Karine Fénelon

Deficiency of Dgcr8, a gene disrupted by the 22q11.2 microdeletion, results in altered short-term plasticity in the prefrontal cortex

Individuals with 22q11.2 microdeletions have cognitive and behavioral impairments and the highest known genetic risk for developing schizophrenia. One gene disrupted by the 22q11.2 microdeletion is DGCR8, a component of the “microprocessor” complex that is essential for microRNA production, resulting in abnormal processing of specific brain miRNAs and working memory deficits. Here, we determine the effect of Dgcr8 deficiency on the structure and function of cortical circuits by assessing their laminar organization, as well as the neuronal morphology, and intrinsic and synaptic properties of layer 5 pyramidal neurons in the prefrontal cortex of Dgcr8+/− mutant mice. We found that heterozygous Dgcr8 mutant mice have slightly fewer cortical layer 2/4 neurons and that the basal dendrites of layer 5 pyramidal neurons have slightly smaller spines. In addition to the modest structural changes, field potential and whole-cell electrophysiological recordings performed in layer 5 of the prefrontal cortex revealed greater short-term synaptic depression during brief stimulation trains applied at 50 Hz to superficial cortical layers. This finding was accompanied by a decrease in the initial phase of synaptic potentiation. Our results identify altered short-term plasticity as a neural substrate underlying the cognitive dysfunction and the increased risk for schizophrenia associated with the 22q11.2 microdeletions.

The 22q11.2 microdeletion: Fifteen years of insights into the genetic and neural complexity of psychiatric disorders

Over the last fifteen years it has become established that 22q11.2 deletion syndrome (22q11DS) is a true genetic risk factor for schizophrenia. Carriers of deletions in chromosome 22q11.2 develop schizophrenia at rate of 25–30% and such deletions account for as many as 1–2% of cases of sporadic schizophrenia in the general population. Access to a relatively homogeneous population of individuals that suffer from schizophrenia as the result of a shared etiological factor and the potential to generate etiologically valid mouse models provides an immense opportunity to better understand the pathobiology of this disease. In this review we survey the clinical literature associated with the 22q11.2 microdeletions with a focus on neuroanatomical changes. Then, we highlight results from work modeling this structural mutation in animals. The key biological pathways disrupted by the mutation are discussed and how these changes impact the structure and function of neural circuits is described.

Molecular Substrates of Altered Axonal Growth and Brain Connectivity in a Mouse Model of Schizophrenia

22q11.2 deletion carriers show specific cognitive deficits, and ∼30% of them develop schizophrenia. One of the disrupted genes is ZDHHC8, which encodes for a palmitoyltransferase. We show that Zdhhc8-deficient mice have reduced palmitoylation of proteins that regulate axonal growth and branching. Analysis of axonal projections of pyramidal neurons from both Zdhhc8-deficient and Df(16)A(+/-) mice, which model the 22q11.2 deletion, revealed deficits in axonal growth and terminal arborization, which can be prevented by reintroduction of active ZDHHC8 protein. Impaired terminal arborization is accompanied by a reduction in the strength of synaptic connections and altered functional connectivity and working memory. The effect of ZDHHC8 is mediated in part via Cdc42-dependent modulation of Akt/Gsk3β signaling at the tip of the axon and can be reversed by pharmacologically decreasing Gsk3β activity during postnatal brain development. Our findings provide valuable mechanistic insights into the cognitive and psychiatric symptoms associated with a schizophrenia-predisposing mutation.

The pattern of cortical dysfunction in a mouse model of a schizophrenia-related microdeletion.

We used a mouse model of the schizophrenia-predisposing 22q11.2 microdeletion to evaluate how this genetic lesion affects cortical neural circuits at the synaptic, cellular, and molecular levels. Guided by cognitive deficits, we demonstrated that mutant mice display robust deficits in high-frequency synaptic transmission and short-term plasticity (synaptic depression and potentiation), as well as alterations in long-term plasticity and dendritic spine stability. Apart from previously reported reduction in dendritic complexity of layer 5 pyramidal neurons, altered synaptic plasticity occurs in the context of relatively circumscribed and often subtle cytoarchitectural changes in neuronal density and inhibitory neuron numbers. We confirmed the pronounced DiGeorge critical region 8 (Dgcr8)-dependent deficits in primary micro-RNA processing and identified additional changes in gene expression and RNA splicing that may underlie the effects of this mutation. Reduction in Dgcr8 levels appears to be a major driver of altered short-term synaptic plasticity in prefrontal cortex and working memory but not of long-term plasticity and cytoarchitecture. Our findings inform the cortical synaptic and neuronal mechanisms of working memory impairment in the context of psychiatric disorders. They also provide insight into the link between micro-RNA dysregulation and genetic liability to schizophrenia and cognitive dysfunction.

The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem

A unilateral activation of the mesencephalic locomotor region (MLR) produces symmetrical bilateral locomotion in all vertebrate species tested to date. How this occurs remains unresolved. This study examined the possibility that the symmetry occurred at the level of the inputs from the MLR to reticulospinal (RS) cells. In lamprey semi-intact preparations, we recorded intracellular responses of pairs of large, homologous RS cells on both sides to stimulation of the MLR on one side. The synaptic responses on both sides were very similar in shape, amplitude, and threshold intensity. Increasing MLR stimulation intensity produced a symmetrical increase in the magnitude of the responses on both sides. Ca2+ imaging confirmed the bilateral activation of smaller-sized RS cells as well. In a high-divalent cation solution, the synaptic responses of homologous RS cells persisted and exhibited a constant latency during high-frequency stimulation. Moreover, during gradual replacement of normal Ringers solution with a Ca2+-free solution, the magnitude of responses showed a gradual reduction with a similar time course in the homologous RS cells. These results support the idea that the MLR projects monosynaptically to RS cells on both sides with symmetrical inputs. During locomotion of the semi-intact preparation, the discharge pattern was also very similar in homologous bilateral RS cells. Anatomical experiments confirmed the presence of MLR neurons projecting ipsilaterally to the reticular formation intermingled with neurons projecting contralaterally. We conclude that the bilaterally symmetrical MLR inputs to RS cells are likely contributors to generating symmetrical locomotor activity.

Evidence for altered hippocampal function in a mouse model of the human 22q11.2 microdeletion.

22q11.2 chromosomal deletions are recurrent copy number mutations that increase the risk of schizophrenia around thirty-fold. Deletion of the orthologous chromosomal region in mice offers an opportunity to characterize changes to neuronal structure and function that may account for the development of this disease. The hippocampus has been implicated in schizophrenia pathogenesis, is reduced in volume in 22q11.2 deletion carriers and displays altered neuronal structure in a mouse model of the mutation (Df(16)A(+/-) mice). Here we investigate hippocampal CA1 physiology, hippocampal-dependent spatial memory and novelty-induced hippocampal activation in Df(16)A(+/-) mice. We found normal spatial reference memory (as assayed by the Morris water maze test) as well as modest but potentially important deficits in physiology. In particular, a reduction in the level of inhibition of CA1 pyramidal neurons was observed, implying a decrease in interneuron activity. Additionally, deficits in LTP were observed using certain induction protocols. Induction of c-Fos expression by exploration of a novel environment suggested a relative sparing of CA1 and dentate gyrus function but showed a robust decrease in the number of activated CA3 pyramidal neurons in Df(16)A(+/-) mice. Overall, experiments performed in this 22q11.2 deletion model demonstrated deficits of various degrees across different regions of the hippocampus, which together may contribute to the increased risk of developing schizophrenia.

The Contribution of Synaptic Inputs to Sustained Depolarizations in Reticulospinal Neurons

Sensory stimulation elicits sustained depolarizations in lamprey reticulospinal (RS) cells for which intrinsic properties were shown to play a crucial role. The depolarizations last up to minutes, and we tested whether the intrinsic properties required the cooperation of synaptic inputs to maintain RS cells depolarized for such long periods of time. Ascending spinal inputs to RS cells were reversibly blocked by applying xylocaine over the rostral spinal cord segments. The duration of the sustained depolarizations was markedly reduced. The membrane potential oscillations in tune with locomotor activity that were present under control condition were also abolished. The contribution of excitatory glutamatergic inputs was then assessed by applying CNQX and AP-5 over one of two simultaneously recorded homologous RS cells on each side of the brainstem. The level of sensory-evoked depolarization decreased significantly in the cell exposed to the antagonists compared with the other RS cell monitored as a control. In contrast, local application of glycine only produced a transient membrane potential hyperpolarization with a marked reduction in the amplitude of membrane potential oscillations. Locally applied strychnine did not change the duration of the sustained depolarizations, suggesting that mechanisms other than glycinergic inhibition are involved in ending the sustained depolarizations in RS cells. It is concluded that excitatory glutamatergic inputs, including ascending spinal feedback, cooperate with intrinsic properties of RS cells to maintain the cells depolarized for prolonged periods, sustaining long bouts of escape swimming.

Role of calsequestrin evaluated from changes in free and total calcium concentrations in the sarcoplasmic reticulum of frog cut skeletal muscle fibres

Calsequestrin is a large‐capacity Ca‐binding protein located in the terminal cisternae of sarcoplasmic reticulum (SR) suggesting a role as a buffer of the concentration of free Ca in the SR ([Ca2+]SR) serving to maintain the driving force for SR Ca2+ release. Essentially all of the functional studies on calsequestrin to date have been carried out on purified calsequestrin or on disrupted muscle preparations such as terminal cisternae vesicles. To obtain information about calsequestrins properties during physiological SR Ca2+ release, experiments were carried out on frog cut skeletal muscle fibres using two optical methods. One – the EGTA–phenol red method – monitored the content of total Ca in the SR ([CaT]SR) and the other used the low affinity Ca indicator tetramethylmurexide (TMX) to monitor the concentration of free Ca in the SR. Both methods relied on a large concentration of the Ca buffer EGTA (20 mm), in the latter case to greatly reduce the increase in myoplasmic [Ca2+] caused by SR Ca2+ release thereby almost eliminating the myoplasmic component of the TMX signal. By releasing almost all of the SR Ca, these optical signals provided information about [CaT]SR versus [Ca2+]SR as [Ca2+]SR varied from its resting level ([Ca2+]SR,R) to near zero. Since almost all of the Ca in the SR is bound to calsequestrin, this information closely resembles the binding curve of the Ca–calsequestrin reaction. Calcium binding to calsequestrin was found to be cooperative (estimated Hill coefficient = 2.95) and to have a very high capacity (at the start of Ca2+ release, 23 times more Ca was estimated to initiate from calsequestrin as opposed to the pool of free Ca in the SR). The latter result contrasts with an earlier report that only ∼25% of released Ca2+ comes from calsequestrin and ∼75% comes from the free pool. The value of [Ca2+]SR,R was close to the KD for calsequestrin, which has a value near 1 mm in in vitro studies. Other evidence indicates that [Ca2+]SR,R is near 1 mm in cut fibres. These results along with the known rapid kinetics of the Ca–calsequestrin binding reaction indicate that calsequestrins properties are optimized to buffer [Ca2+]SR during rapid, physiological SR Ca2+ release. Although the results do not entirely rule out a more active role in the excitation–contraction coupling process, they do indicate that passive buffering of [Ca2+]SR is a very important function of calsequestrin.

Extra activation component of calcium release in frog muscle fibres

In addition to activating more Ca2+ release sites via voltage sensors in the t‐tubular membranes, it has been proposed that more depolarised voltages enhance activation of Ca2+ release channels via a voltage‐dependent increase in Ca‐induced Ca2+ release (CICR). To test this, release permeability signals in response to voltage‐clamp pulses to two voltages, –60 and –45 mV, were compared when Δ[Ca2+] was decreased in two kinds of experiments. (1) Addition of 8 mm of the fast Ca2+ buffer BAPTA to the internal solution decreased release permeability at –45 mV by > 2‐fold and did not significantly affect Ca2+ release at –60 mV. Although some of this decrease may have been due to a decrease in voltage activation at –45 mV – as assessed from measurements of intramembranous charge movement – the results do tend to support a Ca‐dependent enhancement with greater depolarisations. (2) Decreasing SR (sarcoplasmic reticulum) Ca content ([CaSR]) should decrease the Ca2+ flux through an open channel and thereby Δ[Ca2+]. Decreasing [CaSR] from > 1000 μm (the physiological range) to < 200 μm decreased release permeability at –45 mV relative to that at –60 mV by > 6‐fold, an effect shown to be reversible and not attributable to a decrease in voltage activation at –45 mV. These results indicate a Ca‐dependent triggering of Ca2+ release at more depolarised voltages in addition to that expected by voltage control alone. The enhanced release probably involves CICR and appears to involve another positive feedback mechanism in which Ca2+ release speeds up the activation of voltage sensors.

Recruitment of Ca2+ release channels by calcium-induced Ca2+ release does not appear to occur in isolated Ca2+ release sites in frog skeletal muscle

Ca2+ release from the sarcoplasmic reticulum (SR) in skeletal muscle in response to small depolarisations (e.g. to ‐60 mV) should be the sum of release from many isolated Ca2+ release sites. Each site has one SR Ca2+ release channel activated by its associated T‐tubular voltage sensor. The aim of this study was to evaluate whether it also includes neighbouring Ca2+ release channels activated by Ca‐induced Ca2+ release (CICR). Ca2+ release in frog cut muscle fibres was estimated with the EGTA/phenol red method. The fraction of SR Ca content ([CaSR]) released by a 400 ms pulse to ‐60 mV (denoted fCa) provided a measure of the average Ca2+ permeability of the SR associated with the pulse. In control experiments, fCa was approximately constant when [CaSR] was 1500‐3000 μm (plateau region) and then increased as [CaSR] decreased, reaching a peak when [CaSR] was 300‐500 μm that was 4.8 times larger on average than the plateau value. With 8 mm of the fast Ca2+ buffer BAPTA in the internal solution, fCa was 5.0‐5.3 times larger on average than the plateau value obtained before adding BAPTA when [CaSR] was 300‐500 μm. In support of earlier results, 8 mm BAPTA did not affect Ca2+ release in the plateau region. At intermediate values of [CaSR], BAPTA resulted in a small, if any, increase in fCa, presumably by decreasing Ca inactivation of Ca2+ release. Since BAPTA never decreased fCa, the results indicate that neighbouring channels are not activated by CICR with small depolarisations when [CaSR] is 300‐3000 μm.