3.3.3. Schizophrenia and the Heterozygous *Reeler* Mouse

Schizophrenia is a ruinous psychiatric condition that affects about 1% of the population. Its main clinical symptoms are hallucinations, delusions, and cognitive disturbances. These symptoms derive from brain dysfunctions that derive from genetic and environmental factors [214]. However, schizophrenia is not strictly a genetic disease, although gene deletions, duplications, and variations may be risk factors for the disorder. At present, the gene(s) that could be involved in the pathology remain elusive for the most (see OMIM #181500), but a microdeletion in a region of chromosome 22, called 22q11, was recently established to be involved in a small percentage of cases [215].

Genetic studies have shown a link between *RELN* and schizophrenia [216] and, over the past ten years, many SNPs in the *RELN* gene loci occurred in parallel with the beginning and/or severity of the clinical signs [217], but results still are under debate and need further verification [218]. One should perhaps emphasize that observations on gene expression have converged to show that the genes implicated in schizophrenia are more highly expressed during fetal than postnatal life [219], thus making more difficult to ascertain their true role in the etiology of the condition.

#### Imaging

Structural MRI findings in schizophrenia have been recently reviewed [220]. There is enough information to propose that the condition is associated with a continuing development of gray matter aberrations, chiefly throughout the first stages of the disease. Reduction of the depth of the cerebral cortex in the superior temporal and inferior frontal regions was reported in individuals that later became psychotic. In patients with first episode psychosis, there was, instead, a reduction in the thickness of the superior and inferior frontal cortex, and in the volume of the thalamus. In chronic schizophrenia, the gray matter decreased further in the frontal and temporal areas, cingulate cortices, and thalamus, particularly in patients with unfortunate outcomes. Structural modifications of the white matter occurred only in a small number of longitudinal studies.

As the human phenotype is still very far from clear, it is not surprising that the few MRI observations in *Reeler* mice are still insufficient to draw any definitive consideration of translational relevance (for MRI data on Reeler see 3.3.2 Imaging).

#### Histopathology

Although gross structural alterations of the brain were lacking, subtle pathological changes in specific populations of neurons and in cell-to-cell communication occurred in schizophrenic patients, see [221] for a recent review. Histopathology mainly consisted in modifications of the number and density of neurons at the level of the whole brain and/or specific neuronal subpopulations, and in morphological and neurochemical alterations of these neurons.

#### a) Cerebral Cortex

As discussed above for autism, the most widely investigated area of the brain has been the PFC that, in general terms, displayed an increased neuronal density and an altered neuroplasticity with age-related differences between normal and schizophrenic subjects. More specifically, a statistical meta-analysis of thirty papers published between 1993 and 2012, concluded that the density of cortical neurons increased with age irrespective of the condition, but the rate of accretion was much slower in the schizophrenics [222]. However, other cortical areas, such as e.g., the dorsal ACC, displayed no changes in neuronal and glial densities after stereological analysis [223].

The above-mentioned meta-analysis [222] has also taken into consideration the density of inhibitory neurons after immunolabeling with GAD67, PV, or calbindin, and found that it was greater in schizophrenic patients compared to controls before the age of 40, but lower thereafter.

Notably, both Reln and GAD67 mRNAs were downregulated in the PFC of schizophrenic subjects with no relation to neuronal damage [224]. In keeping with these observations, it appeared that in the PFC there was a vulnerability of the inhibitory circuits, with markers of the inhibitory interneurons showing some of the more consistent alterations [225]. More precisely, these alterations consisted in a reduction in the levels of the GAD67 mRNA and protein in subsets of GABAergic basket cells containing PV [222,226,227] or cholecystokinin (CCK) [226]. Notably, these two populations of basket cells are responsible of the inhibition of the pyramidal neurons giving rise, respectively, to the cortical θ and γ oscillations altered in schizophrenia. In addition, the pyramidal neurons targeted by the PV+ basket cells expressed lower levels of the GABAA receptor α1 [226].

It is also interesting that the levels of RELN and GAD67 mRNAs in microdissected GABAergic neurons of PFC layer 1 were lower in schizophrenics, but unchanged in layer 5 of the same patients [228]. In addition, in the dorsolateral division of the PFC, the GABAergic chandelier neurons targeting the axon initial segment of the pyramidal neurons displayed remarkable neurochemical alterations. These changes were particularly evident in layers 2/3, where immunoreactivity for the GABA membrane

transporter GAT1 diminished, in parallel with an increase of the GABAA receptor α2 subunit in the axons of their target pyramidal neurons [229].

Occurrence of dendritic spine pathology was another prominent feature of the schizophrenic human brain [230,231]. Spine loss mainly affected the smaller spines of the pyramidal neurons in layer 3 of the neocortex and arose during development, possibly because of altered mechanisms of generation, pruning, and/or upkeep [230].

As already mentioned in the section dedicated to autism, investigations on the cerebral cortex of the heterozygous *Reeler* reported a reduction in the levels of GAD67 [53,177], in full accordance with the human studies. A study carried out on mice whose mothers were stressed during pregnancy showed that the downregulation of Reln and GAD67 was associated with a hypermethylation of their promoters [232], this being one the mechanisms in support for the contribution of an altered epigenetic control in the down-regulation of RELN expression in schizophrenia, see [233] for review.

#### b) Hippocampus

The human hippocampal pathology in schizophrenia is by far less clear than in the cortex. Some initial studies have, in fact, reported a decrease in area or overall volume of the hippocampus, or in the number, size, and density of neurons, as well as a disarray of the pyramidal cells, with greatest differences affecting the pyramidal cell density in left CA4; however, several other subsequent surveys were negative, see [234] for review. In any case, hippocampal alterations in schizophrenics are not specific, as they display several common traits with those in autism.

We have previously discussed the histological changes in the hippocampus of the heterozygous *Reeler* mice in relation to autism. These modifications recall, in toto or in part, those in schizophrenia. Additional information detailed, in individual hippocampal layers, the decrease of neuronal GAD67 in CA1, CA2 and dentate gyrus, and the reduction of PV immunoreactive interneurons in CA1 and CA2, in the perspective to validate these mice as a model of schizophrenia [111]. In translational terms, the aforementioned impairment of LTP in heterozygous mice [182] is of interest, as it also occurs in schizophrenic patients [235].
