EphB1 controls long-range cortical axon guidance through a cell non-autonomous role in GABAergic cells.

EphB1 is required for proper guidance of cortical axon projections during brain development, but how EphB1 regulates this process remains unclear. We show here that EphB1 conditional knockout (cKO) in GABAergic cells (Vgat-Cre), but not in cortical excitatory neurons (Emx1-Cre), reproduced the cortical axon guidance defects observed in global EphB1 KO mice. Interestingly, in EphB1 cKOVgat mice, the misguided axon bundles contained co-mingled striatal GABAergic and somatosensory cortical glutamatergic axons. In wild-type mice, somatosensory axons also co-fasciculated with striatal axons, notably in the globus pallidus, suggesting that a subset of glutamatergic cortical axons normally follows long-range GABAergic axons to reach their targets. Surprisingly, the ectopic axons in EphB1 KO mice were juxtaposed to major blood vessels. However, conditional loss of EphB1 in endothelial cells (Tie2-Cre) did not produce the axon guidance defects, suggesting that EphB1 in GABAergic neurons normally promotes avoidance of these ectopic axons from the developing brain vasculature. Together, our data reveal a new role for EphB1 in GABAergic neurons to influence proper cortical glutamatergic axon guidance during brain development.

MEF2C Hypofunction in GABAergic Cells Alters Sociability and Prefrontal Cortex Inhibitory Synaptic Transmission in a Sex-Dependent Manner.

Background: Heterozygous mutations or deletions of MEF2C cause a neurodevelopmental disorder termed MEF2C haploinsufficiency syndrome (MCHS), characterized by autism spectrum disorder and neurological symptoms. In mice, global Mef2c heterozygosity has produced multiple MCHS-like phenotypes. MEF2C is highly expressed in multiple cell types of the developing brain, including GABAergic (gamma-aminobutyric acidergic) inhibitory neurons, but the influence of MEF2C hypofunction in GABAergic neurons on MCHS-like phenotypes remains unclear. Methods: We employed GABAergic cell type-specific manipulations to study mouse Mef2c heterozygosity in a battery of MCHS-like behaviors. We also performed electroencephalography, single-cell transcriptomics, and patch-clamp electrophysiology and optogenetics to assess the impact of Mef2c haploinsufficiency on gene expression and prefrontal cortex microcircuits. Results: Mef2c heterozygosity in developing GABAergic cells produced female-specific deficits in social preference and altered approach-avoidance behavior. In female, but not male, mice, we observed that Mef2c heterozygosity in developing GABAergic cells produced 1) differentially expressed genes in multiple cell types, including parvalbumin-expressing GABAergic neurons, 2) baseline and social-related frontocortical network activity alterations, and 3) reductions in parvalbumin cell intrinsic excitability and inhibitory synaptic transmission onto deep-layer pyramidal neurons. Conclusions: MEF2C hypofunction in female, but not male, developing GABAergic cells is important for typical sociability and approach-avoidance behaviors and normal parvalbumin inhibitory neuron function in the prefrontal cortex of mice. While there is no apparent sex bias in autism spectrum disorder symptoms of MCHS, our findings suggest that GABAergic cell-specific dysfunction in females with MCHS may contribute disproportionately to sociability symptoms.

NPAS4 in the medial prefrontal cortex mediates chronic social defeat stress-induced anhedonia-like behavior and reductions in excitatory synapses.

Chronic stress can produce reward system deficits (i.e., anhedonia) and other common symptoms associated with depressive disorders, as well as neural circuit hypofunction in the medial prefrontal cortex (mPFC). However, the molecular mechanisms by which chronic stress promotes depressive-like behavior and hypofrontality remain unclear. We show here that the neuronal activity-regulated transcription factor, NPAS4, in the mPFC is regulated by chronic social defeat stress (CSDS), and it is required in this brain region for CSDS-induced changes in sucrose preference and natural reward motivation in the mice. Interestingly, NPAS4 is not required for CSDS-induced social avoidance or anxiety-like behavior. We also find that mPFC NPAS4 is required for CSDS-induced reductions in pyramidal neuron dendritic spine density, excitatory synaptic transmission, and presynaptic function, revealing a relationship between perturbation in excitatory synaptic transmission and the expression of anhedonia-like behavior in the mice. Finally, analysis of the mice mPFC tissues revealed that NPAS4 regulates the expression of numerous genes linked to glutamatergic synapses and ribosomal function, the expression of upregulated genes in CSDS-susceptible animals, and differentially expressed genes in postmortem human brains of patients with common neuropsychiatric disorders, including depression. Together, our findings position NPAS4 as a key mediator of chronic stress-induced hypofrontal states and anhedonia-like behavior.

Repeated methamphetamine administration produces cognitive deficits through augmentation of GABAergic synaptic transmission in the prefrontal cortex.

Methamphetamine (METH) abuse is associated with the emergence of cognitive deficits and hypofrontality, a pathophysiological marker of many neuropsychiatric disorders that is produced by altered balance of local excitatory and inhibitory synaptic transmission. However, there is a dearth of information regarding the cellular and synaptic mechanisms underlying METH-induced cognitive deficits and associated hypofrontal states. Using PV-Cre transgenic rats that went through a METH sensitization regime or saline (SAL) followed by 7-10 days of home cage abstinence combined with cognitive tests, chemogenetic experiments, and whole-cell patch recordings on the prelimbic prefrontal cortex (PFC), we investigated the cellular and synaptic mechanisms underlying METH-induce hypofrontality. We report here that repeated METH administration in rats produces deficits in working memory and increases in inhibitory synaptic transmission onto pyramidal neurons in the PFC. The increased PFC inhibition is detected by an increase in spontaneous and evoked inhibitory postsynaptic synaptic currents (IPSCs), an increase in GABAergic presynaptic function, and a shift in the excitatory-inhibitory balance onto PFC deep-layer pyramidal neurons. We find that pharmacological blockade of D1 dopamine receptor function reduces the METH-induced augmentation of IPSCs, suggesting a critical role for D1 dopamine signaling in METH-induced hypofrontality. In addition, repeated METH administration increases the intrinsic excitability of parvalbumin-positive fast spiking interneurons (PV + FSIs), a key local interneuron population in PFC that contributes to the control of inhibitory tone. Using a cell type-specific chemogenetic approach, we show that increasing PV + FSIs activity in the PFC is necessary and sufficient to cause deficits in temporal order memory similar to those induced by METH. Conversely, reducing PV + FSIs activity in the PFC of METH-exposed rats rescues METH-induced temporal order memory deficits. Together, our findings reveal that repeated METH exposure increases PFC inhibitory tone through a D1 dopamine signaling-dependent potentiation of inhibitory synaptic transmission, and that reduction of PV + FSIs activity can rescue METH-induced cognitive deficits, suggesting a potential therapeutic approach to treating cognitive symptoms in patients suffering from METH use disorder.

Targeted in utero electroporation of the ventro-temporal mouse retina.

Techniques enabling DNA delivery into mouse retinal cells using in utero electroporation are available. However, these techniques target the central retina and do not enable the electroporation of the ventro-temporal retina where ipsilateral retinal ganglion cells are located. Here, we describe a protocol to specifically electroporate the ventro-temporal retina, a critical approach to manipulate ipsilaterally projecting retinal ganglion cells and contralaterally projecting neurons located in the same region of the retina. The procedure is adaptable to target other retinal quadrants. For complete details on the use and execution of this protocol, please refer to Louail et al. (2020).

Sex-dependent role for EPHB2 in brain development and autism-associated behavior.

Autism spectrum disorder (ASD) is characterized by impairments in social communication and interaction and restricted, repetitive behaviors. It is frequently associated with comorbidities, such as attention-deficit hyperactivity disorder, altered sensory sensitivity, and intellectual disability. A de novo nonsense mutation in EPHB2 (Q857X) was discovered in a female patient with ASD [13], revealing EPHB2 as a candidate ASD risk gene. EPHB2 is a receptor tyrosine kinase implicated in axon guidance, synaptogenesis, and synaptic plasticity, positioning it as a plausible contributor to the pathophysiology of ASD and related disorders. In this study, we show that the Q857X mutation produced a truncated protein lacking forward signaling and that global disruption of one EphB2 allele (EphB2+/-) in mice produced several behavioral phenotypes reminiscent of ASD and common associated symptoms. EphB2+/- female, but not male, mice displayed increased repetitive behavior, motor hyperactivity, and learning and memory deficits, revealing sex-specific effects of EPHB2 hypofunction. Moreover, we observed a significant increase in the intrinsic excitability, but not excitatory/inhibitory ratio, of motor cortex layer V pyramidal neurons in EphB2+/- female, but not male, mice, suggesting a possible mechanism by which EPHB2 hypofunction may contribute to sex-specific motor-related phenotypes. Together, our findings suggest that EPHB2 hypofunction, particularly in females, is sufficient to produce ASD-associated behaviors and altered cortical functions in mice.

cAMP-Dependent Co-stabilization of Axonal Arbors from Adjacent Developing Neurons.

Axonal arbors in many neuronal networks are exuberant early during development and become refined by activity-dependent competitive mechanisms. Theoretical work proposed non-competitive interactions between co-active axons to co-stabilize their connections, but the demonstration of such interactions is lacking. Here, we provide experimental evidence that reducing cyclic AMP (cAMP) signaling in a subset of retinal ganglion cells favors the elimination of thalamic projections from neighboring neurons, pointing to a cAMP-dependent interaction that promotes axon stabilization.

MEF2C Hypofunction in Neuronal and Neuroimmune Populations Produces MEF2C Haploinsufficiency Syndrome-like Behaviors in Mice.

BACKGROUND: Microdeletions of the MEF2C gene are linked to a syndromic form of autism termed MEF2C haploinsufficiency syndrome (MCHS). MEF2C hypofunction in neurons is presumed to underlie most of the symptoms of MCHS. However, it is unclear in which cell populations MEF2C functions to regulate neurotypical development. METHODS: Multiple biochemical, molecular, electrophysiological, behavioral, and transgenic mouse approaches were used to characterize MCHS-relevant synaptic, behavioral, and gene expression changes in mouse models of MCHS. RESULTS: We showed that MCHS-associated missense mutations cluster in the conserved DNA binding domain and disrupt MEF2C DNA binding. DNA binding-deficient global Mef2c heterozygous mice (Mef2c-Het) displayed numerous MCHS-related behaviors, including autism-related behaviors, changes in cortical gene expression, and deficits in cortical excitatory synaptic transmission. We detected hundreds of dysregulated genes in Mef2c-Het cortex, including significant enrichments of autism risk and excitatory neuron genes. In addition, we observed an enrichment of upregulated microglial genes, but this was not due to neuroinflammation in the Mef2c-Het cortex. Importantly, conditional Mef2c heterozygosity in forebrain excitatory neurons reproduced a subset of the Mef2c-Het phenotypes, while conditional Mef2c heterozygosity in microglia reproduced social deficits and repetitive behavior. CONCLUSIONS: Taken together, our findings show that mutations found in individuals with MCHS disrupt the DNA-binding function of MEF2C, and DNA binding-deficient Mef2c global heterozygous mice display numerous MCHS-related phenotypes, including excitatory neuron and microglia gene expression changes. Our findings suggest that MEF2C regulates typical brain development and function through multiple cell types, including excitatory neuronal and neuroimmune populations.

Opposing Regulation of Cocaine Seeking by Glutamate and GABA Neurons in the Ventral Pallidum.

Projections from the nucleus accumbens to the ventral pallidum (VP) regulate relapse in animal models of addiction. The VP contains GABAergic (VPGABA) and glutamatergic (VPGlu) neurons, and a subpopulation of GABAergic neurons co-express enkephalin (VPPenk). Rabies tracing reveals that VPGlu and VPPenk neurons receive preferential innervation from upstream D1- relative to D2-expressing accumbens neurons. Chemogenetic stimulation of VPGlu neurons inhibits, whereas stimulation of VPGABA and VPPenk neurons potentiates cocaine seeking in mice withdrawn from intravenous cocaine self-administration. Calcium imaging reveals cell type-specific activity patterns when animals learn to suppress drug seeking during extinction training versus engaging in cue-induced cocaine seeking. During cued seeking, VPGABA neurons increase their overall activity, and VPPenk neurons are selectively activated around nose pokes for cocaine. In contrast, VPGlu neurons increase their spike rate following extinction training. These data show that VP subpopulations differentially encode and regulate cocaine seeking, with VPPenk and VPGABA neurons facilitating and VPGlu neurons inhibiting cocaine seeking.

Emerging roles for MEF2 in brain development and mental disorders.

The MEF2 family of transcription factors regulate large programs of gene expression important for the development and maintenance of many tissues, including the brain. MEF2 proteins are regulated by neuronal synaptic activity, and they recruit several epigenetic enzymes to influence chromatin structure and gene expression during development and throughout adulthood. Here, we provide a brief review of the recent literature reporting important roles for MEF2 during early brain development and function, and we highlight emerging roles for MEF2 as a risk factor for multiple neurodevelopmental disorders and mental illnesses, such as autism, intellectual disability, and schizophrenia.

RIM1/2 in retinal ganglion cells are required for the refinement of ipsilateral axons and eye-specific segregation.

Neural activity is crucial for the refinement of neuronal connections during development, but the contribution of synaptic release mechanisms is not known. In the mammalian retina, spontaneous neural activity controls the refinement of retinal projections to the dorsal lateral geniculate nucleus (dLGN) and the superior colliculus (SC) to form appropriate topographic and eye-specific maps. To evaluate the role of synaptic release, the rab-interacting molecules (RIMs), a family of active zone proteins that play a central role in calcium-triggered release, were conditionally ablated in a subset of retinal ganglion cells (RGCs). We found that this deletion is sufficient to reduce presynaptic release probability onto dLGN neurons. Furthermore, eye-specific segregation in the dLGN and topographic refinement of ipsilateral axons in the SC and the dLGN, are impaired in RIM1/2 conditional knock-out (Rim-cDKO) mice. These defects are similar to those found when retinal activity is globally disturbed. However, reduction in synaptic release had no effect on eye-specific lamination in the SC nor on the retinotopic refinement of contralateral axons in the SC. This study highlights a potential distinction between synaptic and non-synaptic roles of neuronal activity for different mapping rules operating in visual system development.

A plasma membrane microdomain compartmentalizes ephrin-generated cAMP signals to prune developing retinal axon arbors.

The development of neuronal circuits is controlled by guidance molecules that are hypothesized to interact with the cholesterol-enriched domains of the plasma membrane termed lipid rafts. Whether such domains enable local intracellular signalling at the submicrometre scale in developing neurons and are required for shaping the nervous system connectivity in vivo remains controversial. Here, we report a role for lipid rafts in generating domains of local cAMP signalling in axonal growth cones downstream of ephrin-A repulsive guidance cues. Ephrin-A-dependent retraction of retinal ganglion cell axons involves cAMP signalling restricted to the vicinity of lipid rafts and is independent of cAMP modulation outside of this microdomain. cAMP modulation near lipid rafts controls the pruning of ectopic axonal branches of retinal ganglion cells in vivo, a process requiring intact ephrin-A signalling. Together, our findings indicate that lipid rafts structure the subcellular organization of intracellular cAMP signalling shaping axonal arbors during the nervous system development.

Activity dependent mechanisms of visual map formation--from retinal waves to molecular regulators.

The refinement of neural connections requires activity-dependent mechanisms in addition to the genetic program initially establishing wiring diagrams. The well-understood organization of the visual system makes it an accessible model for analyzing the contribution of activity in the formation of connectivity. Prior to visual experience, patterned spontaneous activity in the form of retinal waves has an important role for the establishment of eye-specific and retinotopic maps by acting on the refinement of axon arborization. In the present review, which focuses on experimental data obtained in mice and ferrets, we highlight the features of retinal activity that are important for visual map formation and question whether synaptic release and Hebbian based competition rules apply to this system. Recent evidence using genetic tools that allowed the manipulation of different features of neural activity have clarified the controversy on whether activity is instructive or permissive for visual map formation. Furthermore, current evidence strongly suggests that different mechanisms are at play for different types of axons (ipsilateral vs. contralateral), maps (eye-specific vs. retinotopic) or targets. Many molecules that either modulate activity or are modulated by activity are important in the formation of the visual map, such as adenylate cyclase 1, serotonin, or molecules from the immune system. Finally, new players in the game include retrograde messengers signaling from the target cell to the retinal axons as well as microglia that could help to eliminate inappropriate synapses.