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Layers II/III of Prefrontal Cortex in Df(h22q11)/+ Mouse Model of the 22q11.2 Deletion Display Loss of Parvalbumin Interneurons and Modulation of Neuronal Morphology and Excitability

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Abdelrahman Alabsi at Aarhus University Hospital
Abdelrahman Alabsi
Per Qvist at Aarhus University
Per Qvist
Samora Okujeni at University of Freiburg
Samora Okujeni
Ahmad Raza Khan at National Brain Research Centre
Ahmad Raza Khan
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Abstract and Figures

The 22q11.2 deletion has been identified as a risk factor for multiple neurodevelopmental disorders. Behavioral and cognitive impairments are common among carriers of the 22q11.2 deletion. Parvalbumin expressing (PV+) interneurons provide perisomatic inhibition of excitatory neuronal circuits through GABAA receptors, and a deficit of PV+ inhibitory circuits may underlie a multitude of the behavioral and functional deficits in the 22q11.2 deletion syndrome. We investigated putative deficits of PV+ inhibitory circuits and the associated molecular, morphological, and functional alterations in the prefrontal cortex (PFC) of the Df(h22q11)/+ mouse model of the 22q11.2 hemizygous deletion. We detected a significant decrease in the number of PV+ interneurons in layers II/III of PFC in Df(h22q11)/+ mice together with a reduction in the mRNA and protein levels of GABAA (α3), a PV+ putative postsynaptic receptor subunit. Pyramidal neurons from the same layers further experienced morphological reorganizations of spines and dendrites. Accordingly, a decrease in the levels of the postsynaptic density protein 95 (PSD95) and a higher neuronal activity in response to the GABAA antagonist bicuculline were measured in these layers in PFC of Df(h22q11)/+ mice compared with their wild-type littermates. Our study shows that a hemizygotic deletion of the 22q11.2 locus leads to deficit in the GABAergic control of network activity and involves molecular and morphological changes in both the inhibitory and excitatory synapses of parvalbumin interneurons and pyramidal neurons specifically in layers II/III PFC.
Loss of PV⁺ interneurons and decreased mRNA and protein levels of GABAA (α3) in PFC of Df(h22q11)/+ mice. a Immunofluorescence staining against PV⁺ interneurons in 50 μm fixed sections from PFC of WT (n = 6) and Df(h22q11)/+ (n = 6) mice. PFC was delineated, and PV⁺ interneurons were identified and counted in layers II/III and V/VI separately using Leica fluorescence microscope with a 63× oil lens. A significant decrease was detected in the number of PV⁺ interneurons in layers II/III (p < 0.05), while no difference was shown in layers V/VI in PFC of Df(h22q11)/+ compared with WT mice. Data were analyzed using Student t test. Results were considered significant when p value <0.05 (*p < 0.05). Data are presented as mean ± S.D. b Quantitative real time PCR was used to quantify the mRNA levels of Gabra1, Gabra2, and Gabra3. mRNA levels of Gabra1 and Gabra2 remained unchanged, while a significant decrease was detected at the Gabra3 mRNA levels (q < 0.05). Data from WT (n = 8) and Df(h22q11)/+ (n = 8) mice were analyzed with multiple t tests. Results were considered significant when Benjamini Krieger Yuketieli corrected p value (q value) < 0.05 (*q < 0.05). Data are presented as mean ± S.D. Western blot was used to immunoblot and quantify the protein levels of GABAA (α1), GABAA (α2), and GABAA (α3) in PFC of Df(h22q11)/+ compared with WT littermates. No significant change was detected in the protein levels of c GABAA (α1) or d GABAA (α2). e Protein levels of GABAA (α3) were significantly reduced in PFC of Df(h22q11)/+ compared with WT mice (q < 0.01). Data from WT (n = 6) and Df(h22q11)/+ (n = 6) mice. Data were analyzed with multiple t tests. Results were considered significant when q < 0.05 (**q < 0.01). Data are presented as mean ± S.D. f High-resolution confocal images of GABAA (α3) positive soma from layers II/III PFC of (n = 5) Df(h22q11)/+ and (n = 5) WT littermates. The number of GABAA (α3) positive puncta around soma was quantified, and a significant decrease was detected for soma from layers II/III PFC of Df(h22q11)/+ compared with WT mice (p < 0.05). Data were analyzed using Student t test. Results were considered significant when p < 0.05 (*p < 0.05). Data are presented as mean ± S.D.
Loss of PV⁺ interneurons and decreased mRNA and protein levels of GABAA (α3) in PFC of Df(h22q11)/+ mice. a Immunofluorescence staining against PV⁺ interneurons in 50 μm fixed sections from PFC of WT (n = 6) and Df(h22q11)/+ (n = 6) mice. PFC was delineated, and PV⁺ interneurons were identified and counted in layers II/III and V/VI separately using Leica fluorescence microscope with a 63× oil lens. A significant decrease was detected in the number of PV⁺ interneurons in layers II/III (p < 0.05), while no difference was shown in layers V/VI in PFC of Df(h22q11)/+ compared with WT mice. Data were analyzed using Student t test. Results were considered significant when p value <0.05 (*p < 0.05). Data are presented as mean ± S.D. b Quantitative real time PCR was used to quantify the mRNA levels of Gabra1, Gabra2, and Gabra3. mRNA levels of Gabra1 and Gabra2 remained unchanged, while a significant decrease was detected at the Gabra3 mRNA levels (q < 0.05). Data from WT (n = 8) and Df(h22q11)/+ (n = 8) mice were analyzed with multiple t tests. Results were considered significant when Benjamini Krieger Yuketieli corrected p value (q value) < 0.05 (*q < 0.05). Data are presented as mean ± S.D. Western blot was used to immunoblot and quantify the protein levels of GABAA (α1), GABAA (α2), and GABAA (α3) in PFC of Df(h22q11)/+ compared with WT littermates. No significant change was detected in the protein levels of c GABAA (α1) or d GABAA (α2). e Protein levels of GABAA (α3) were significantly reduced in PFC of Df(h22q11)/+ compared with WT mice (q < 0.01). Data from WT (n = 6) and Df(h22q11)/+ (n = 6) mice. Data were analyzed with multiple t tests. Results were considered significant when q < 0.05 (**q < 0.01). Data are presented as mean ± S.D. f High-resolution confocal images of GABAA (α3) positive soma from layers II/III PFC of (n = 5) Df(h22q11)/+ and (n = 5) WT littermates. The number of GABAA (α3) positive puncta around soma was quantified, and a significant decrease was detected for soma from layers II/III PFC of Df(h22q11)/+ compared with WT mice (p < 0.05). Data were analyzed using Student t test. Results were considered significant when p < 0.05 (*p < 0.05). Data are presented as mean ± S.D.
… 
Morphological reorganization of pyramidal neurons and reduced levels of PSD95 in layers II/III PFC of Df(h22q11)/+ mice. a-b Golgi staining and 3D reconstruction of layers II/III pyramidal neurons in 100 μm thick PFC sections from WT (n = 7) and Df(h22q11)/+ (n = 7) mice. a A significant decrease (p < 0.05) was found in spine density of layers II/III pyramidal neurons from PFC of Df(h22q11)/+ mice compared with WT mice. b A significant decrease (p < 0.05) was detected in the total intersection number of dendritic branches of layers II/III pyramidal neurons from PFC of Df(h22q11)/+ compared with WT mice. c Immunofluorescence staining against the excitatory post synaptic marker PSD95. The number of PSD95 positive puncta was quantified in layers II/III PFC in sections from WT (n = 5) and Df(h22q11)/+ (n = 5) mice. A significant decrease was detected in the density of PSD95 puncta in Df(h22q11)/+ compared with WT mice (p < 0.01). Data were analyzed using Student t test. Results were considered significant when p value <0.05 (*p < 0.05,**p < 0.01). Data are presented as mean ± S.D.
Morphological reorganization of pyramidal neurons and reduced levels of PSD95 in layers II/III PFC of Df(h22q11)/+ mice. a-b Golgi staining and 3D reconstruction of layers II/III pyramidal neurons in 100 μm thick PFC sections from WT (n = 7) and Df(h22q11)/+ (n = 7) mice. a A significant decrease (p < 0.05) was found in spine density of layers II/III pyramidal neurons from PFC of Df(h22q11)/+ mice compared with WT mice. b A significant decrease (p < 0.05) was detected in the total intersection number of dendritic branches of layers II/III pyramidal neurons from PFC of Df(h22q11)/+ compared with WT mice. c Immunofluorescence staining against the excitatory post synaptic marker PSD95. The number of PSD95 positive puncta was quantified in layers II/III PFC in sections from WT (n = 5) and Df(h22q11)/+ (n = 5) mice. A significant decrease was detected in the density of PSD95 puncta in Df(h22q11)/+ compared with WT mice (p < 0.01). Data were analyzed using Student t test. Results were considered significant when p value <0.05 (*p < 0.05,**p < 0.01). Data are presented as mean ± S.D.
… 
Higher neuronal activity in layers II/III PFC of Df(h22q11)/+ mice after the application of bicuculline. Recording of the extracellular activity using HD-MEA in 300 μm thickness PFC acute brain slices from WT (n = 6) and Df(h22q11)/+ (n = 6) mice before the application [(−) bicuculline] and after the application [(+) bicuculline] of 10 μM of GABAA antagonist bicuculline. a Representative spike activity recorded in layers II/III PFC of WT and Df(h22q11)/+ mice before and after the application of bicuculline. The blue arrows indicate successfully detected spikes. b Average spiking rate from layers II/III PFC of Df(h22q11)/+ mice compared with WT mice before (p > 0.05) and after (p < 0.01) the incubation with bicuculline. c Schematic representation of the recording area from layers II/III PFC of WT and Df(h22q11)/+ mice before and after the application of bicuculline. A blue dot represents a channel with no-detected activity. A red dot represents a channel with detected activity. d Number of active units in Df(h22q11)/+ slices compared with WT slices before (p > 0.05) and after (p < 0.05) the application of bicuculline
Higher neuronal activity in layers II/III PFC of Df(h22q11)/+ mice after the application of bicuculline. Recording of the extracellular activity using HD-MEA in 300 μm thickness PFC acute brain slices from WT (n = 6) and Df(h22q11)/+ (n = 6) mice before the application [(−) bicuculline] and after the application [(+) bicuculline] of 10 μM of GABAA antagonist bicuculline. a Representative spike activity recorded in layers II/III PFC of WT and Df(h22q11)/+ mice before and after the application of bicuculline. The blue arrows indicate successfully detected spikes. b Average spiking rate from layers II/III PFC of Df(h22q11)/+ mice compared with WT mice before (p > 0.05) and after (p < 0.01) the incubation with bicuculline. c Schematic representation of the recording area from layers II/III PFC of WT and Df(h22q11)/+ mice before and after the application of bicuculline. A blue dot represents a channel with no-detected activity. A red dot represents a channel with detected activity. d Number of active units in Df(h22q11)/+ slices compared with WT slices before (p > 0.05) and after (p < 0.05) the application of bicuculline
… 
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ORIGINAL ARTICLE
Layers II/III of Prefrontal Cortex in Df(h22q11)/+ Mouse Model
of the 22q11.2 Deletion Display Loss of Parvalbumin Interneurons
and Modulation of Neuronal Morphology and Excitability
Abdel-Rahman Al-Absi
1
&Per Qvist
2,3,4,5
&Samora Okujeni
6
&Ahmad Raza Khan
7,8
&
Simon Glerup
2
&Connie Sanchez
9
&Jens R. Nyengaard
1
Received: 19 May 2020 /Accepted: 9 August 2020
#Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The 22q11.2 deletion has been identified as a risk factor for multiple neurodevelopmental disorders. Behavioral and cognitive
impairments are common among carriers of the 22q11.2 deletion. Parvalbumin expressing (PV
+
) interneurons provide
perisomatic inhibition of excitatory neuronal circuits through GABA
A
receptors, and a deficit of PV
+
inhibitory circuits may
underlie a multitude of the behavioral and functional deficits in the 22q11.2 deletion syndrome. We investigated putative deficits
of PV
+
inhibitory circuits and the associated molecular, morphological, and functional alterations in the prefrontal cortex (PFC)
of the Df(h22q11)/+ mouse model of the 22q11.2 hemizygous deletion. We detected a significant decrease in the number of PV
+
interneurons in layers II/III of PFC in Df(h22q11)/+ mice together with a reduction in the mRNA and protein levels of GABA
A
(α3), a PV
+
putative postsynaptic receptor subunit. Pyramidal neurons from the same layers further experienced morphological
reorganizations of spines and dendrites. Accordingly, a decrease in the levels of the postsynaptic density protein 95 (PSD95) and
a higher neuronal activity in response to the GABA
A
antagonist bicuculline were measured in these layers in PFC of
Df(h22q11)/+ mice compared with their wild-type littermates. Our study shows that a hemizygotic deletion of the 22q11.2 locus
leads to deficit in the GABAergic control of network activity and involves molecular and morphological changes in both the
inhibitory and excitatory synapses of parvalbumin interneurons and pyramidal neurons specifically in layers II/III PFC.
Keywords Df(h22q11)/+mouse model .Prefrontal cortex .Parvalbumin interneurons .GABA
A
(α3)receptorsubunit .Pyramidal
neurons
Introduction
The 22q11.2 deletion is a genetic condition that involves 1.5–
3 M bases encompassing 30–50 genes and has an approximate
prevalence of 1 in 3000–4000 live births [1]. This deletion
represents the highest known individual genetic risk factor
for the emergence of schizophrenia [2], and it confers high
risk for other neurodevelopmental disorders [3]. Carriers of
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s12035-020-02067-1) contains supplementary
material, which is available to authorized users.
*Abdel-Rahman Al-Absi
abd.alabsi@clin.au.dk
Per Qvist
per.q@biomed.au.dk
Samora Okujeni
samora.okujeni@biologie.uni-freiburg.de
Ahmad Raza Khan
110ahmadkhan@gmail.com
Simon Glerup
glerup@biomed.au.dk
Connie Sanchez
connie_sanchez@clin.au.dk
Jens R. Nyengaard
jrnyengaard@clin.au.dk
Extended author information available on the last page of the article
https://doi.org/10.1007/s12035-020-02067-1
/ Published online: 20 August 2020
Molecular Neurobiology (2020) 57:4978–4988
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
... We have recently reported on deficit of GABA A receptor expression in the prefrontal cortex of Df(h22q11)/+ mice that also exhibited decreased gamma oscillatory synchrony and reduced synaptic plasticity in the cortical-hippocampal networks (Nilsson et al., 2018;Al-Absi et al., 2020;Tripathi et al., 2020). Df(h22q11)/+ mice further display deficits in multiple cognitive and behavioral domains that resemble core neurodevelopmental symptoms of 22q11.2DS ...
... In our previous study, we reported multi-site deficits affecting the GABAergic inhibitory circuits in the prefrontal cortex of Df(h22q11)/+ mice, involving altered levels of GABA A (α3) receptors (Al-Absi et al., 2020). Interestingly, treatment of 22q11.2 ...
... locus is further reported to disrupt the brain GABAergic transmission (Crabtree et al., 2016). Results from our previous study (Al-Absi et al., 2020), where we observed alterations in the levels of GABA A receptors in the prefrontal cortex, in addition to the results from this study may provide supporting evidence in this direction. The loss of GAD67 + interneurons in CA1 of Df(h22q11)/+ mice can also contribute to the reduction of GABA A receptor binding levels in this brain subregion. ...
Df(h22q11)/+ mouse model exhibits reduced binding levels of GABAA receptors and structural and functional dysregulation in the inhibitory and excitatory networks of hippocampus
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  • Aug 2022
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The 22q11.2 hemizygous deletion confers high risk for multiple neurodevelopmental disorders. Inhibitory signaling, largely regulated through GABAA receptors, is suggested to serve a multitude of brain functions that are disrupted in the 22q11.2 deletion syndrome. We investigated the putative deficit of GABAA receptors and the potential substrates contributing to the inhibitory and excitatory dysregulations in hippocampal networks of the Df(h22q11)/+ mouse model of the 22q11.2 hemizygous deletion. The Df(h22q11)/+ mice exhibited impairments in several hippocampus-related functional domains, represented by impaired spatial memory and sensory gating functions. Autoradiography using the [³H]muscimol tracer revealed a significant reduction in GABAA receptor binding in the CA1 and CA3 subregions, together with a loss of GAD67⁺ interneurons in CA1 of Df(h22q11)/+ mice. Furthermore, electrophysiology recordings exhibited significantly higher neuronal activity in CA3, in response to the GABAA receptor antagonist, bicuculline, as compared with wild type mice. Density and volume of dendritic spines in pyramidal neurons were reduced and Sholl analysis also showed a reduction in the complexity of basal dendritic tree in CA1 and CA3 subregions of Df(h22q11)/+ mice. Overall, our findings demonstrate that hemizygous deletion in the 22q11.2 locus leads to dysregulations in the inhibitory circuits, involving reduced binding levels of GABAA receptors, in addition to functional and structural modulations of the excitatory networks of hippocampus.
View
... A great amount of high-quality work has been conducted in animal models of NDDs, many of which use an induced CNV to model disease processes. Whilst animal research does tend to support findings in human populations [144][145][146]; these studies were also excluded for two reasons. Firstly, like cellular models, animal models cannot accurately represent human NDD symptoms, for example hallucinations or theory of mind. ...
Neural excitation/inhibition imbalance and neurodevelopmental pathology in human copy number variant syndromes: a systematic review
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  • Jun 2025
  • J NEURODEV DISORD
Cumulative evidence suggests neurodevelopmental disorders are closely related. The risk of these disorders is increased by a series of copy number variant syndromes – phenotypically heterogeneous genetic disorders, present in a minority of the population. Recent models suggest that a disruption in the balance between excitatory and inhibitory neural activity may contribute to the aetiology of neurodevelopmental disorders, and may be additionally disturbed in copy number variant syndromes. In this systematic review, the databases PubMed, Embase, and Scopus were searched for studies of excitation/inhibition imbalance in relation to neurodevelopmental disorders in human copy number variant samples. A total of 53 studies were included, representing a variety of copy number variants and research methodologies. The resulting data suggests excitation/inhibition balance is indeed disrupted in different copy number variant populations, providing insight into a putative mechanism of both idiopathic and genetic neurodevelopmental disorders. However, the high level of heterogeneity in the data set, alongside emerging techniques for excitation/inhibition assessment, prompts further investigation of this field.
View
... More generally, deficits in inhibitory signaling have been reported in the prefrontal cortex and hippocampus in other mouse models of 22q11.2DS [43,44], and human 22q11.2DS cerebral cortical organoids have been found to display increased spontaneous firing [45]. ...
Auditory evoked-potential abnormalities in a mouse model of 22q11.2 Deletion Syndrome and their interactions with hearing impairment
Article
Full-text available
  • Jan 2025
The 22q11.2 deletion is a risk factor for multiple psychiatric disorders including schizophrenia and also increases vulnerability to middle-ear problems that can cause hearing impairment. Up to 60% of deletion carriers experience hearing impairment and ~30% develop schizophrenia in adulthood. It is not known if these risks interact. Here we used the Df1/+ mouse model of the 22q11.2 deletion to investigate how hearing impairment might interact with increased genetic vulnerability to psychiatric disease to affect brain function. We measured brain function using cortical auditory evoked potentials (AEPs), which are commonly measured non-invasively in humans. After identifying one of the simplest and best-validated methods for AEP measurement in mice from the diversity of previous approaches, we measured peripheral hearing sensitivity and cortical AEPs in Df1/+ mice and their WT littermates. We exploited large inter-individual variation in hearing ability among Df1/+ mice to distinguish effects of genetic background from effects of hearing impairment. Central auditory gain and adaptation were quantified by comparing brainstem activity and cortical AEPs and by analyzing the growth of cortical AEPs with increasing sound level or inter-tone interval duration. We found that level-dependent AEP growth was abnormally large in Df1/+ mice regardless of hearing impairment, but other AEP measures of central auditory gain and adaptation depended on both genotype and hearing phenotype. Our results demonstrate the relevance of comorbid hearing loss to auditory brain dysfunction in 22q11.2DS and also identify potential biomarkers for psychiatric disease that are robust to hearing impairment.
View
... deletion [54]. Studies in both humans with the deletion and 2p11.2 deletion mouse models also support disturbed E-I neurotransmitter system balance because of the deletion, with alterations in both glutamate and GABAergic neurotransmission supported [55][56][57], although effects on glutamate are not always found in patients [58]. While several studies have characterised the impact of 22q11.2 ...
Progress towards understanding risk factor mechanisms in the development of autism spectrum disorders
Article
Full-text available
  • Sep 2024
  • BIOCHEM SOC T
Autism spectrum disorders (ASD) are a heterogenous set of syndromes characterised by social impairment and cognitive symptoms. Currently, there are limited treatment options available to help people with ASD manage their symptoms. Understanding the biological mechanisms that result in ASD diagnosis and symptomatology is an essential step in developing new interventional strategies. Human genetic studies have identified common gene variants of small effect and rare risk genes and copy number variants (CNVs) that substantially increase the risk of developing ASD. Reverse translational studies using rodent models based on these genetic variants provide new insight into the biological basis of ASD. Here we review recent findings from three ASD associated CNV mouse models (16p11.2, 2p16.3 and 22q11.2 deletion) that show behavioural and cognitive phenotypes relevant to ASD. These models have identified disturbed excitation-inhibition neurotransmitter balance, evidenced by dysfunctional glutamate and GABA signalling, as a key aetiological mechanism. These models also provide emerging evidence for serotoninergic neurotransmitter system dysfunction, although more work is needed to clarify the nature of this. At the brain network level, prefrontal cortex (PFC) dysfunctional connectivity is also evident across these models, supporting disturbed PFC function as a key nexus in ASD aetiology. Overall, published data highlight the utility and valuable insight gained into ASD aetiology from preclinical CNV mouse models. These have identified key aetiological mechanisms that represent putative novel therapeutic targets for the treatment of ASD symptoms, making them useful translational models for future drug discovery, development and validation.
View
... To generate biologically-informed hypotheses from rs-fMRI, we relate spatial maps of case-control differences to brain maps from multiple published sources, including metabolic positron emission tomography (PET), and post-mortem gene expression (36)(37)(38)(39). This approach can help explore human phenotypes relating to findings from pre-clinical 22qDel studies, such as altered mitochondria (40)(41)(42)(43), neurovascular development (44)(45)(46)(47), and cell type markers (48)(49)(50). ...
View
... deletion model. 94 General Factors (Age of Evaluation, Species, Sex, Experimental Bias) SCZ is often diagnosed in late adolescence and early adulthood. Along with the illness onset, patients show deficits in GABAergic transmission and impairment in cortical gamma frequency oscillations, which are thought to depend upon the normal functioning of PV interneurons. 1 We found that all animal models significantly affected PV interneurons in both brain areas when PV markers were evaluated in adulthood. ...
Prefrontal and Hippocampal Parvalbumin Interneurons in Animal Models for Schizophrenia: A Systematic Review and Meta-analysis
Article
  • Aug 2023
  • SCHIZOPHRENIA BULL
Background Consistent with postmortem findings in patients, most animal models for schizophrenia (SCZ) present abnormal levels of parvalbumin (PV), a marker of fast-spiking GABAergic interneurons, in the prefrontal cortex (PFC) and hippocampus (HIP). However, there are discrepancies in the literature. PV reductions lead to a functional loss of PV interneurons, which is proposed to underly SCZ symptoms. Given its complex etiology, different categories of animal models have been developed to study SCZ, which may distinctly impact PV levels in rodent brain areas. Study Design We performed a quantitative meta-analysis on PV-positive cell number/density and expression levels in the PFC and HIP of animal models for SCZ based on pharmacological, neurodevelopmental, and genetic manipulations. Results Our results confirmed that PV levels are significantly reduced in the PFC and HIP regardless of the animal model. By categorizing into subgroups, we found that all pharmacological models based on NMDA receptor antagonism decreased PV-positive cell number/density or PV expression levels in both brain areas examined. In neurodevelopmental models, abnormal PV levels were confirmed in both brain areas in maternal immune activation models and HIP of the methylazoxymethanol acetate model. In genetic models, negative effects were found in neuregulin 1 and ERBB4 mutant mice in both brain regions and the PFC of dysbindin mutant mice. Regarding sex differences, male rodents exhibited PV reductions in both brain regions only in pharmacological models, while few studies have been conducted in females. Conclusion Overall, our findings support deficits in prefrontal and hippocampal PV interneurons in animal models for SCZ.
View
... Plastic changes of interneuron structure and function, whether through disease-relevant genetic insult, exogenous neuronal activation, receptor modulation, or experience-induced alterations, are poised to remodel computations within mPFC, and the routing of circuit-specific information through mPFC, to sculpt behavior. Numerous interneuron adaptations have been reported following each of these types of manipulations, manifesting as changes to mesoscopic and microscopic interneuron structure (e.g., Boksa et al., 2016;Al-Absi et al., 2020;Gildawie et al., 2020;Bueno-Fernandez et al., 2021), protein expression (e.g., Stedehouder et al., 2018;Mukherjee et al., 2019;Reichelt et al., 2021), and intrinsic physiology (e.g., Campanac and Hoffman, 2013;Dao et al., 2020;Zorrilla de San Martin et al., 2020). Here, we highlight some recent advances in our understanding of how plasticity in the synaptic and circuit connectivity of mPFC interneurons can reshape typical and disordered mPFC function and associated behavior (see also Yang et al., 2021). ...
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View
... locus have been developed to better understand the contribution of single and multiple 22q11.2 genes to developmental, cell-specific, and behavioral deviations relative to wild-type littermates [54][55][56][57][58][59]. For example, Tbx1 heterozygous mice showed significant alterations of myelinated axons in the fimbria, lower mRNA levels of oligodendrocyte-related genes, and postnatal progenitor cells from the subventricular zone produced fewer oligodendrocytes in vitro [60]. ...
A genetics-first approach to understanding autism and schizophrenia spectrum disorders: the 22q11.2 deletion syndrome
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  • MOL PSYCHIATR
Recently, increasing numbers of rare pathogenic genetic variants have been identified that are associated with variably elevated risks of a range of neurodevelopmental outcomes, notably including Autism Spectrum Disorders (ASD), Schizophrenia Spectrum Disorders (SSD), and Intellectual Disability (ID). This review is organized along three main questions: First, how can we unify the exclusively descriptive basis of our current psychiatric diagnostic classification system with the recognition of an identifiable, highly penetrant genetic risk factor in an increasing proportion of patients with ASD or SSD? Second, what can be learned from studies of individuals with ASD or SSD who share a common genetic basis? And third, what accounts for the observed variable penetrance and pleiotropy of neuropsychiatric phenotypes in individuals with the same pathogenic variant? In this review, we focus on findings of clinical and preclinical studies of the 22q11.2 deletion syndrome (22q11DS). This particular variant is not only one of the most common among the increasing list of known rare pathogenic variants, but also one that benefits from a relatively long research history. Consequently, 22q11DS is an appealing model as it allows us to: (1) elucidate specific genotype–phenotype associations, (2) prospectively study behaviorally defined classifications, such as ASD or SSD, in the context of a known, well-characterized genetic basis, and (3) elucidate mechanisms underpinning variable penetrance and pleiotropy, phenomena with far-reaching ramifications for research and clinical practice. We discuss how findings from animal and in vitro studies relate to observations in human studies and can help elucidate factors, including genetic, environmental, and stochastic, that impact the expression of neuropsychiatric phenotypes in 22q11DS, and how this may inform mechanisms underlying neurodevelopmental expression in the general population. We conclude with research priorities for the field, which may pave the way for novel therapeutics.
View
... does harbor a reduction in GABA-ergic parvalbumin containing cortical interneurons in 22q11.2DS (Al-Absi et al., 2020). Therefore, a potential mechanism of altered sleep and sleep-associated EEG oscillations may involve neurodevelopmental changes to cortical structure and GABAergic signaling in cortical inhibitory networks in 22q11.2DS. ...
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  • Aug 2022
  • eLife
Background Young people living with 22q11.2 Deletion Syndrome (22q11.2DS) are at increased risk of schizophrenia, intellectual disability, attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). In common with these conditions, 22q11.2DS is also associated with sleep problems. We investigated whether abnormal sleep or sleep-dependent network activity in 22q11.2DS reflects convergent, early signatures of neural circuit disruption also evident in associated neurodevelopmental conditions. Methods In a cross-sectional design, we recorded high-density sleep EEG in young people (6–20 years) with 22q11.2DS (n=28) and their unaffected siblings (n=17), quantifying associations between sleep architecture, EEG oscillations (spindles and slow waves) and psychiatric symptoms. We also measured performance on a memory task before and after sleep. Results 22q11.2DS was associated with significant alterations in sleep architecture, including a greater proportion of N3 sleep and lower proportions of N1 and REM sleep than in siblings. During sleep, deletion carriers showed broadband increases in EEG power with increased slow-wave and spindle amplitudes, increased spindle frequency and density, and stronger coupling between spindles and slow-waves. Spindle and slow-wave amplitudes correlated positively with overnight memory in controls, but negatively in 22q11.2DS. Mediation analyses indicated that genotype effects on anxiety, ADHD and ASD were partially mediated by sleep EEG measures. Conclusions This study provides a detailed description of sleep neurophysiology in 22q11.2DS, highlighting alterations in EEG signatures of sleep which have been previously linked to neurodevelopment, some of which were associated with psychiatric symptoms. Sleep EEG features may therefore reflect delayed or compromised neurodevelopmental processes in 22q11.2DS, which could inform our understanding of the neurobiology of this condition and be biomarkers for neuropsychiatric disorders. Funding This research was funded by a Lilly Innovation Fellowship Award (UB), the National Institute of Mental Health (NIMH 5UO1MH101724; MvdB), a Wellcome Trust Institutional Strategic Support Fund (ISSF) award (MvdB), the Waterloo Foundation (918-1234; MvdB), the Baily Thomas Charitable Fund (2315/1; MvdB), MRC grant Intellectual Disability and Mental Health: Assessing Genomic Impact on Neurodevelopment (IMAGINE) (MR/L011166/1; JH, MvdB and MO), MRC grant Intellectual Disability and Mental Health: Assessing Genomic Impact on Neurodevelopment 2 (IMAGINE-2) (MR/T033045/1; MvdB, JH and MO); Wellcome Trust Strategic Award ‘Defining Endophenotypes From Integrated Neurosciences’ Wellcome Trust (100202/Z/12/Z MO, JH). NAD was supported by a National Institute for Health Research Academic Clinical Fellowship in Mental Health and MWJ by a Wellcome Trust Senior Research Fellowship in Basic Biomedical Science (202810/Z/16/Z). CE and HAM were supported by Medical Research Council Doctoral Training Grants (C.B.E. 1644194, H.A.M MR/K501347/1). HMM and UB were employed by Eli Lilly & Co during the study; HMM is currently an employee of Boehringer Ingelheim Pharma GmbH & Co KG. The views and opinions expressed are those of the author(s), and not necessarily those of the NHS, the NIHR or the Department of Health funders.
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Cognition- and circuit-based dysfunction in a mouse model of 22q11.2 microdeletion syndrome: effects of stress
Article
Full-text available
  • Jan 2020
Genetic microdeletion at the 22q11 locus is associated with very high risk for schizophrenia. The 22q11.2 microdeletion (Df(h22q11)/+) mouse model shows cognitive deficits observed in this disorder, some of which can be linked to dysfunction of the prefrontal cortex (PFC). We used behavioral (n = 10 per genotype), electrophysiological (n = 7 per genotype per group), and neuroanatomical (n = 5 per genotype) techniques to investigate schizophrenia-related pathology of Df(h22q11)/+ mice, which showed a significant decrease in the total number of parvalbumin positive interneurons in the medial PFC. The Df(h22q11)/+ mice when tested on PFC-dependent behavioral tasks, including gambling tasks, perform significantly worse than control animals while exhibiting normal behavior on hippocampus-dependent tasks. They also show a significant decrease in hippocampus-medial Prefrontal cortex (H-PFC) synaptic plasticity (long-term potentiation, LTP). Acute platform stress almost abolished H-PFC LTP in both wild-type and Df(h22q11)/+ mice. H-PFC LTP was restored to prestress levels by clozapine (3 mg/kg i.p.) in stressed Df(h22q11)/+ mice, but the restoration of stress-induced LTP, while significant, was similar between wild-type and Df(h22q11)/+ mice. A medial PFC dysfunction may underlie the negative and cognitive symptoms in human 22q11 deletion carriers, and these results are relevant to the current debate on the utility of clozapine in such subjects.
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Inhibitory Synapse Formation at the Axon Initial Segment
Article
Full-text available
  • Nov 2019
The axon initial segment (AIS) is the site of action potential (AP) initiation in most neurons and is thus a critical site in the regulation of neuronal excitability. Normal function within the discrete AIS compartment requires intricate molecular machinery to ensure the proper concentration and organization of voltage-gated and ligand-gated ion channels; in humans, dysfunction at the AIS due to channel mutations is commonly associated with epileptic disorders. In this review, we will examine the molecular mechanisms underlying the formation of the only synapses found at the AIS: synapses containing γ-aminobutyric type A receptors (GABAARs). GABAARs are heteropentamers assembled from 19 possible subunits and are the primary mediators of fast synaptic inhibition in the brain. Although the total GABAAR population is incredibly heterogeneous, only one specific GABAAR subtype—the α2-containing receptor—is enriched at the AIS. These AIS synapses are innervated by GABAergic chandelier cells, and this inhibitory signaling is thought to contribute to the tight control of AP firing. Here, we will summarize the progress made in understanding the regulation of GABAAR synapse formation, concentrating on post-translational modifications of subunits and on interactions with intracellular proteins. We will then discuss subtype-specific synapse formation, with a focus on synapses found at the AIS, and how these synapses influence neuronal excitation.
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Continuous performance test impairment in a 22q11.2 microdeletion mouse model: improvement by amphetamine
Article
Full-text available
  • Nov 2018
The 22q11.2 deletion syndrome (22q11.2DS) confers high risk of neurodevelopmental disorders such as schizophrenia and attention-deficit hyperactivity disorder. These disorders are associated with attentional impairment, the remediation of which is important for successful therapeutic intervention. We assessed a 22q11.2DS mouse model (Df(h22q11)/+) on a touchscreen rodent continuous performance test (rCPT) of attention and executive function that is analogous to human CPT procedures. Relative to wild-type littermates, Df(h22q11)/+ male mice showed impaired attentional performance as shown by decreased correct response ratio (hit rate) and a reduced ability to discriminate target stimuli from non-target stimuli (discrimination sensitivity, or d’). The Df(h22q11)/+ model exhibited decreased prefrontal cortical-hippocampal oscillatory synchrony within multiple frequency ranges during quiet wakefulness, which may represent a biomarker of cognitive dysfunction. The stimulant amphetamine (0–1.0 mg/kg, i.p.) dose-dependently improved d’ in Df(h22q11)/+ mice whereas the highest dose of modafinil (40 mg/kg, i.p.) exacerbated their d’ impairment. This is the first report to directly implicate attentional impairment in a 22q11.2DS mouse model, mirroring a key endophenotype of the human disorder. The capacity of the rCPT to detect performance impairments in the 22q11.2DS mouse model, and improvement following psychostimulant-treatment, highlights the utility and translational potential of the Df(h22q11)/+ model and this automated behavioral procedure.
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Spatiotemporal Distribution of GABAA Receptor Subunits Within Layer II of Mouse Medial Entorhinal Cortex: Implications for Grid Cell Excitability
Article
Full-text available
  • Jun 2018
GABAergic parvalbumin-expressing (PV+) interneurons provide powerful inhibitory modulation of grid cells in layer II of the medial entorhinal cortex (MEC LII). However, the molecular machinery through which PV+ cells regulate grid cell activity is poorly defined. PV+ interneurons impart inhibitory modulation primarily via GABA-A receptors (GABAARs). GABAARs are pentameric ion channels assembled from a repertoire of 19 subunits. Multiple subunit combinations result in a variety of receptor subtypes mediating functionally diverse postsynaptic inhibitory currents. Whilst the broad expression patterns of GABAAR subunits within the EC have been reported, those expressed by individual MEC LII cell types, in particular grid cells candidates, stellate and pyramidal cells, are less well described. Stellate and pyramidal cells are distinguished by their selective expression of reelin (RE+) and calbindin (CB+) respectively. Thus, the overall aim of this study was to provide a high resolution analysis of the major (α and γ) GABAAR subunits expressed in proximity to somato-dendritic PV+ boutons, on RE+ and CB+ cells, using immunohistochemistry, confocal microscopy and quantitative RT-PCR (qPCR). Clusters immunoreactive for the α1 and γ2 subunits decorated the somatic membranes of both RE+ and CB+ cells and were predominantly located in apposition to clusters immunoreactive for PV and vesicular GABA transporter (VGAT), suggesting expression in GABAergic synapses innervated by PV interneurons. Although intense α2 subunit-immunopositive clusters were evident in hippocampal fields located in close proximity to the EC, no specific signal was detected in MEC LII RE+ and CB+ profiles. Immunoreactivity for the α3 subunit was detected in all RE+ somata. In contrast, only a sub-population of CB+ cells was α3 immunopositive. These included CB-α3 cells which were both PV+ and PV−. Furthermore, α3 subunit mRNA and immunofluorescence decreased significantly between P 15 and P 25, a period implicated in the functional maturation of grid cells. Finally, α5 subunit immunoreactivity was detectable only on CB+ cells, not on RE+ cells. The present data demonstrates that physiologically distinct GABAAR subtypes are selectively expressed by CB+ and RE+ cells. This suggests that PV+ interneurons could utilize distinct postsynaptic signaling mechanisms to regulate the excitability of these different, candidate grid cell sub-populations.
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PV Interneurons: Critical Regulators of E/I Balance for Prefrontal Cortex-Dependent Behavior and Psychiatric Disorders
Article
Full-text available
  • May 2018
Elucidating the prefrontal cortical microcircuit has been challenging, given its role in multiple complex behaviors, including working memory, cognitive flexibility, attention, social interaction and emotional regulation. Additionally, previous methodological limitations made it difficult to parse out the contribution of certain neuronal subpopulations in refining cortical representations. However, growing evidence supports a fundamental role of fast-spiking parvalbumin (PV) GABAergic interneurons in regulating pyramidal neuron activity to drive appropriate behavioral responses. Further, their function is heavily diminished in the prefrontal cortex (PFC) in numerous psychiatric diseases, including schizophrenia and autism. Previous research has demonstrated the importance of the optimal balance of excitation and inhibition (E/I) in cortical circuits in maintaining the efficiency of cortical information processing. Although we are still unraveling the mechanisms of information representation in the PFC, the E/I balance seems to be crucial, as pharmacological, chemogenetic and optogenetic approaches for disrupting E/I balance induce impairments in a range of PFC-dependent behaviors. In this review, we will explore two key hypotheses. First, PV interneurons are powerful regulators of E/I balance in the PFC, and help optimize the representation and processing of supramodal information in PFC. Second, diminishing the function of PV interneurons is sufficient to generate an elaborate symptom sequelae corresponding to those observed in a range of psychiatric diseases. Then, using this framework, we will speculate on whether this circuitry could represent a platform for the development of therapeutic interventions in disorders of PFC function.
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A spike sorting toolbox for up to thousands of electrodes validated with ground truth recordings in vitro and in vivo
Article
Full-text available
  • Mar 2018
  • eLife
In recent years, multielectrode arrays and large silicon probes have been developed to record simultaneously between hundreds and thousands of electrodes packed with a high density. However, they require novel methods to extract the spiking activity of large ensembles of neurons. Here we developed a new toolbox to sort spikes from these large-scale extracellular data. To validate our method, we performed simultaneous extracellular and loose patch recordings in rodents to obtain 'ground truth' data, where the solution to this sorting problem is known for one cell. The performance of our algorithm was always close to the best expected performance, over a broad range of signal to noise ratios, in vitro and in vivo. The algorithm is entirely parallelized and has been successfully tested on recordings with up to 4225 electrodes. Our toolbox thus offers a generic solution to sort accurately spikes for up to thousands of electrodes.
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Rare GABRA3 variants are associated with epileptic seizures, encephalopathy and dysmorphic features
Article
Full-text available
  • Oct 2017
  • BRAIN
Genetic epilepsies are caused by mutations in a range of different genes, many of them encoding ion channels, receptors or transporters. While the number of detected variants and genes increased dramatically in the recent years, pleiotropic effects have also been recognized, revealing that clinical syndromes with various degrees of severity arise from a single gene, a single mutation, or from different mutations showing similar functional defects. Accordingly, several genes coding for GABAA receptor subunits have been linked to a spectrum of benign to severe epileptic disorders and it was shown that a loss of function presents the major correlated pathomechanism. Here, we identified six variants in GABRA3 encoding the α3-subunit of the GABAA receptor. This gene is located on chromosome Xq28 and has not been previously associated with human disease. Five missense variants and one microduplication were detected in four families and two sporadic cases presenting with a range of epileptic seizure types, a varying degree of intellectual disability and developmental delay, sometimes with dysmorphic features or nystagmus. The variants co-segregated mostly but not completely with the phenotype in the families, indicating in some cases incomplete penetrance, involvement of other genes, or presence of phenocopies. Overall, males were more severely affected and there were three asymptomatic female mutation carriers compared to only one male without a clinical phenotype. X-chromosome inactivation studies could not explain the phenotypic variability in females. Three detected missense variants are localized in the extracellular GABA-binding NH2-terminus, one in the M2-M3 linker and one in the M4 transmembrane segment of the α3-subunit. Functional studies in Xenopus laevis oocytes revealed a variable but significant reduction of GABA-evoked anion currents for all mutants compared to wild-type receptors. The degree of current reduction correlated partially with the phenotype. The microduplication disrupted GABRA3 expression in fibroblasts of the affected patient. In summary, our results reveal that rare loss-of-function variants in GABRA3 increase the risk for a varying combination of epilepsy, intellectual disability/developmental delay and dysmorphic features, presenting in some pedigrees with an X-linked inheritance pattern.
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Long-Lasting Rescue of Network and Cognitive Dysfunction in a Genetic Schizophrenia Model
Article
  • Aug 2019
  • CELL
Here we demonstrate that adult LgDel+/− mice, a genetic model of schizophrenia, exhibit deficits in PV neuron recruitment and associated chronic PV neuron plasticity together with network and cognitive dysfunctions. All these deficits can be permanently rescued by chemogenetic activation of PV neurons or local D2R antagonist treatments, specifically in the ventral hippocampus (vH) or medial-prefrontal cortex during a late-adolescence-sensitive time window. Therefore, progression to disease in schizophrenia-model mice can be prevented by treatments supporting vH-mPFC PV network function during a sensitive time window late in adolescence, suggesting a novel therapeutic strategies to prevent the outbreak of schizophrenia in a susceptible background.
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Evaluation of semi-automatic 3D reconstruction for studying geometry of dendritic spines
Article
  • Oct 2018
  • J CHEM NEUROANAT
Background: Spine geometry is considered to reflect synapse function. An accurate and fast method for 3D reconstruction of spines is considered a valuable tool for the purpose of studying spine geometry. Currently, most studies employ manual or automatic reconstruction methods, which still suffer from either poor accuracy or extreme time-consumption. The semi-automatic reconstruction method has previously been described as a time-economic and accurate tool for spine number counting. The purpose of this study is to further validate the semi-automatic method with regards to spine geometry investigation, by comparing it with the manual method as well as with the automatic method. Methods: In this study, dendritic trees of six pyramidal neurons that belong to layers II/III of mouse frontal cortex are stained using the Golgi method. Thereafter, spines from 42 dendritic branches are 3D reconstructed by manual, semi-automatic and automatic methods using Imaris software. Spine features, including spine volume, spine area, spine length and spine neck length, and the relative distribution of classified stubby, mushroom and thin spines are compared between the semi-automatic method and the two other methods. Results: Results from the semi-automatic and the manual reconstruction methods are in line with respect to all measured spine geometric features as well as spine classes. However, significant difference has been detected between the two methods and the automatic method in spine length, spine neck length and spine volume. Compared to the manual method, both the semi-automatic and the automatic methods have significantly reduced the spine reconstruction time. Conclusion: These findings suggest that the semi-automatic method may represent both a time-economic and reliable option for the purpose of studying spine geometry.
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PSD95: A synaptic protein implicated in schizophrenia or autism?
Article
  • Nov 2017
The molecular components of the postsynaptic density (PSD) in excitatory synapses of the brain are currently being investigated as one of the major etiologies of neurodevelopmental disorders such as schizophrenia (SCZ) and autism. Postsynaptic density protein-95 (PSD-95) is a major regulator of synaptic maturation by interacting, stabilizing and trafficking N-methyl-d-aspartic acid receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isox-azoleproprionic acid receptors (AMPARs) to the postsynaptic membrane. Recently, there has been overwhelming evidence that associates PSD-95 disruption with cognitive and learning deficits observed in SCZ and autism. For instance, recent genomic and sequencing studies of psychiatric patients highlight the aberrations at the PSD of glutamatergic synapses that include PSD-95 dysfunction. In animal studies, PSD-95 deficiency shows alterations in NMDA and AMPA-receptor composition and function in specific brain regions that may contribute to phenotypes observed in neuropsychiatric pathologies. In this review, we describe the role of PSD-95 as an essential scaffolding protein during synaptogenesis and neurodevelopment. More specifically, we discuss its interactions with NMDA receptor subunits that potentially affect glutamate transmission, and the formation of silent synapses during critical time points of neurodevelopment. Furthermore, we describe how PSD-95 may alter dendritic spine morphologies, thus regulating synaptic function that influences behavioral phenotypes in SCZ versus autism. Understanding the role of PSD-95 in the neuropathologies of SCZ and autism will give an insight of the cellular and molecular attributes in the disorders, thus providing treatment options in patients affected.
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