotic experiences: findings Frontiers in Cell and Developmental Biology | www.frontiersin.org 16 July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 17 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model from the national comorbidity survey replication. Schizophr. Res. 171, 74–78. doi: 10.1016/j.schres.2016.01.018 Oliver, P. L., and Davies, K. E. (2009). Interaction between environmental and genetic factors modulates schizophrenic endophenotypes in the Snap-25 mouse mutant blind-drunk. Hum. Mol. Genet. 18, 4576–4589. doi: 10.1093/hmg/ ddp425 Oliver, P. L., Sobczyk, M. V., Maywood, E. S., Edwards, B., Lee, S., Livieratos, A., et al. (2012). Disrupted circadian rhythms in a mouse model of schizophrenia. Curr. Biol. 22, 314–319. doi: 10.1016/j.cub.2011.12.051 Ozdemir, H., Ertugrul, A., Basar, K., and Saka, E. (2012). Corrigendum to “Differential Effects of Antipsychotics on Hippocampal Presynaptic Protein Expressions and Recognition Memory in a Schizophrenia Model in Mice” [Prog Neuropsychopharmacol Biol Psychiatry 39 (2012) 1–218]. Prog. Neuropsychopharmacol. Biol. Psychiatry 39:388. doi: 10.1016/j.pnpbp.2012. 08.006 Paus, T. (2001). Primate anterior cingulate cortex: Where motor control, drive and cognition interface. Nat. Rev. Neurosci. 2, 417–424. doi: 10.1038/35077500 Pei, J.-C., Liu, C.-M., and Lai, W.-S. (2014). Distinct phenotypes of new transmembrane-domain neuregulin 1 mutant mice and the rescue effects of valproate on the observed schizophrenia-related cognitive deficits. Front. Behav. Neurosci. 8:126. doi: 10.3389/fnbeh.2014.00126 Picard, N., and Strick, P. L. (1996). Motor areas of the medial wall: a review of their location and functional activation. Cereb. Cortex 6, 342–353. doi: 10.1093/ cercor/6.3.342 Pietersen, C. Y., Bosker, F. J., Doorduin, J., Jongsma, M. E., Postema, F., Haas, J. V., et al. (2007). An animal model of emotional blunting in schizophrenia. PLoS One 2:e1360. doi: 10.1371/journal.pone.0001360 Piggins, H. (2003). The roles of vasoactive intestinal polypeptide in the mammalian circadian clock. J. Endocrinol. 177, 7–15. doi: 10.1677/joe.0.1770007 Pilz, D. T., Macha, M. E., Precht, K. S., Smith, A. C., Dobyns, W. B., and Ledbetter, D. H. (1998). Fluorescence in situ hybridization analysis with LIS1 specific probes reveals a high deletion mutation rate in isolated lissencephaly sequence. Genet. Med. 1, 29–33. doi: 10.1097/00125817-199811000-00007 Post, R. M. (1992). Transduction of psychosocial stress into the neurobiology of recurrent affective disorder. Am. J. Psychiatry 149, 999–1010. doi: 10.1176/ajp. 149.8.999 Ramirez-Amaya, V., Marrone, D. F., Gage, F. H., Worley, P. F., and Barnes, C. A. (2006). Integration of new neurons into functional neural networks. J. Neurosci. 26, 12237–12241. doi: 10.1523/jneurosci.2195-06.2006 Reiner, O. (2000). LIS1. Neuron 28, 633–636. doi: 10.1016/s0896-6273(00)00142-2 Reiner, O., Albrecht, U., Gordon, M., Chianese, K. A., Wong, C., Gal-Gerber, O., et al. (1995). Lissencephaly gene (LIS1) expression in the CNS suggests a role in neuronal migration. J. Neurosci. 15, 3730–3738. doi: 10.1523/jneurosci.15-0503730.1995 Reiner, O., Cahana, A., Escamez, T., and Martinez, S. (2002). LIS1-no more no less. Mol. Psychiatry 7, 12–16. doi: 10.1038/sj.mp.4000975 Reynolds, G. P., Abdul-Monim, Z., Neill, J. C., and Zhang, Z.-J. (2004). Calcium binding protein markers of GABA deficits in schizophrenia — post mortem studies and animal models. Neurotox. Res. 6, 57–61. doi: 10.1007/bf03033297 Ridderinkhof, K. R., Ullsperger, M., Crone, E. A., and Nieuwenhuis, S. (2004). The role of the medial frontal cortex in cognitive control. Science 306, 443–447. doi: 10.1126/science.1100301 Ross, C. A., Margolis, R. L., Reading, S. A. J., Pletnikov, M., and Coyle, J. T. (2006). Neurobiology of schizophrenia. Neuron 52, 139–153. Rushworth, M. F. S., Buckley, M. J., Behrens, T. E. J., Walton, M. E., and Bannerman, D. M. (2007). Functional organization of the medial frontal cortex. Curr. Opin. Neurobiol. 17, 220–227. doi: 10.1016/j.conb.2007.03.001 Sagar, S. M., Sharp, F. R., and Curran, T. (1988). Expression of c-fos protein in brain: metabolic mapping at the cellular level. Science 240, 1328–1331. doi: 10.1126/science.3131879 Santana, N., Troyano-Rodriguez, E., Mengod, G., Celada, P., and Artigas, F. (2011). Activation of thalamocortical networks by the N-methyl-D-aspartate receptor antagonist phencyclidine: reversal by clozapine. Biol. Psychiatry 69, 918–927. doi: 10.1016/j.biopsych.2010.10.030 Sato, D., Umino, A., Kaneda, K., Takigawa, M., and Nishikawa, T. (1997). Developmental changes in distribution patterns of phencyclidine-induced c-Fos in rat forebrain. Neurosci. Lett. 239, 21–24. doi: 10.1016/s0304-3940(97) 00879-3 Satvat, E., Gheidi, A., Voll, S., Odintsova, I. V., and Marrone, D. F. (2012). Location is everything: neurons born during fluoxetine treatment accumulate in regions that do not support spatial learning. Neuropharmacology 62, 1627–1633. doi: 10.1016/j.neuropharm.2011.11.025 Scharfman, H. E., Sollas, A. L., Smith, K. L., Jackson, M. B., and Goodman, J. H. (2002). Structural and functional asymmetry in the normal and epileptic rat dentate gyrus. J. Comp. Neurol. 454, 424–439. doi: 10.1002/cne.10449 Schmidt, B., Marrone, D. F., and Markus, E. J. (2012). Disambiguating the similar: the dentate gyrus and pattern separation. Behav. Brain Res. 226, 56–65. doi: 10.1016/j.bbr.2011.08.039 Sharp, F. R., Jasper, P., Hall, J., Noble, L., and Sagar, S. M. (1991). MK-801 and ketamine induce heat shock protein HSP72 in injured neurons in posterior cingulate and retrosplenial cortex. Ann. Neurol. 30, 801–809. doi: 10.1002/ana. 410300609 Stafforini, D. M., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (2003). Platelet-activating factor, a pleiotrophic mediator of physiological and pathological processes. Crit. Rev. Clin. Lab. Sci. 40, 643–672. doi: 10.1080/ 714037693 Stansfield, K. H., Ruby, K. N., Soares, B. D., McGlothan, J. L., Liu, X., and Guilarte, T. R. (2015). Early-life lead exposure recapitulates the selective loss of parvalbumin-positive GABAergic interneurons and subcortical dopamine system hyperactivity present in schizophrenia. Transl. Psychiatry 5:e522. doi: 10.1038/tp.2014.147 Stefansson, H., Steinthorsdottir, V., Thorgeirsson, T. E., Gulcher, J. R., and Stefansson, K. (2004). Neuregulin 1 and schizophrenia. Ann. Med. 36, 62–71. Sullivan, E. M., and O’Donnell, P. (2012). Inhibitory interneurons, oxidative stress, and schizophrenia. Schizophr. Bull. 38, 373–376. doi: 10.1093/schbul/s bs052 Tabarés-Seisdedos, R., Escámez, T., Martínez-Giménez, J. A., Balanzá, V., Salazar, J., Selva, G., et al. (2006). Variations in genes regulating neuronal migration predict reduced prefrontal cognition in schizophrenia and bipolar subjects from mediterranean Spain: a preliminary study. Neuroscience 139, 1289–1300. doi: 10.1016/j.neuroscience.2006.01.054 Tabarés-Seisdedos, R., Mata, I., Escámez, T., Vieta, E., López-Ilundain, J. M., Salazar, J., et al. (2008). Evidence for association between structural variants in lissencephaly-related genes and executive deficits in schizophrenia or bipolar patients from a Spanish isolate population. Psychiatr. Genet. 18, 313–317. doi: 10.1097/ypg.0b013e3283118725 Tam, S. K. E., Pritchett, D., Brown, L. A., Foster, R. G., Bannerman, D. M., and Peirson, S. N. (2015). Sleep and circadian rhythm disruption and recognition memory in schizophrenia. Methods Enzymol. 552, 325–349. doi: 10.1016/bs. mie.2014.10.008 Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J.-I., Obata, K., and Kaneko, T. (2003). Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol. 467, 60–79. doi: 10.1002/cne.10905 Tavares, R. F., and Corrêa, F. M. A. (2006). Role of the medial prefrontal cortex in cardiovascular responses to acute restraint in rats. Neuroscience 143, 231–240. doi: 10.1016/j.neuroscience.2006.07.030 Thoma, P., Zoppelt, D., Wiebel, B., and Daum, I. (2006). Recollection and familiarity in negative schizophrenia. Neuropsychologia 44, 430–435. doi: 10. 1016/j.neuropsychologia.2005.05.017 Thomases, D. R., Cass, D. K., and Tseng, K. Y. (2013). Periadolescent exposure to the NMDA receptor antagonist MK-801 impairs the functional maturation of local GABAergic circuits in the adult prefrontal cortex. J. Neurosci. 33, 26–34. doi: 10.1523/jneurosci.4147-12.2013 Thomsen, M. S., Hansen, H. H., and Mikkelsen, J. D. (2010). Opposite effect of phencyclidine on activity-regulated cytoskeleton-associated protein (Arc) in juvenile and adult limbic rat brain regions. Neurochem. Int. 56, 270–275. doi: 10.1016/j.neuint.2009.10.011 Ting, A. K., Chen, Y., Wen, L., Yin, D.-M., Shen, C., Tao, Y., et al. (2011). Neuregulin 1 promotes excitatory synapse development and function in GABAergic interneurons. J. Neurosci. 31, 15–25. doi: 10.1523/jneurosci.253810.2011 Valdés-Sánchez, L., Escámez, T., Echevarria, D., Ballesta, J. J., Tabarés-Seisdedos, R., Reiner, O., et al. (2007). Postnatal alterations of the inhibitory synaptic responses recorded from cortical pyramidal neurons in the Lis1/sLis1 mutant mouse. Mol. Cell. Neurosci. 35, 220–229. doi: 10.1016/j.mcn.2007.02.017 Frontiers in Cell and Developmental Biology | www.frontiersin.org 17 July 2021 | Volume 9 | Article 693919 fcell-09-693919 July 8, 2021 Time: 17:35 # 18 Garcia-Lopez et al. Lis1/sLis1 a Schizophrenia Mouse Model Vertes, R. P. (2006). Interactions among the medial prefrontal cortex, hippocampus and midline thalamus in emotional and cognitive processing in the rat. Neuroscience 142, 1–20. doi: 10.1016/j.neuroscience.2006.06.027 Vogt, B. A. (2005). Pain and emotion interactions in subregions of the cingulate gyrus. Nat. Rev. Neurosci. 6, 533–544. doi: 10.1038/nrn1704 Wakuda, T., Iwata, K., Iwata, Y., Anitha, A., Takahashi, T., Yamada, K., et al. (2015). Perinatal asphyxia alters neuregulin-1 and COMT gene expression in the medial prefrontal cortex in rats. Prog. Neuropsychopharmacol. Biol. Psychiatry 56, 149–154. doi: 10.1016/j.pnpbp.2014.08.002 Wang, Y., and Baraban, S. C. (2008). Aberrant dentate gyrus cytoarchitecture and fiber lamination in Lis1 mutant mice. Hippocampus 18, 758–765. doi: 10.1002/hipo.20434 Warburton, E. C., and Brown, M. W. (2010). Findings from animals concerning when interactions between perirhinal cortex, hippocampus and medial prefrontal cortex are necessary for recognition memory. Neuropsychologia 48, 2262–2272. doi: 10.1016/j.neuropsychologia.2009.12.022 Williams, N. M., Preece, A., Spurlock, G., Norton, N., Williams, H. J., Zammit, S., et al. (2003). Support for genetic variation in neuregulin 1 and susceptibility to schizophrenia. Mol. Psychiatry 8, 485–487. doi: 10.1038/sj.mp.4001348 Winters, B. D. (2005). Glutamate receptors in perirhinal cortex mediate encoding, retrieval, and consolidation of object recognition memory. J. Neurosci. 25, 4243–4251. doi: 10.1523/jneurosci.0480-05.2005 Woo, T.-U. W., Kim, A. M., and Viscidi, E. (2008). Disease-specific alterations in glutamatergic neurotransmission on inhibitory interneurons in the prefrontal cortex in schizophrenia. Brain Res. 1218, 267–277. doi: 10.1016/j.brainres.2008. 03.092 Wulff, K., Dijk, D.-J., Middleton, B., Foster, R. G., and Joyce, E. M. (2011). Sleep and circadian rhythm disruption in schizophrenia. Br. J. Psychiatry 200, 308–316. doi: 10.1192/bjp.bp.111.096321 Wynshaw-Boris, A. (2007). Lissencephaly and LIS1: insights into the molecular mechanisms of neuronal migration and development. Clin. Genet. 72, 296–304. doi: 10.1111/j.1399-0004.2007.00888.x Yang, K., Trepanier, C. H., Li, H., Beazely, M. A., Lerner, E. A., Jackson, M. F., et al. (2009). Vasoactive intestinal peptide acts via multiple signal pathways to regulate hippocampal NMDA receptors and synaptic transmission. Hippocampus 19, 779–789. doi: 10.1002/hipo. 20559 Zhang, Z. J., and Reynolds, G. P. (2002). A selective decrease in the relative density of parvalbumin-immunoreactive neurons in the hippocampus in schizophrenia. Schizophr. Res. 55, 1–10. doi: 10.1016/s0920-9964(01)0 0188-8 Zola-Morgan, S., Squire, L. R., Alvarez-Royo, P., and Clower, R. P. (1991). Independence of memory functions and emotional behavior: separate contributions of the hippocampal formation and the amygdala. Hippocampus 1, 207–220. doi: 10.1002/hipo.45001 0208 Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2021 Garcia-Lopez, Pombero, Estirado, Geijo-Barrientos and Martinez. 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