Probes for GAD2

While apneas are associated with multiple pathological and fatal conditions, the underlying molecular mechanisms remain elusive. We report that a mutated form of the transcription factor Mafa (Mafa4A) that prevents phosphorylation of the Mafa protein leads to an abnormally high incidence of breath holding apneas and death in newborn Mafa4A/4A mutant mice. This apneic breathing is phenocopied by restricting the mutation to central GABAergic inhibitory neurons and by activation of inhibitory Mafa neurons while reversed by inhibiting GABAergic transmission centrally. We find that Mafa activates the Gad2 promoter in vitro and that this activation is enhanced by the mutation that likely results in increased inhibitory drives onto target neurons. We also find that Mafa inhibitory neurons are absent from respiratory, sensory (primary and secondary) and pontine structures but are present in the vicinity of the hypoglossal motor nucleus including premotor neurons that innervate the geniohyoid muscle, to control upper airway patency. Altogether, our data reveal a role for Mafa phosphorylation in regulation of GABAergic drives and suggest a mechanism whereby reduced premotor drives to upper airway muscles may cause apneic breathing at birth.

The lateral habenula (LHb) sends complex projections to several areas of the mesopontine tegmentum, the raphe, and the hypothalamus. However, few markers have been available to distinguish subsets of LHb neurons that may serve these pathways. In order to address this complexity, we examined the mouse and rat LHb for neurons that express the GABA biosynthesis enzymes glutamate decarboxylase 1 and 2 (GAD1, GAD2), and the vesicular GABA transporter (VGAT). The mouse LHb contains a population of neurons that express GAD2, while the rat LHb contains discrete populations of neurons that express GAD1 and VGAT. However, we could not detect single neurons in either species that co-express a GABA synthetic enzyme and VGAT, suggesting that these LHb neurons do not use GABA for conventional synaptic transmission. Instead, all of the neuronal types expressing a GABAergic marker in both species showed co-expression of the glutamate transporter VGluT2. Anterograde tract-tracing of the projections of GAD2-expressing LHb neurons in Gad2Cre mice, combined with retrograde tracing from selected downstream nuclei, show that LHb-GAD2 neurons project selectively to the midline structures in the mesopontine tegmentum, including the median raphe and nucleus incertus, and only sparsely innervate the hypothalamus, rostromedial tegmental nucleus, and ventral tegmental area. Postsynaptic recording of LHb-GAD2 neuronal input to tegmental neurons confirms that glutamate, not GABA, is the fast neurotransmitter in this circuit. Thus GAD2 expression can serve as a marker for functional studies of excitatory neurons serving specific LHb output pathways in mice.SIGNFICANCE STATEMENT The lateral habenula provides a major link between subcortical forebrain areas and the dopamine (DA) and serotonin (5HT) systems of the midbrain and pons, and it has been implicated in reward mechanisms and the regulation of mood states. Few markers have been available for the specific cell types and complex output pathways of the lateral habenula. Here we examined the expression of genes mediating GABAergic and glutamatergic transmission in the mouse and rat LHb, where no neurons in either species expressed a full complement of GABAergic markers, and all expressed the glutamatergic marker VGluT2. Consistent with this, in mice the LHb GAD2 neurons are excitatory and appear to use only glutamate for fast synaptic transmission.

Early Alzheimer's disease (AD) is associated with hippocampal hyperactivity and decreased sleep quality. Here we show that homeostatic mechanisms transiently counteract the increased excitatory drive to CA1 neurons in AppNL-G-F mice, but that this mechanism fails in older mice. Spatial transcriptomics analysis identifies Pmch as part of the adaptive response in AppNL-G-F mice. Pmch encodes melanin-concentrating hormone (MCH), which is produced in sleep-active lateral hypothalamic neurons that project to CA1 and modulate memory. We show that MCH downregulates synaptic transmission, modulates firing rate homeostasis in hippocampal neurons and reverses the increased excitatory drive to CA1 neurons in AppNL-G-F mice. AppNL-G-F mice spend less time in rapid eye movement (REM) sleep. AppNL-G-F mice and individuals with AD show progressive changes in morphology of CA1-projecting MCH axons. Our findings identify the MCH system as vulnerable in early AD and suggest that impaired MCH-system function contributes to aberrant excitatory drive and sleep defects, which can compromise hippocampus-dependent functions.

A great deal of evidence supports the inevitable importance of spinal glycinergic inhibition in the development of chronic pain conditions. However, it remains unclear how glycinergic neurons contribute to the formation of spinal neural circuits underlying pain-related information processing. Thus, we intended to explore the synaptic targets of spinal glycinergic neurons in the pain processing region (laminae I-III) of the spinal dorsal horn by combining transgenic technology with immunocytochemistry and in situ hybridization accompanied by light and electron microscopy. First, our results suggest that, in addition to neurons in laminae I-III, glycinergic neurons with cell bodies in lamina IV may contribute substantially to spinal pain processing. On the one hand, we show that glycine transporter 2 immunostained glycinergic axon terminals target almost all types of excitatory and inhibitory interneurons identified by their neuronal markers in laminae I-III. Thus, glycinergic postsynaptic inhibition, including glycinergic inhibition of inhibitory interneurons, must be a common functional mechanism of spinal pain processing. On the other hand, our results demonstrate that glycine transporter 2 containing axon terminals target only specific subsets of axon terminals in laminae I-III, including nonpeptidergic nociceptive C fibers binding IB4 and nonnociceptive myelinated A fibers immunoreactive for type 1 vesicular glutamate transporter, indicating that glycinergic presynaptic inhibition may be important for targeting functionally specific subpopulations of primary afferent inputs.

Sevoflurane has been the most widely used inhaled anesthetics with a favorable recovery profile; however, the precise mechanisms underlying its anesthetic action are still not completely understood. Here the authors show that sevoflurane activates a cluster of urocortin 1 (UCN1+ )/cocaine- and amphetamine-regulated transcript (CART+ ) neurons in the midbrain involved in its anesthesia. Furthermore, growth hormone secretagogue receptor (GHSR) is highly enriched in sevoflurane-activated UCN1+ /CART+ cells and is necessary for sleep induction. Blockade of GHSR abolishes the excitatory effect of sevoflurane on UCN1+ /CART+ neurons and attenuates its anesthetic effect. Collectively, their data suggest that anesthetic action of sevoflurane necessitates the GHSR activation in midbrain UCN1+ /CART+ neurons, which provides a novel target including the nucleus and receptor in the field of anesthesia.

As the discovery of cellular diversity in the brain accelerates, so does the need for functional tools that target cells based on multiple features, such as gene expression and projection target. By selectively driving recombinase expression in a feature-specific manner, one can utilize intersectional strategies to conditionally promote payload expression only where multiple features overlap. We developed Conditional Viral Expression by Ribozyme Guided Degradation (ConVERGD), a single-construct intersectional targeting strategy that combines a self-cleaving ribozyme with traditional FLEx switches. ConVERGD offers benefits over existing platforms, such as expanded intersectionality, the ability to accommodate larger and more complex payloads, and a vector design that is easily modified to better facilitate rapid toolkit expansion. To demonstrate its utility for interrogating neural circuitry, we employed ConVERGD to target an unexplored subpopulation of norepinephrine (NE)-producing neurons within the rodent locus coeruleus (LC) identified via single-cell transcriptomic profiling to co-express the stress-related endogenous opioid gene prodynorphin ( Pdyn ). These studies showcase ConVERGD as a versatile tool for targeting diverse cell types and reveal Pdyn -expressing NE + LC neurons as a small neuronal subpopulation capable of driving anxiogenic behavioral responses in rodents.

Myelin transcription factor 1 like (Myt1l), a zinc-finger transcription factor, promotes neuronal differentiation and is implicated in autism spectrum disorder (ASD) and intellectual disability. However, it remains unclear whether Myt1l promotes neuronal differentiation in vivo and its deficiency in mice leads to disease-related phenotypes. Here, we report that Myt1l-heterozygous mutant (Myt1l-HT) mice display postnatal age-differential ASD-related phenotypes: newborn Myt1l-HT mice, with strong Myt1l expression, show ASD-like transcriptomic changes involving decreased synaptic gene expression and prefrontal excitatory synaptic transmission and altered righting reflex. Juvenile Myt1l-HT mice, with markedly decreased Myt1l expression, display reverse ASD-like transcriptomes, increased prefrontal excitatory transmission, and largely normal behaviors. Adult Myt1l-HT mice show ASD-like transcriptomes involving astrocytic and microglial gene upregulation, increased prefrontal inhibitory transmission, and behavioral deficits. Therefore, Myt1l haploinsufficiency leads to ASD-related phenotypes in newborn mice, which are temporarily normalized in juveniles but re-appear in adults, pointing to continuing phenotypic changes long after a marked decrease of Myt1l expression in juveniles.

Nicotine stimulates the dopamine (DA) system, which is essential for its rewarding effect. Nicotine is also aversive at high doses; yet, our knowledge about nicotine's dose-dependent effects on DA circuits remains limited. Here, we demonstrate that high doses of nicotine, which induce aversion-related behavior in mice, cause biphasic inhibitory and excitatory responses in VTA DA neurons that can be dissociated by distinct projections to lateral and medial nucleus accumben subregions, respectively. Guided by computational modeling, we performed a pharmacological investigation to establish that inhibitory effects of aversive nicotine involve desensitization of α4β2 and activation of α7 nicotinic acetylcholine receptors. We identify α7-dependent activation of upstream GABA neurons in the laterodorsal tegmentum (LDT) as a key regulator of heterogeneous DA release following aversive nicotine. Finally, inhibition of LDT GABA terminals in VTA prevents nicotine aversion. Together, our findings provide a mechanistic circuit-level understanding of nicotine's dose-dependent effects on reward and aversion.

Leigh syndrome is a mitochondrial disease characterized by neurodegeneration, neuroinflammation, and early death. Mice lacking NDUFS4, a mitochondrial complex I subunit (Ndufs4 KO mice), have been established as a good animal model for studying human pathology associated with Leigh syndrome. As the disease progresses, there is an increase in neurodegeneration and neuroinflammation, thereby leading to deteriorating neurological symptoms, including motor deficits, breathing alterations, and eventually, death of the animal. However, despite the magnitude of neuroinflammation associated with brain lesions, the role of neuroinflammatory pathways and their main cellular components have not been addressed directly as relevant players in the disease pathology. Here, we investigate the role of microglial cells, the main immune cells of the CNS, in Leigh-like syndrome pathology, by pharmacologically depleting them using the colony-stimulating factor 1 receptor antagonist PLX3397. Microglial depletion extended lifespan and delayed motor symptoms in Ndufs4 KO mice, likely by preventing neuronal loss. Next, we investigated the role of the major cytokine interleukin-6 (IL-6) in the disease progression. IL-6 deficiency partially rescued breathing abnormalities and modulated gliosis but did not extend the lifespan or rescue motor decline in Ndufs4 KO mice. The present results show that microglial accumulation is pathogenic, in a process independent of IL-6, and hints toward a contributing role of neuroinflammation in the disease of Ndufs4 KO mice and potentially in patients with Leigh syndrome.

The interpeduncular nucleus (IPN) plays a key role in nicotine dependence and is involved in regulation of fear responses, affective states, and novelty processing. IPN neurons express nicotinic acetylcholine receptors (nAChR) and receive strong cholinergic innervation from the ventral medial habenula. Dorsal medial habenula neurons are primarily peptidergic, releasing substance P (SP) mainly onto IPN neurons in the lateral subnucleus (IPL). IPL neurons are sensitive to SP, but it is not known if they are involved in cholinergic transmission like other IPN neurons. We examined nAChR subunit gene expression in IPL neurons, revealing that Chrna7 (α7 nAChR subunit) is expressed in a subset of GABAergic IPL neurons. In patch-clamp recordings from IPL neurons, ACh-evoked inward currents were attenuated by methyllycaconitine (α7 nAChR antagonist) and potentiated by NS1738 (α7 Type I positive allosteric modulator). We confirmed α7 functional expression in IPL neurons by also showing that ACh-evoked currents were potentiated by PNU-120596 (Type II positive allosteric modulator). Additional pharmacological experiments show that IPN neurons expressing α7 nAChRs also express α3β4 nAChRs. Finally, we used 2-photon laser scanning microscopy and nicotine uncaging to directly examine the morphology of IPL neurons that express α7 nAChRs. These results highlight a novel aspect of α7 nAChR neurobiology, adding to the complexity of cholinergic modulation by nAChRs in the IPN.

Birdsong is a precisely timed animal behavior. The connectivity of song premotor neural networks has been proposed to underlie the temporal patterns of neuronal activity that control vo-cal muscle movements during singing. Although the connectivity of premotor nuclei via chemical synapses has been characterized, electrical synapses and their molecular identity remain unex-plored. We show with in situ hybridizations that GJD2 mRNA, coding for the major channel-form-ing electrical synapse protein in mammals, connexin 36, is expressed in the two nuclei that control song production, HVC and RA from canaries and zebra finches. In canaries’ HVC, GJD2 mRNA is extensively expressed in GABAergic and only a fraction of glutamatergic cells. By contrast, in RA, GJD2 mRNA expression is widespread in glutamatergic and GABAergic neurons. Remarkably, GJD2 expression is similar in song nuclei and their respective embedding brain regions, revealing the widespread expression of GJD2 in the avian brain. Inspection of a single-cell sequencing data-base from zebra and Bengalese finches generalizes the distributions of electrical synapses across cell types and song nuclei that we found in HVC and RA from canaries, reveals a differential GJD2 mRNA expression in HVC glutamatergic subtypes and its transient increase along the neurogenic lineage. We propose that songbirds are a suitable model to investigate the contribution of electrical synapses to motor skill learning and production.