Background
Cognitive control depends on neural synchrony that maintains a balanced excitation and inhibition in different brain regions[
1]. GABAergic interneurons are critical for providing inhibitory control over pyramidal neurons and modulating synchronized oscillations[
2]. Interneuron deficits have been one of the most consistent findings in human post-mortem schizophrenia studies, including reductions in glutamic acid decarboxylase-67 (GAD67) expression, and parvalbumin (PV) mRNA expression and immunoreactivity[
3‐
5]. Different interneuron subtypes have distinct electrophysiological and synaptic characteristics[
6]. In schizophrenia, GAD67 reduction appears to be restricted to PV-interneurons[
7,
8]. This is of particular relevance as recent optogenetic studies on animal models have shown that PV-interneurons are required for generating gamma-frequency oscillations[
9,
10], that are critical for cognition[
11,
12]. Consistent with this notion, schizophrenia patients display abnormal neural oscillations and synchronizations[
13,
14]. Furthermore, rodents with loss of PV-interneurons and impaired gamma activity show selective cognitive deficits reminiscent of schizophrenia symptoms[
15,
16].
Disrupted-in-schizophrenia 1 (
DISC1) is a strong susceptibility gene for schizophrenia and other mental disorders[
17]. DISC1 functions as a scaffold protein and regulates a wide-range of neurodevelopmental processes[
18‐
20]. Different mutant DISC1 mouse models have displayed selective reductions in PV interneurons[
21‐
24] and alterations in their laminar distribution[
22]. Recently, Steinecke
et al. demonstrated that DISC1 also regulates interneuron tangential migration[
25], further supporting a possible role for DISC1 in modulating interneuron development.
Our group previously described a mutant mouse line,
Disc1-L100P that has behavioral and cognitive abnormalities related to schizophrenia[
26], consistent with four other publications[
27]. Given the accumulating evidence for DISC1 and interneuron abnormalities in schizophrenia, we undertook a comprehensive histological analysis of interneurons in the
Disc1-L100P mutants. Our findings suggest that
Disc1 mutations may have distinct spatial and temporal effects in different interneuron subtypes. Overall, our study provides evidence for the effects of
Disc1 SNPs on interneuron development that represent a starting point for further investigations into developmental and pathophysiological mechanisms in schizophrenia.
Discussion
There is substantial evidence for an association between
DISC1 and several major mental illnesses. However, the mechanism by which
DISC1 gene variants produce both cellular and behavioral abnormalities is still unclear. In this study, we examined embryonic interneuron tangential migratory position and adult histology of two interneuron subtypes (CB and PV) in a mouse with a point mutation in the
Disc1 gene (L100P), which has been previously shown to have behaviors relevant to schizophrenia[
26].
A recent study suggests that DISC1 is necessary for proper tangential migration of cortical interneurons[
25]. Therefore, we examined the tangential migratory pathway of interneurons at E14 and E16, as an indicator of migration. Consistent with the putative role for DISC1 in interneuron development, our study revealed that the
Disc1-L100P mutants displayed abnormal tangential migration. This was further supported by our findings that PV-interneurons remained in the lateral adult cortex and that there were fewer interneurons overall in the mPFC. A plausible explanation is that the L100P mutation disrupts specific DISC1 protein interactions and results in mis-regulated downstream signals. ErbB4, and its substrate Neuregulin-1 (NRG1), have been extensively studied for their role in interneuron tangential migration[
33‐
35]. DISC1 has been hypothesized to converge with NRG1-ErbB4 cascades in modulating migration[
28]. However, interneuron tangential migration deficits are likely to arise through the simultaneous dysregulation of not just one, but several protein interactions including cytoskeletal proteins[
31], dysbindin[
2], neurotrophins[
36] and transcription factors[
37‐
39]. Future research addressing how DISC1 can affect these various pathways will help to elucidate the precise molecular mechanisms by which DISC1 affects interneuron tangential migration.
Next we examined the number and positioning of interneurons in adult
Disc1-L100P mice and found changes consistent with human post-mortem schizophrenia studies including reductions in PV immunoreactivity and abnormal laminar distribution patterns[
4,
8,
30]. Interestingly, other mutant
Disc1 mouse models exhibit similar reductions of PV-interneurons in the PFC and aberrant cortical positioning[
21‐
23]. This suggests that DISC1 protein disruptions may overlap among these different mouse models, with a common effect on interneuron genesis and incorporation of PV-interneurons into proper cortical layers. Interneuron genesis in the ganglionic eminence is likely to be controlled by different transcription factors[
40], but the relationship between DISC1 and interneuron production remains to be determined.
Another theory is based on the pyramidal interneuron network gamma (PING) model, which suggests that PV-interneurons are recruited by glutamatergic inputs from pyramidal neurons[
41]. Previously, misplaced cortical pyramidal neurons and reduced spine densities within layers III and V pyramidal neurons were found in the
Disc1-L100P mutants[
42]. Consequently, incorrect guidance cues and weakened excitatory drive may lead to less recruitment of PV-interneurons and aberrant cortical lamination[
43].
Interestingly, the increase in CB immunoreactivity within the DLFC and PV-immunoreactivity within the CA1 and CA2/3 subfields of the hippocampus did not parallel those observed in post-mortem schizophrenia studies[
44‐
46] and a truncated
Disc1 mouse model[
22]. Despite the inconsistent findings in the literature, an increase in CB mRNA expression and immunoreactivity in the PFC has been reported in several post-mortem studies[
47,
48]. Compared to PV subpopulations, CB-interneurons are less extensively studied and thus their features in schizophrenia remain unclear. CB-interneurons may affect pyramidal neuron activity in a different way than PV-interneurons, since the two interneuron types have different electrophysiological and synaptic characteristics[
6]. Furthermore, the increase in CB-interneurons may be a compensatory response to PV-interneuron reductions[
47]. Moreover, DISC1 can have differential regional effects between the cortex and hippocampus, as evident from opposing neuronal migration and outgrowth effects in previous DISC1 knockdown studies[
49,
50]. Multiple pathways are likely to be involved in determining interneuron fate. Further research is required to elucidate the precise relationship between DISC1-related pathways and to understand the specific roles of DISC1 in different interneuron subpopulations.
As mentioned previously, reduced GAD67 expression in PV-expressing cells has been consistently reported in post-mortem brains of schizophrenia patients[
7,
8]. Here, we provide novel evidence of diminished GAD67/PV co-localization in
Disc1-L100P mutants when compared to WT controls. Immunohistochemical analyses have confirmed the co-expression of DISC1 and GAD67 in GABAergic interneurons[
51]. The
Disc1 L100P mutation may affect specific downstream transcription control of GAD67 enzyme levels, or GAD67 reduction may be a compensatory response to reduced PV immunoreactivity. Furthermore, western blots of GAD67 can provide information on whether GAD67 protein levels are changed in our
Disc1 mutants. The causes and functional relationship between DISC1 and GAD67 remain to be determined. Our findings provide a starting point for future research to elucidate the role of DISC1 in GABAergic signaling.
As mentioned previously, the immunoreactivity and distribution patterns of PV-immunostained cells have been extensively studied in human post-mortem and animal studies. However, the histological relationship between different interneuron subpopulations has not been examined. Given that our Disc1-L100P mutants displayed selective alterations in density and distribution of both PV- and CB-immunostained cells, future double interneuron immunolabeling experiments would provide important insights about whether the density and distribution of one interneuron subtype is associated with the other.
In conclusion, the results presented in this study support the notion that DISC1 plays a role in interneuron development. But whether DISC1 mutations are a primary cause of aberrant interneuron development through direct disruption of interactions with relevant proteins and transcription factors or produce secondary effects from disturbed pyramidal neuron positioning, remains to be determined. Moreover, investigating electrophysiological properties of the
Disc1-L100P mouse cortex and the hippocampus[
52] would be useful in addressing functional outcomes of these histological abnormalities. Nonetheless, we have provided an overview of interneuron histology and development in an
N-ethyl-
N-nitrosourea (
ENU)-induced
Disc1-L100P mouse line, which supplements our previous work in characterizing cortical abnormalities of pyramidal neurons[
42]. Our findings further support the role of DISC1 in interneuron development and provide additional insights about how
Disc1 mutations can lead to the brain and cognitive abnormalities associated with schizophrenia. More importantly, this study represents a starting point for further investigation of DISC1-related mechanistic pathways in interneuron development.
Competing interests
All authors declare that they have no competing financial interests.
Authors’ contributions
Albert Wong, Sabine Cordes, John Roder and Frankie Lee designed the study. Frankie Lee and Clement Zai performed all experiments and statistical analysis. Frankie Lee and Albert Wong prepared the first draft of the manuscript. All authors contributed to and have approved the final manuscript.