Chronic intermittent hypoxia reveals role of the Postinspiratory Complex in the mediation of normal swallow production.

Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder that results in multiple bouts of intermittent hypoxia. OSA has many neurological and systemic comorbidities, including dysphagia, or disordered swallow, and discoordination with breathing. However, the mechanism in which chronic intermittent hypoxia (CIH) causes dysphagia is unknown. Recently, we showed the postinspiratory complex (PiCo) acts as an interface between the swallow pattern generator (SPG) and the inspiratory rhythm generator, the preBötzinger complex, to regulate proper swallow-breathing coordination (Huff et al., 2023). PiCo is characterized by interneurons co-expressing transporters for glutamate (Vglut2) and acetylcholine (ChAT). Here we show that optogenetic stimulation of ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2 mice exposed to CIH does not alter swallow-breathing coordination, but unexpectedly disrupts swallow behavior via triggering variable swallow motor patterns. This suggests that glutamatergic-cholinergic neurons in PiCo are not only critical for the regulation of swallow-breathing coordination, but also play an important role in the modulation of swallow motor patterning. Our study also suggests that swallow disruption, as seen in OSA, involves central nervous mechanisms interfering with swallow motor patterning and laryngeal activation. These findings are crucial for understanding the mechanisms underlying dysphagia, both in OSA and other breathing and neurological disorders.

Mesoaccumbal glutamate neurons drive reward via glutamate release but aversion via dopamine co-release.

Ventral tegmental area (VTA) projections to the nucleus accumbens (NAc) drive reward-related motivation. Although dopamine neurons are predominant, a substantial glutamatergic projection is also present, and a subset of these co-release both dopamine and glutamate. Optogenetic stimulation of VTA glutamate neurons not only supports self-stimulation but can also induce avoidance behavior, even in the same assay. Here, we parsed the selective contribution of glutamate or dopamine co-release from VTA glutamate neurons to reinforcement and avoidance. We expressed channelrhodopsin-2 (ChR2) in mouse VTA glutamate neurons in combination with CRISPR-Cas9 to disrupt either the gene encoding vesicular glutamate transporter 2 (VGLUT2) or tyrosine hydroxylase (Th). Selective disruption of VGLUT2 abolished optogenetic self-stimulation but left real-time place avoidance intact, whereas CRISPR-Cas9 deletion of Th preserved self-stimulation but abolished place avoidance. Our results demonstrate that glutamate release from VTA glutamate neurons is positively reinforcing but that dopamine release from VTA glutamate neurons can induce avoidance behavior.

Loss of glutamatergic signalling from MCH neurons reduced anxiety-like behaviours in novel environments.

Melanin-concentrating hormone (MCH) neurons within the hypothalamus are heterogeneous and can coexpress additional neuropeptides and transmitters. The majority of MCH neurons express vesicular transporters to package glutamate for synaptic release, and MCH neurons can directly innervate downstream neurons via glutamate release. Although glutamatergic signalling from MCH neurons may support physiological and behavioural roles that are independent of MCH (e.g., in glucose homeostasis and nutrient-sensing), it can also mediate similar roles to MCH in the regulation of energy balance. In addition to energy balance, the MCH system has also been implicated in mood disorders, as MCH receptor antagonists have anxiolytic and anti-depressive effects. However, the contribution of glutamatergic signalling from MCH neurons to mood-related functions have not been investigated. We crossed Mch-cre mice with floxed-Vglut2 mice to delete the expression of the vesicular glutamate transporter 2 (Vglut2) and disable glutamatergic signalling specifically from MCH neurons. The resulting Mch-Vglut2-KO mice showed Vglut2 deletion from over 75% of MCH neurons, and although we did not observe changes in depressive-like behaviours, we found that Mch-Vglut2-KO mice displayed anxiety-like behaviours. Mch-Vglut2-KO mice showed reduced exploratory activity when placed in a new cage and were quicker to consume food placed in the centre of a novel open arena. These findings showed that Vglut2 deletion from MCH neurons resulted in anxiolytic actions and suggested that the anxiogenic effects of glutamate are similar to those of the MCH peptide. Taken together, these findings suggest that glutamate and MCH may synergize to regulate and promote anxiety-like behaviour.

KDM6B cooperates with Tau and regulates synaptic plasticity and cognition via inducing VGLUT1/2.

The excitatory neurotransmitter glutamate shapes learning and memory, but the underlying epigenetic mechanism of glutamate regulation in neuron remains poorly understood. Here, we showed that lysine demethylase KDM6B was expressed in excitatory neurons and declined in hippocampus with age. Conditional knockout of KDM6B in excitatory neurons reduced spine density, synaptic vesicle number and synaptic activity, and impaired learning and memory without obvious effect on brain morphology in mice. Mechanistically, KDM6B upregulated vesicular glutamate transporter 1 and 2 (VGLUT1/2) in neurons through demethylating H3K27me3 at their promoters. Tau interacted and recruited KDM6B to the promoters of Slc17a7 and Slc17a6, leading to a decrease in local H3K27me3 levels and induction of VGLUT1/2 expression in neurons, which could be prevented by loss of Tau. Ectopic expression of KDM6B, VGLUT1, or VGLUT2 restored spine density and synaptic activity in KDM6B-deficient cortical neurons. Collectively, these findings unravel a fundamental mechanism underlying epigenetic regulation of synaptic plasticity and cognition.

Atomistic insight into the luminal allosteric regulation of vesicular glutamate transporter 2 by chloride and protons: An all-atom molecular dynamics simulation study.

Vesicular glutamate transporters (VGLUTs) are essential components of synaptic transmission in the brain. Synaptic vesicles' luminal chloride and low pH regulate VGLUTs allosterically in a cooperative way. The luminal allosteric regulation of VGLUTs by chloride (Cl- ) and proton (H+ ) is possible through the collective work of luminal Cl- and H+ binding site residues. However, precise atomistic details about the luminal Cl- binding to the luminal Cl- binding site and the role of allosteric activation by H+ in VGLUTs are unknown. Using all-atom molecular dynamics simulations, this study demonstrates the critical role of Cl- binding site residues, details about Cl- binding to the luminal Cl- binding site, and the role of allosteric regulation of VGLUT2 by H+ at an atomistic level. By point mutations, we found out that Arginine (R184), Histidine (H128), and Glutamate (E191) are critical residues in the allosteric regulation of VGLUT2, R184 is the luminal Cl- binding site residue, and H128 and R88 support Cl- binding to R184. Furthermore, we found out that the protonation of H128 and E191 is important in Cl- binding to the luminal Cl- binding site. Furthermore, we investigated the essential interactions between Cl- and H+ binding site residues. Our results can give atomistic evidence for a previous experimental hypothesis about the VGLUTs luminal allosteric regulation by H+ and Cl- .

Dopamine neurons exhibit emergent glutamatergic identity in Parkinson's disease.

Loss of midbrain dopamine neurons causes the cardinal symptoms of Parkinson's disease. However, not all dopamine neurons are equally vulnerable and a better understanding of the cell-type specific properties relating to selective dopamine neuron degeneration is needed. Most midbrain dopamine neurons express the vesicular glutamate transporter VGLUT2 during development and a subset continue to express low levels of VGLUT2 in adulthood, enabling the co-release of glutamate. Moreover, VGLUT2 expression in dopamine neurons can be neuroprotective since its genetic disruption was shown to sensitize dopamine neurons to neurotoxins. Here, we show that in response to toxic insult, and in two distinct models of alpha-synuclein stress, VGLUT2 dopamine neurons were resilient to degeneration. Dopamine neurons expressing VGLUT2 were enriched whether or not insult induced dopamine neuron loss, suggesting that while VGLUT2 dopamine neurons are more resilient, VGLUT2 expression can also be transcriptionally upregulated by injury. Finally, we observed that VGLUT2 expression was enhanced in surviving dopamine neurons from post-mortem Parkinson's disease individuals. These data indicate that emergence of a glutamatergic identity in dopamine neurons may be part of a neuroprotective response in Parkinson's disease.

Theta-Burst Stimulation of Primary Afferents Drives Long-Term Potentiation in the Spinal Cord and Persistent Pain via α2δ-1-Bound NMDA Receptors.

Long-term potentiation (LTP) and long-term depression (LTD) in the spinal dorsal horn reflect activity-dependent synaptic plasticity and central sensitization in chronic pain. Tetanic high-frequency stimulation is commonly used to induce LTP in the spinal cord. However, primary afferent nerves often display low-frequency, rhythmic bursting discharges in painful conditions. Here, we determined how theta-burst stimulation (TBS) of primary afferents impacts spinal cord synaptic plasticity and nociception in male and female mice. We found that TBS induced more LTP, whereas tetanic stimulation induced more LTD, in mouse spinal lamina II neurons. TBS triggered LTP, but not LTD, in 50% of excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2). By contrast, TBS induced LTD and LTP in 12-16% of vesicular GABA transporter (VGAT)-expressing inhibitory neurons. Nerve injury significantly increased the prevalence of TBS-induced LTP in VGluT2-expressing, but not VGAT-expressing, lamina II neurons. Blocking NMDARs, inhibiting α2δ-1 with gabapentin, or α2δ-1 knockout abolished TBS-induced LTP in lamina II neurons. Also, disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide prevented TBS-induced LTP in VGluT2-expressing neurons. Furthermore, TBS of the sciatic nerve induced long-lasting allodynia and hyperalgesia in wild-type, but not α2δ-1 knockout, mice. TBS significantly increased the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. In addition, treatment with NMDAR antagonists, gabapentin, or α2δ-1Tat peptide reversed TBS-induced pain hypersensitivity. Therefore, TBS-induced primary afferent input causes a neuropathic pain-like phenotype and LTP predominantly in excitatory dorsal horn neurons via α2δ-1-dependent NMDAR activation. α2δ-1-bound NMDARs may be targeted for reducing chronic pain development at the onset of tissue/nerve injury.SIGNIFICANCE STATEMENT Spinal dorsal horn synaptic plasticity is a hallmark of chronic pain. Although sensory nerves display rhythmic bursting discharges at theta frequencies during painful conditions, the significance of this naturally occurring firing activity in the induction of spinal synaptic plasticity is largely unknown. In this study, we found that theta-burst stimulation (TBS) of sensory nerves induced LTP mainly in excitatory dorsal horn neurons and that the prevalence of TBS-induced LTP was potentiated by nerve injury. This TBS-driven synaptic plasticity required α2δ-1 and its interaction with NMDARs. Furthermore, TBS of sensory nerves induced persistent pain, which was maintained by α2δ-1-bound NMDARs. Thus, TBS-induced LTP at primary afferent-dorsal horn neuron synapses is an appropriate cellular model for studying mechanisms of chronic pain.

Impaired Kv7 channel activity in the central amygdala contributes to elevated sympathetic outflow in hypertension.

AIMS: Elevated sympathetic outflow is associated with primary hypertension. However, the mechanisms involved in heightened sympathetic outflow in hypertension are unclear. The central amygdala (CeA) regulates autonomic components of emotions through projections to the brainstem. The neuronal Kv7 channel is a non-inactivating voltage-dependent K+ channel encoded by KCNQ2/3 genes involved in stabilizing the neuronal membrane potential and regulating neuronal excitability. In this study, we investigated if altered Kv7 channel activity in the CeA contributes to heightened sympathetic outflow in hypertension. METHODS AND RESULTS: The mRNA and protein expression levels of Kv7.2/Kv7.3 in the CeA were significantly reduced in spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto (WKY) rats. Lowering blood pressure with coeliac ganglionectomy in SHRs did not alter Kv7.2 and Kv7.3 channel expression levels in the CeA. Fluospheres were injected into the rostral ventrolateral medulla (RVLM) to retrogradely label CeA neurons projecting to the RVLM (CeA-RVLM neurons). Kv7 channel currents recorded from CeA-RVLM neurons in brain slices were much smaller in SHRs than in WKY rats. Furthermore, the basal firing activity of CeA-RVLM neurons was significantly greater in SHRs than in WKY rats. Bath application of specific Kv7 channel blocker 10, 10-bis (4-pyridinylmethyl)-9(10H)-anthracnose (XE-991) increased the excitability of CeA-RVLM neurons in WKY rats, but not in SHRs. Microinjection of XE-991 into the CeA increased arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA), while microinjection of Kv7 channel opener QO-58 decreased ABP and RSNA, in anaesthetized WKY rats but not SHRs. CONCLUSIONS: Our findings suggest that diminished Kv7 channel activity in the CeA contributes to elevated sympathetic outflow in primary hypertension. This novel information provides new mechanistic insight into the pathogenesis of neurogenic hypertension.

Activation of parabrachial nucleus - ventral tegmental area pathway underlies the comorbid depression in chronic neuropathic pain in mice.

Depression symptoms are often found in patients suffering from chronic pain, a phenomenon that is yet to be understood mechanistically. Here, we systematically investigate the cellular mechanisms and circuits underlying the chronic-pain-induced depression behavior. We show that the development of chronic pain is accompanied by depressive-like behaviors in a mouse model of trigeminal neuralgia. In parallel, we observe increased activity of the dopaminergic (DA) neuron in the midbrain ventral tegmental area (VTA), and inhibition of this elevated VTA DA neuron activity reverses the behavioral manifestations of depression. Further studies establish a pathway of glutamatergic projections from the spinal trigeminal subnucleus caudalis (Sp5C) to the lateral parabrachial nucleus (LPBN) and then to the VTA. These glutamatergic projections form a direct circuit that controls the development of the depression-like behavior under the state of the chronic neuropathic pain.

Dysfunctions of the paraventricular hypothalamic nucleus induce hypersomnia in mice.

Hypersomnolence disorder (HD) is characterized by excessive sleep, which is a common sequela following stroke, infection, or tumorigenesis. HD is traditionally thought to be associated with lesions of wake-promoting nuclei. However, lesions of a single wake-promoting nucleus, or even two simultaneously, did not exert serious HD. Therefore, the specific nucleus and neural circuitry for HD remain unknown. Here, we observed that the paraventricular nucleus of the hypothalamus (PVH) exhibited higher c-fos expression during the active period (23:00) than during the inactive period (11:00) in mice. Therefore, we speculated that the PVH, in which most neurons are glutamatergic, may represent one of the key arousal-controlling centers. By using vesicular glutamate transporter 2 (vglut2Cre) mice together with fiber photometry, multichannel electrophysiological recordings, and genetic approaches, we found that PVHvglut2 neurons were most active during wakefulness. Chemogenetic activation of PVHvglut2 neurons induced wakefulness for 9 hr, and photostimulation of PVHvglut2→parabrachial complex/ventral lateral septum circuits immediately drove transitions from sleep to wakefulness. Moreover, lesioning or chemogenetic inhibition of PVHvglut2 neurons dramatically decreased wakefulness. These results indicate that the PVH is critical for arousal promotion and maintenance.

Posterodorsal Medial Amygdala Regulation of Female Social Behavior: GABA versus Glutamate Projections.

Social behaviors, including reproductive behaviors, often display sexual dimorphism. Lordosis, the measure of female sexual receptivity, is one of the most apparent sexually dimorphic reproductive behaviors. Lordosis is regulated by estrogen and progesterone (P4) acting within a hypothalamic-limbic circuit, consisting of the arcuate, medial preoptic, and ventromedial nuclei of the hypothalamus. Social cues are integrated into the circuit through the amygdala. The posterodorsal part of the medial amygdala (MeApd) is involved in sexually dimorphic social and reproductive behaviors, and sends projections to hypothalamic neuroendocrine regions. GABA from the MeApd appears to facilitate social behaviors, while glutamate may play the opposite role. To test these hypotheses, adult female vesicular GABA transporter (VGAT)-Cre and vesicular glutamate transporter 2 (VGluT2)-Cre mice were transfected with halorhodopsin (eNpHR)-expressing or channelrhodopsin-expressing adeno-associated viruses (AAVs), respectively, in the MeApd. The lordosis quotient (LQ) was measured following either photoinhibition of VGAT or photoexcitation of VGluT2 neurons, and brains were assessed for c-Fos immunohistochemistry (IHC). Photoinhibition of VGAT neurons in the MeApd decreased LQ, and decreased c-Fos expression within VGAT neurons, within the MeApd as a whole, and within the ventrolateral part of the ventromedial nucleus (VMHvl). Photoexcitation of VGluT2 neurons did not affect LQ, but did increase time spent self-grooming, and increased c-Fos expression within VGluT2 neurons in the MeApd. Neither condition altered c-Fos expression in the medial preoptic nucleus (MPN) or the arcuate nucleus (ARH). These data support a role for MeApd GABA in the facilitation of lordosis. Glutamate from the MeApd does not appear to be directly involved in the lordosis circuit, but appears to direct behavior away from social interactions.SIGNIFICANCE STATEMENT Lordosis, the measure of female sexual receptivity, is a sexually dimorphic behavior regulated within a hypothalamic-limbic circuit. Social cues are integrated through the amygdala, and the posterodorsal part of the medial amygdala (MeApd) is involved in sexually dimorphic social and reproductive behaviors. Photoinhibition of GABAergic neurons in the MeApd inhibited lordosis, while photoactivation of glutamate neurons had no effect on lordosis, but increased self-grooming. These data support a role for MeApd GABA in the facilitation of social behaviors and MeApd glutamate projections in anti-social interactions.

Maturation of Purkinje cell firing properties relies on neurogenesis of excitatory neurons.

Preterm infants that suffer cerebellar insults often develop motor disorders and cognitive difficulty. Excitatory granule cells, the most numerous neuron type in the brain, are especially vulnerable and likely instigate disease by impairing the function of their targets, the Purkinje cells. Here, we use regional genetic manipulations and in vivo electrophysiology to test whether excitatory neurons establish the firing properties of Purkinje cells during postnatal mouse development. We generated mutant mice that lack the majority of excitatory cerebellar neurons and tracked the structural and functional consequences on Purkinje cells. We reveal that Purkinje cells fail to acquire their typical morphology and connectivity, and that the concomitant transformation of Purkinje cell firing activity does not occur either. We also show that our mutant pups have impaired motor behaviors and vocal skills. These data argue that excitatory cerebellar neurons define the maturation time-window for postnatal Purkinje cell functions and refine cerebellar-dependent behaviors.

Preterm infants have a higher risk of developing movement difficulties and neurodevelopmental conditions like autism spectrum disorder. This is likely caused by injuries to a part of the brain called the cerebellum. The cerebellum is important for movement, language and social interactions. During the final weeks of pregnancy, the cerebellum grows larger and develops a complex pattern of folds. Tiny granule cells, which are particularly vulnerable to harm, drive this development. Exactly how damage to granule cells causes movement difficulties and other conditions is unclear. One potential explanation may be that granule cells are important for the development of Purkinje cells in the brain. The Purkinje cells send and receive messages and are very important for coordinating movement. To learn more, van der Heijden et al. studied Purkinje cells in mice during a period that corresponds with the third trimester of pregnancy in humans. During this time, the pattern of electrical signals sent by the Purkinje cells changed from slow and irregular to fast and rhythmic with long pauses between bursts. However, mice that had been genetically engineered to lack most of their granule cells showed a completely different pattern of Purkinje cell development. The pattern of electrical signals emitted by these Purkinje cells stayed slow and irregular. Mice that lacked granule cells also had movement difficulties, tremors, and abnormal vocalizations. The experiments confirm that granule cells are essential for normal brain development. Without enough granule cells, the Purkinje cells become stuck in an immature state. This discovery may help physicians identify preterm infants with motor disorders and other conditions earlier. It may also lead to changes in the care of preterm infants designed to protect their granule cells.

Elimination of glutamatergic transmission from Hb9 interneurons does not impact treadmill locomotion.

The spinal cord contains neural circuits that can produce the rhythm and pattern of locomotor activity. It has previously been postulated that a population of glutamatergic neurons, termed Hb9 interneurons, contributes to locomotor rhythmogenesis. These neurons were identified by their expression of the homeobox gene, Hb9, which is also expressed in motor neurons. We developed a mouse line in which Cre recombinase activity is inducible in neurons expressing Hb9. We then used this line to eliminate vesicular glutamate transporter 2 from Hb9 interneurons, and found that there were no deficits in treadmill locomotion. We conclude that glutamatergic neurotransmission by Hb9 interneurons is not required for locomotor behaviour. The role of these neurons in neural circuits remains elusive.

DNA methylation of Vesicular Glutamate Transporters in the mesocorticolimbic brain following early-life stress and adult ethanol exposure-an explorative study.

DNA methylation and gene expression can be altered by early life stress (ELS) and/or ethanol consumption. The present study aimed to investigate whether DNA methylation of the Vesicular Glutamate Transporters (Vglut)1-3 is related to previously observed Vglut1-3 transcriptional differences in the ventral tegmental area (VTA), nucleus accumbens (Acb), dorsal striatum (dStr) and medial prefrontal cortex (mPFC) of adult rats exposed to ELS, modelled by maternal separation, and voluntary ethanol consumption. Targeted next-generation bisulfite sequencing was performed to identify the methylation levels on 61 5'-cytosine-phosphate-guanosine-3' sites (CpGs) in potential regulatory regions of Vglut1, 53 for Vglut2, and 51 for Vglut3. In the VTA, ELS in ethanol-drinking rats was associated with Vglut1-2 CpG-specific hypomethylation, whereas bidirectional Vglut2 methylation differences at single CpGs were associated with ELS alone. Exposure to both ELS and ethanol, in the Acb, was associated with lower promoter and higher intronic Vglut3 methylation; and in the dStr, with higher and lower methylation in 26% and 43% of the analyzed Vglut1 CpGs, respectively. In the mPFC, lower Vglut2 methylation was observed upon exposure to ELS or ethanol. The present findings suggest Vglut1-3 CpG-specific methylation signatures of ELS and ethanol drinking, underlying previously reported Vglut1-3 transcriptional differences in the mesocorticolimbic brain.

Characterization of basal forebrain glutamate neurons suggests a role in control of arousal and avoidance behavior.

The basal forebrain (BF) is involved in arousal, attention, and reward processing but the role of individual BF neuronal subtypes is still being uncovered. Glutamatergic neurons are the least well-understood of the three main BF neurotransmitter phenotypes. Here we analyzed the distribution, size, calcium-binding protein content and projections of the major group of BF glutamatergic neurons expressing the vesicular glutamate transporter subtype 2 (vGluT2) and tested the functional effect of activating them. Mice expressing Cre recombinase under the control of the vGluT2 promoter were crossed with a reporter strain expressing the red fluorescent protein, tdTomato, to generate vGluT2-cre-tdTomato mice. Immunohistochemical staining for choline acetyltransferase and a cross with mice expressing green fluorescent protein selectively in GABAergic neurons confirmed that cholinergic, GABAergic and vGluT2+ neurons represent distinct BF subpopulations. Subsets of BF vGluT2+ neurons expressed the calcium-binding proteins calbindin or calretinin, suggesting that multiple subtypes of BF vGluT2+ neurons exist. Anterograde tracing using adeno-associated viral vectors expressing channelrhodopsin2-enhanced yellow fluorescent fusion proteins revealed major projections of BF vGluT2+ neurons to neighboring BF cholinergic and parvalbumin neurons, as well as to extra-BF areas involved in the control of arousal or aversive/rewarding behavior such as the lateral habenula and ventral tegmental area. Optogenetic activation of BF vGluT2+ neurons elicited a striking avoidance of the area where stimulation was given, whereas stimulation of BF parvalbumin or cholinergic neurons did not. Together with previous optogenetic findings suggesting an arousal-promoting role, our findings suggest that BF vGluT2 neurons play a dual role in promoting wakefulness and avoidance behavior.

Parafacial neurons in the human brainstem express specific markers for neurons of the retrotrapezoid nucleus.

The retrotrapezoid nucleus (RTN) is a hub for respiratory chemoregulation in the mammal brainstem that integrates chemosensory information from peripheral sites and central relays. Chemosensitive neurons of the RTN express specific genetic and molecular determinants, which have been used to identify RTN precise location within the brainstem of rodents and nonhuman primates. Based on a comparative approach, we hypothesized that among mammals, neurons exhibiting the same specific molecular and genetic signature would have the same function. The co-expression of preprogalanin (PPGAL) and SLC17A6 (VGluT2) mRNAs with duplex in situ hybridization has been studied in formalin fixed paraffin-embedded postmortem human brainstems. Two specimens were processed and analyzed in line with RTN descriptions in adult rats and macaques. Double-labeled PPGAL+/SLC17A6+ neurons were only identified in the parafacial region of the brainstem. These neurons were found surrounding the nucleus of the facial nerve, located ventrally to the nucleus VII on caudal sections, and slightly more dorsally on rostral sections. The expression of neuromedin B (NMB) mRNA as a single marker of chemosensitive RTN neurons has not been confirmed in humans. The location of the RTN in human adults is provided. This should help to develop investigation tools combining anatomic high-resolution imaging and respiratory functional investigations to explore the pathogenic role of the RTN in congenital or acquired neurodegenerative diseases.

Input-specific modulation of murine nucleus accumbens differentially regulates hedonic feeding.

Hedonic feeding is driven by the "pleasure" derived from consuming palatable food and occurs in the absence of metabolic need. It plays a critical role in the excessive feeding that underlies obesity. Compared to other pathological motivated behaviors, little is known about the neural circuit mechanisms mediating excessive hedonic feeding. Here, we show that modulation of prefrontal cortex (PFC) and anterior paraventricular thalamus (aPVT) excitatory inputs to the nucleus accumbens (NAc), a key node of reward circuitry, has opposing effects on high fat intake in mice. Prolonged high fat intake leads to input- and cell type-specific changes in synaptic strength. Modifying synaptic strength via plasticity protocols, either in an input-specific optogenetic or non-specific electrical manner, causes sustained changes in high fat intake. These results demonstrate that input-specific NAc circuit adaptations occur with repeated exposure to a potent natural reward and suggest that neuromodulatory interventions may be therapeutically useful for individuals with pathologic hedonic feeding.

Glutamatergic Neurons in the Preoptic Hypothalamus Promote Wakefulness, Destabilize NREM Sleep, Suppress REM Sleep, and Regulate Cortical Dynamics.

Clinical and experimental data from the last nine decades indicate that the preoptic area of the hypothalamus is a critical node in a brain network that controls sleep onset and homeostasis. By contrast, we recently reported that a group of glutamatergic neurons in the lateral and medial preoptic area increases wakefulness, challenging the long-standing notion in sleep neurobiology that the preoptic area is exclusively somnogenic. However, the precise role of these subcortical neurons in the control of behavioral state transitions and cortical dynamics remains unknown. Therefore, in this study, we used conditional expression of excitatory hM3Dq receptors in these preoptic glutamatergic (Vglut2+) neurons and show that their activation initiates wakefulness, decreases non-rapid eye movement (NREM) sleep, and causes a persistent suppression of rapid eye movement (REM) sleep. We also demonstrate, for the first time, that activation of these preoptic glutamatergic neurons causes a high degree of NREM sleep fragmentation, promotes state instability with frequent arousals from sleep, decreases body temperature, and shifts cortical dynamics (including oscillations, connectivity, and complexity) to a more wake-like state. We conclude that a subset of preoptic glutamatergic neurons can initiate, but not maintain, arousals from sleep, and their inactivation may be required for NREM stability and REM sleep generation. Further, these data provide novel empirical evidence supporting the hypothesis that the preoptic area causally contributes to the regulation of both sleep and wakefulness.SIGNIFICANCE STATEMENT Historically, the preoptic area of the hypothalamus has been considered a key site for sleep generation. However, emerging modeling and empirical data suggest that this region might play a dual role in sleep-wake control. We demonstrate that chemogenetic stimulation of preoptic glutamatergic neurons produces brief arousals that fragment sleep, persistently suppresses REM sleep, causes hypothermia, and shifts EEG patterns toward a "lighter" NREM sleep state. We propose that preoptic glutamatergic neurons can initiate, but not maintain, arousal from sleep and gate REM sleep generation, possibly to block REM-like intrusions during NREM-to-wake transitions. In contrast to the long-standing notion in sleep neurobiology that the preoptic area is exclusively somnogenic, we provide further evidence that preoptic neurons also generate wakefulness.

Neuron-specific spinal cord translatomes reveal a neuropeptide code for mouse dorsal horn excitatory neurons.

The spinal dorsal horn harbors a sophisticated and heterogeneous network of excitatory and inhibitory neurons that process peripheral signals encoding different sensory modalities. Although it has long been recognized that this network is crucial both for the separation and the integration of sensory signals of different modalities, a systematic unbiased approach to the use of specific neuromodulatory systems is still missing. Here, we have used the translating ribosome affinity purification (TRAP) technique to map the translatomes of excitatory glutamatergic (vGluT2+) and inhibitory GABA and/or glycinergic (vGAT+ or Gad67+) neurons of the mouse spinal cord. Our analyses demonstrate that inhibitory and excitatory neurons are not only set apart, as expected, by the expression of genes related to the production, release or re-uptake of their principal neurotransmitters and by genes encoding for transcription factors, but also by a differential engagement of neuromodulator, especially neuropeptide, signaling pathways. Subsequent multiplex in situ hybridization revealed eleven neuropeptide genes that are strongly enriched in excitatory dorsal horn neurons and display largely non-overlapping expression patterns closely adhering to the laminar and presumably also functional organization of the spinal cord grey matter.

Adult mouse dorsal root ganglia neurons form aberrant glutamatergic connections in dissociated cultures.

Cultured sensory neurons can exhibit complex activity patterns following stimulation in terms of increased excitability and interconnected responses of multiple neurons. Although these complex activity patterns suggest a network-like configuration, research so far had little interest in synaptic network formation ability of the sensory neurons. To identify interaction profiles of Dorsal Root Ganglia (DRG) neurons and explore their putative connectivity, we developed an in vitro experimental approach. A double transgenic mouse model, expressing genetically encoded calcium indicator (GECI) in their glutamatergic neurons, was produced. Dissociated DRG cultures from adult mice were prepared with a serum-free protocol and no additional growth factors or cytokines were utilized for neuronal sensitization. DRG neurons were grown on microelectrode arrays (MEA) to induce stimulus-evoked activity with a modality-free stimulation strategy. With an almost single-cell level electrical stimulation, spontaneous and evoked activity of GCaMP6s expressing neurons were detected under confocal microscope. Typical responses were analyzed, and correlated calcium events were detected across individual DRG neurons. Next, correlated responses were successfully blocked by glutamatergic receptor antagonists, which indicated functional synaptic coupling. Immunostaining confirmed the presence of synapses mainly in the axonal terminals, axon-soma junctions and axon-axon intersection sites. Concisely, the results presented here illustrate a new type of neuron-to-neuron interaction in cultured DRG neurons conducted through synapses. The developed assay can be a valuable tool to analyze individual and collective responses of the cultured sensory neurons.