ABSTRACT
Schizophrenia is a severely debilitating neurodevelopmental disorder. Establishing a causal link between circuit dysfunction and particular behavioural traits relevant to schizophrenia is crucial to shed new light on the mechanisms underlying the pathology. Here we studied an animal model of the 22q11 deletion syndrome, which is the highest genetic risk to develop the pathology. We report a desynchronization of hippocampal neuronal assemblies that resulted from parvalbumin interneuron hypoexcitability. Rescuing parvalbumin interneuron excitability with pharmacological or chemogenetic approaches is sufficient to restore wild type-like network dynamics and behaviour during adulthood. In conclusion, our data provide mechanistic insights underlying network dysfunction relevant to schizophrenia and demonstrate the potential of reverse engineering in fostering new therapeutic strategies to alleviate the burden of neurodevelopmental disorders.
Main text
Alterations of network dynamics have been proposed to be instrumental in schizophrenia1-3. A specific population of inhibitory neurons, the parvalbumin interneurons (PVIs), plays a key role in regulating network dynamics4-7 and may be involved in the pathology8-11. Although specific manipulations of PVI can reproduce behavioural phenotypes relevant to schizophrenia in rodents12,13, it remains unclear whether PVI dysfunction is causally linked to network dysfunction and pathological behaviour associated with schizophrenia. More importantly, it is not known whether manipulating PVI could restore altered physiology.
Among various genetic alterations, the specific deletion of ~30 genes on chromosome 22 that leads to the 22q11 deletion syndrome (22q11DS), is the highest identified genetic risk to develop schizophrenia14,15. We used a genetically engineered mouse bearing a hemizygous deletion on chromosome 16, termed Lgdel/+, which replicates the chromosomal alteration of the human 22q11DS16. In the CA1 area of the hippocampus, mouse models of 22q11DS differ from wild-type (WT) animals regarding their structural17-19 and electrophysiological properties20, and their functional connectivity with distant brain areas3. We first tested whether those differences were accompanied by intrinsic differences in network dynamics. Neural activity was monitored in hippocampal slices using the genetically encoded calcium indicator GCaMP6s expressed by CA1 neurons following adeno-associated viral (AAV) vector transfection (Fig. 1a,b). Network dynamics were induced by bath application of carbachol (50 μM), which triggered spontaneous calcium activity in individual neurons of wild type (WT) mice21,22 (Fig. 1c,d). Likewise, individual CA1 neurons of Lgdel/+ mice exhibited spontaneous calcium activity during the duration of the recording. Neither the fraction of active neurons (Fig. 1e), nor the mean frequency (Fig. 1f), the mean amplitude (Fig. 1g) and the mean duration (Fig. 1h) of calcium transients were significantly different between genotypes. In contrast, CA1 ensemble dynamics was strongly altered in Lgdel/+ mice. Indeed, neurons were largely desynchronized in respect to each other and the oscillations observed at a population level were largely reduced in Lgdel/+ mice (Fig. 1c,d). We thus quantified population activity and neuronal correlations and compared these quantities to data randomly shuffled in time. This enabled to control for the influence of neuronal co-activation occurring by chance (Supplementary Figs. 2 and 3). Lgdel/+ mice displayed less co-active cells (Fig. 1i), fewer occurrences of these co-activations (Fig. 1j) and less correlated neurons (Fig. 1k,l) in respect to WT animals. In summary, brain slices from Lgdel/+ mice exhibited a strong desynchronization of the CA1 hippocampal network.
To gain further insights into the mechanism underlying hippocampal desynchronization, we performed patch-clamp recordings of various neuronal populations in the CA1 region. First, we recorded spontaneous excitatory and inhibitory post-synaptic currents (sEPSCs and sIPSCs, respectively) in pyramidal cells (Fig. 2a). Consistent with a previous report20, CA1 pyramids recorded from Lgdel/+ mice had a comparable number of sEPSCs but less sIPSCs per second compared to CA1 pyramids in WT mice (Fig. 2a-d). In addition, no difference in sPSC amplitude (Supplementary Fig. 4a-c), in mEPSC frequency (Supplementary Fig. 4d) and in mIPSC frequency (Fig. 2e) was observed between the two genotypes. These results provide evidence that hippocampal pyramids in Lgdel/+ mice were characterized by hypoinhibition, likely to originate from the firing activity of GABAergic neurons.
We further tested the latter hypothesis by recording genetically labelled PVIs (Fig. 2f), which exert an inhibitory control over pyramidal cells23,24 in the hippocampal region. An analysis of input/output function in current-clamp experiments revealed that CA1 PVIs were less excitable in Lgdel/+ than in WT animals when recorded in artificial cerebrospinal fluid (Fig. 2g,h). Interestingly, such firing difference disappeared when GABAA, AMPA and NMDA receptors were blocked pharmacologically (Fig. 2i). Thus, the PVI hypoexcitability may rather reflect differences in PVI synaptic inputs than changes of their intrinsic properties. Supporting this notion, CA1 PVIs recorded from Lgdel/+ mice received similar number of sEPSCs but more sIPSCs per second than CA1 PVI neurons recorded in WT animals (Fig. 2j,k). No differences in sPSC amplitude (Supplementary Fig. 4G-I), mEPSC frequency (Supplementary Fig. 4j) and mIPSC frequency (Fig. 2n) were observed. Taken together, our data show that CA1 pyramidal cells of Lgdel/+ mice are hypoinhibited and that PVI are hypoexcitable.
Could network desynchronization be due to PVI hypoexcitability? We used a cell-autonomous chemogenetic approach by specifically infecting CA1 PVIs from Pvalbcre/+;+/+ mice with AAV enabling the conditional expression of the inhibitory designer receptor exclusively activated by designer drug (DREADD) hM4Gi, which is selectively activated by the inert drug clozapine-N-oxide (CNO)25. Effectively, reducing PVI excitability desynchronized CA1 assemblies in WT mice (Supplementary Fig. 5). Thus, could hippocampal network dynamics be rescued in Lgdel/+ mice by increasing PVI excitability? First, we tested a pharmacological approach by using a Neuregulin 1 peptide (NRG1), which increases PVI excitability in WT mice26,27. Second, we specifically expressed the excitatory DREADD hM3Gq in CA1 PVIs from Pvalbcre/+;Lgdel/+ mice. During path-clamp recordings of hippocampal slices, both strategies (Fig. 3a-f) raised PVI excitability and restored input/output functions comparable to the ones recorded from WT mice (difference between WT and treatments in Lgdel/+: two-way repeated measures ANOVA F1,29 = 0.44, P = 0.51 and F1,28 = 0.12, P = 0.73 for NRG1 and CNO treatment, respectively). We then tested the effect of the two treatments on CA1 network dynamics by pre-incubating hippocampal slices with either NRG1 or CNO prior to calcium imaging. Strikingly, both strategies increased neuronal correlations (Fig. 3g-j) and co-activations (Supplementary Fig. 6) in Lgdel/+ mice to a level comparable to WT littermates. Thus, counterbalancing the PVI hypoexcitability of Lgdel/+ mice was sufficient to extinguish the network desynchronization observed in brain slices.
Although 22q11DS is considered to be a neurodevelopmental disorder, we investigated whether the same pharmacological and chemogenetic treatments are efficient in adult animals. We first performed in vivo electrophysiological recordings in the dorsal CA1 area (dCA1) of awake mice (Fig. 4a and Supplementary Fig. 7a). A spectral analysis of the local field potential (LFP) revealed lower power in Lgdel/+ mice compared to WT animals in the theta frequency band (5-8Hz; Fig. 4a,b and Supplementary Fig. 7b, Kolmogorov-Smirnov test P = 8.1 x 10−4). Neuronal oscillations in the theta frequency band are crucial to hippocampal functions28 and their strength can be modulated by PVIs activations5. Interestingly, NRG1 injections increased the power of theta oscillations to levels close to WT mice (Fig. 4a-c, Kolmogorov-Smirnov test P = 0.1). To improve the specificity of our manipulation, we infected dCA1 PVI of Pvalbcre/+;Lgdel/+ mice with an AAV enabling the conditional expression of the hM3Gq DREADD (Supplementary Fig. 7c). CNO injections also increased the LFP power up to levels comparable to WT mice (Fig. 4a-c, Kolmogorov-Smirnov test P = 0.67). In summary, NRG1 injection and chemogenetic excitation of PVIs in adult animals were sufficient to establish WT-like network dynamics in awake Lgdel/+ mice.
Could the same pharmacological and chemogenetic manipulations also affect hippocampal-dependent behaviours? As observed in various animal models of schizophrenia29 including the 22q11DS mouse model18, adult Lgdel/+ mice exhibit hyper-locomotor activity in comparison to their WT littermates in the open field test (Fig. 4d,e). Intraperitoneal injections of NRG1 in Lgdel/+ mice (30-60 min prior to open field testing) and selective excitation of dCA1 PVI expressing the hM3D DREADD led to a reduction in the distance travelled to levels similar to the ones reached by WT littermates (Fig. 4d,e; Mann-Whitney test P = 0.15 and P = 0.55, respectively). Therefore increasing PVI excitability in adult Lgdel/+ mice was sufficient to induce a behavioural pattern similar to WT littermates, though the converse is not true. Indeed, when we selectively inhibited dCA1 PVI expressing the hM4D DREADD, the locomotion of WT mice was not significantly affected12 (Supplementary Fig. 8a,b).
In conclusion, our findings provide a mechanistic explanation of the alterations observed in an animal model of 22q11DS at the cellular and network levels. Our work suggests that the hypo-excitability of inhibitory neurons such as PVI leads to an alteration of the neuronal synchronization in the hippocampus and probably in other brain areas30. Although the 22q11DS is a neurodevelopmental disorder, our data demonstrate that selective neuronal manipulations during adulthood are sufficient to restore functional brain dynamics and typical behavioural patterns. Furthermore, our results highlight PVIs as a valuable therapeutical target for 22q11DS and similar neuropsychiatric disorders.
AUTHOR CONTRIBUTIONS
T.M., D.M. and A.C. carried out the study conceptualization. T.M., R.S., C.B., S.M., M.D.R., I.R., D.M. and A.C. contributed to the experimental design. TM performed the calcium imaging experiments and their analyses. C.B. carried out the in vitro electrophysiological experiments. R.S. performed the in vivo electrophysiological recordings and developed most of the MATLAB-based programs used for the analysis of the calcium imaging and the electrophysiological recordings. M.D.R. developed some MATLAB-based scripts used to analyze calcium imaging data. T.M. performed behavioral experiments with the help of S.M. A.C., T.M., R.S. and I.R. wrote and edited the manuscript with comments from all other authors.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
ACKNOWLEDGEMENTS
This paper is dedicated to the memory of Dominique Müller, whose ideas inspired this study and will continue to inspire all of us. We thank Raju Kucherlapati for generously providing the Lgdel/+ mice. We thank Lorena Jourdain and MariePriscille Hervé, for their technical support. We thank Yann Bernardelli, Pablo Mendez Garcia, Thomas Stefanelli, Stéphane Eliez, Pico Caroni and other members of the NCCR SYNAPSY for helpful discussions and/or comments on the manuscript. This research was supported by the University of Geneva, the Swiss National Science Foundation (grant numbers: 31003A_172878 to A.C., 310030B_144080 to D.M. and 310030E_135910 to I.R.), the National Center of Competence in Research (NCCR) “SYNAPSY - The Synaptic Bases of Mental Diseases” financed by the Swiss National Science Foundation (grant 51NF40-158776, D.M. and A.C.) and the Lejeune Foundation (T.M.).
Footnotes
↵† Deceased on 29 April 2015.