Signaling through the nicotinic acetylcholine receptor in the liver protects against the development of metabolic dysfunction-associated steatohepatitis.

Metabolic dysfunction-associated steatohepatitis (MASH) is the progressive form of liver steatosis, the most common liver disease, and substantially increases the mortality rate. However, limited therapies are currently available to prevent MASH development. Identifying potential pharmacological treatments for the condition has been hampered by its heterogeneous and complex nature. Here, we identified a hepatic nonneuronal cholinergic signaling pathway required for metabolic adaptation to caloric overload. We found that cholinergic receptor nicotinic alpha 2 subunit (CHRNA2) is highly expressed in hepatocytes of mice and humans. Further, CHRNA2 is activated by a subpopulation of local acetylcholine-producing macrophages during MASH development. The activation of CHRNA2 coordinates defensive programs against a broad spectrum of MASH-related pathogenesis, including steatosis, inflammation, and fibrosis. Hepatocyte-specific loss of CHRNA2 signaling accelerates the disease onset in different MASH mouse models. Activation of this pathway via pharmacological inhibition of acetylcholine degradation protects against MASH development. Our study uncovers a hepatic nicotinic cholinergic receptor pathway that constitutes a cell-autonomous self-defense route against prolonged metabolic stress and holds therapeutic potential for combatting human MASH.

Functional specialization of hippocampal somatostatin-expressing interneurons.

Hippocampal somatostatin-expressing (Sst) GABAergic interneurons (INs) exhibit considerable anatomical and functional heterogeneity. Recent single-cell transcriptome analyses have provided a comprehensive Sst-IN subpopulations census, a plausible molecular ground truth of neuronal identity whose links to specific functionality remain incomplete. Here, we designed an approach to identify and access subpopulations of Sst-INs based on transcriptomic features. Four mouse models based on single or combinatorial Cre- and Flp- expression differentiated functionally distinct subpopulations of CA1 hippocampal Sst-INs that largely tiled the morpho-functional parameter space of the Sst-INs superfamily. Notably, the Sst;;Tac1 intersection revealed a population of bistratified INs that preferentially synapsed onto fast-spiking interneurons (FS-INs) and were sufficient to interrupt their firing. In contrast, the Ndnf;;Nkx2-1 intersection identified a population of oriens lacunosum-moleculare INs that predominantly targeted CA1 pyramidal neurons, avoiding FS-INs. Overall, our results provide a framework to translate neuronal transcriptomic identity into discrete functional subtypes that capture the diverse specializations of hippocampal Sst-INs.

A flowchart for adequate controls in virus-based monosynaptic tracing experiments identified Cre-independent leakage of the TVA receptor in RΦGT mice.

BACKGROUND: A pseudotyped modified rabies virus lacking the rabies glycoprotein (G-protein), which is crucial for transsynaptic spread, can be used for monosynaptic retrograde tracing. By coupling the pseudotyped virus with transgene expression of the G-protein and the avian leukosis and sarcoma virus subgroup A receptor (TVA), which is necessary for cell entry of the virus, researchers can investigate specific neuronal populations. Responder mouse lines, like the RΦGT mouse line, carry the genes encoding the G-protein and TVA under Cre-dependent expression. These mouse lines are valuable tools because they reduce the number of viral injections needed compared to when using helper viruses. Since RΦGT mice do not express Cre themselves, introducing the pseudotyped rabies virus into their brain should not result in viral cell entry or spread. RESULTS: We present a straightforward flowchart for adequate controls in tracing experiments, which we employed to demonstrate Cre-independent expression of TVA in RΦGT mice. CONCLUSIONS: Our observations revealed TVA leakage, indicating that RΦGT mice should be used with caution for transgene expression of TVA. Inaccurate tracing outcomes may occur if TVA is expressed in the absence of Cre since background leakage leads to nonspecific cell entry. Moreover, conducting appropriate control experiments can identify the source of potential caveats in virus-based neuronal tracing experiments.

A loss-of-function mutation in RORB disrupts saltatorial locomotion in rabbits.

Saltatorial locomotion is a type of hopping gait that in mammals can be found in rabbits, hares, kangaroos, and some species of rodents. The molecular mechanisms that control and fine-tune the formation of this type of gait are unknown. Here, we take advantage of one strain of domesticated rabbits, the sauteur d'Alfort, that exhibits an abnormal locomotion behavior defined by the loss of the typical jumping that characterizes wild-type rabbits. Strikingly, individuals from this strain frequently adopt a bipedal gait using their front legs. Using a combination of experimental crosses and whole genome sequencing, we show that a single locus containing the RAR related orphan receptor B gene (RORB) explains the atypical gait of these rabbits. We found that a splice-site mutation in an evolutionary conserved site of RORB results in several aberrant transcript isoforms incorporating intronic sequence. This mutation leads to a drastic reduction of RORB-positive neurons in the spinal cord, as well as defects in differentiation of populations of spinal cord interneurons. Our results show that RORB function is required for the performance of saltatorial locomotion in rabbits.

The alpha2 nicotinic acetylcholine receptor, a subunit with unique and selective expression in inhibitory interneurons associated with principal cells.

Nicotinic acetylcholine receptors (nAChRs) play crucial roles in various human disorders, with the α7, α4, α6, and α3-containing nAChR subtypes extensively studied in relation to conditions such as Alzheimer's disease, Parkinson's disease, nicotine dependence, mood disorders, and stress disorders. In contrast, the α2-nAChR subunit has received less attention due to its more restricted expression and the scarcity of specific agonists and antagonists for studying its function. Nevertheless, recent research has shed light on the unique expression pattern of the Chrna2 gene, which encodes the α2-nAChR subunit, and its involvement in distinct populations of inhibitory interneurons. This review highlights the structure, pharmacology, localization, function, and disease associations of α2-containing nAChRs and points to the unique expression pattern of the Chrna2 gene and its role in different inhibitory interneuron populations. These populations, including the oriens lacunosum moleculare (OLM) cells in the hippocampus, Martinotti cells in the neocortex, and Renshaw cells in the spinal cord, share common features and contribute to recurrent inhibitory microcircuits. Thus, the α2-nAChR subunit's unique expression pattern in specific interneuron populations and its role in recurrent inhibitory microcircuits highlight its importance in various physiological processes. Further research is necessary to uncover the comprehensive functionality of α2-containing nAChRs, delineate their specific contributions to neuronal circuits, and investigate their potential as therapeutic targets for related disorders.

The polyamine transporter Slc18b1(VPAT) is important for both short and long time memory and for regulation of polyamine content in the brain.

SLC18B1 is a sister gene to the vesicular monoamine and acetylcholine transporters, and the only known polyamine transporter, with unknown physiological role. We reveal that Slc18b1 knock out mice has significantly reduced polyamine content in the brain providing the first evidence that Slc18b1 is functionally required for regulating polyamine levels. We found that this mouse has impaired short and long term memory in novel object recognition, radial arm maze and self-administration paradigms. We also show that Slc18b1 KO mice have altered expression of genes involved in Long Term Potentiation, plasticity, calcium signalling and synaptic functions and that expression of components of GABA and glutamate signalling are changed. We further observe a partial resistance to diazepam, manifested as significantly lowered reduction in locomotion after diazepam treatment. We suggest that removal of Slc18b1 leads to reduction of polyamine contents in neurons, resulting in reduced GABA signalling due to long-term reduction in glutamatergic signalling.

EphA4 Is Required for Neural Circuits Controlling Skilled Reaching.

Skilled forelimb movements are initiated by feedforward motor commands conveyed by supraspinal motor pathways. The accuracy of reaching and grasping relies on internal feedback pathways that update ongoing motor commands. In mice lacking the axon guidance molecule EphA4, axonal misrouting of the corticospinal tract and spinal interneurons is manifested, leading to a hopping gait in hindlimbs. Moreover, mice with a conditional forebrain deletion of EphA4, display forelimb hopping in adaptive locomotion and exploratory reaching movements. However, it remains unclear how loss of EphA4 signaling disrupts function of forelimb motor circuit and skilled reaching and grasping movements. Here we investigated how neural circuits controlling skilled reaching were affected by the loss of EphA4. Both male and female C57BL/6 wild-type, heterozygous EphA4+/-, and homozygous EphA4-/- mice were used in behavioral and in vivo electrophysiological investigations. We found that EphA4 knock-out (-/-) mice displayed impaired goal-directed reaching movements. In vivo intracellular recordings from forelimb motor neurons demonstrated increased corticoreticulospinal excitation, decreased direct reticulospinal excitation, and reduced direct propriospinal excitation in EphA4 knock-out mice. Cerebellar surface recordings showed a functional perturbation of the lateral reticular nucleus-cerebellum internal feedback pathway in EphA4 knock-out mice. Together, our findings provide in vivo evidence at the circuit level that loss of EphA4 disrupts the function of both feedforward and feedback motor pathways, resulting in deficits in skilled reaching.SIGNIFICANCE STATEMENT The central advances of this study are the demonstration that null mutation in the axon guidance molecule EphA4 gene impairs the ability of mice to perform skilled reaching, and identification of how these behavioral deficits correlates with discrete neurophysiological changes in central motor pathways involved in the control of reaching. Our findings provide in vivo evidence at the circuit level that loss of EphA4 disrupts both feedforward and feedback motor pathways, resulting in deficits in skilled reaching. This analysis of motor circuit function may help to understand the pathophysiological mechanisms underlying movement disorders in humans.

Characterization of Dmrt3-Derived Neurons Suggest a Role within Locomotor Circuits.

Neuronal networks within the spinal cord, collectively known as the central pattern generator (CPG), coordinate rhythmic movements underlying locomotion. The transcription factor doublesex and mab-3-related transcription factor 3 (DMRT3) is involved in the differentiation of the dorsal interneuron 6 class of spinal cord interneurons. In horses, a non-sense mutation in the Dmrt3 gene has major effects on gaiting ability, whereas mice lacking the Dmrt3 gene display impaired locomotor activity. Although the Dmrt3 gene is necessary for normal spinal network formation and function in mice, a direct role for Dmrt3-derived neurons in locomotor-related activities has not been demonstrated. Here we present the characteristics of the Dmrt3-derived spinal cord interneurons. Using transgenic mice of both sexes, we characterized interneurons labeled by their expression of Cre driven by the endogenous Dmrt3 promoter. We used molecular, retrograde tracing and electrophysiological techniques to examine the anatomical, morphological, and electrical properties of the Dmrt3-Cre neurons. We demonstrate that inhibitory Dmrt3-Cre neurons receive extensive synaptic inputs, innervate surrounding CPG neurons, intrinsically regulate CPG neuron's electrical activity, and are rhythmically active during fictive locomotion, bursting at frequencies independent to the ventral root output. The present study provides novel insights on the character of spinal Dmrt3-derived neurons, data demonstrating that these neurons participate in locomotor coordination.SIGNIFICANCE STATEMENT In this work, we provide evidence for a role of the Dmrt3 interneurons in spinal cord locomotor circuits as well as molecular and functional insights on the cellular and microcircuit level of the Dmrt3-expressing neurons in the spinal cord. Dmrt3 neurons provide the first example of an interneuron population displaying different oscillation frequencies. This study presents novel findings on an under-reported population of spinal cord neurons, which will aid in deciphering the locomotor network and will facilitate the design and development of therapeutics for spinal cord injury and motor disorders.

Chrna2-Martinotti Cells Synchronize Layer 5 Type A Pyramidal Cells via Rebound Excitation.

Martinotti cells are the most prominent distal dendrite-targeting interneurons in the cortex, but their role in controlling pyramidal cell (PC) activity is largely unknown. Here, we show that the nicotinic acetylcholine receptor α2 subunit (Chrna2) specifically marks layer 5 (L5) Martinotti cells projecting to layer 1. Furthermore, we confirm that Chrna2-expressing Martinotti cells selectively target L5 thick-tufted type A PCs but not thin-tufted type B PCs. Using optogenetic activation and inhibition, we demonstrate how Chrna2-Martinotti cells robustly reset and synchronize type A PCs via slow rhythmic burst activity and rebound excitation. Moreover, using optical feedback inhibition, in which PC spikes controlled the firing of surrounding Chrna2-Martinotti cells, we found that neighboring PC spike trains became synchronized by Martinotti cell inhibition. Together, our results show that L5 Martinotti cells participate in defined cortical circuits and can synchronize PCs in a frequency-dependent manner. These findings suggest that Martinotti cells are pivotal for coordinated PC activity, which is involved in cortical information processing and cognitive control.

Chrna2-OLM interneurons display different membrane properties and h-current magnitude depending on dorsoventral location.

The hippocampus is an extended structure displaying heterogeneous anatomical cell layers along its dorsoventral axis. It is known that dorsal and ventral regions show different integrity when it comes to functionality, innervation, gene expression, and pyramidal cell properties. Still, whether hippocampal interneurons exhibit different properties along the dorsoventral axis is not known. Here, we report electrophysiological properties of dorsal and ventral oriens lacunosum moleculare (OLM) cells from coronal sections of the Chrna2-cre mouse line. We found dorsal OLM cells to exhibit a significantly more depolarized resting membrane potential compared to ventral OLM cells, while action potential properties were similar between the two groups. We found ventral OLM cells to show a higher initial firing frequency in response to depolarizing current injections but also to exhibit a higher spike-frequency adaptation than dorsal OLM cells. Additionally, dorsal OLM cells displayed large membrane sags in response to negative current injections correlating with our results showing that dorsal OLM cells have more hyperpolarization-activated current (Ih ) compared to ventral OLM cells. Immunohistochemical examination indicates the h-current to correspond to hyperpolarization-activated cyclic nucleotide-gated subunit 2 (HCN2) channels. Computational studies suggest that Ih in OLM cells is essential for theta oscillations in hippocampal circuits, and here we found dorsal OLM cells to present a higher membrane resonance frequency than ventral OLM cells. Thus, our results highlight regional differences in membrane properties between dorsal and ventral OLM cells allowing this interneuron to differently participate in the generation of hippocampal theta rhythms depending on spatial location along the dorsoventral axis of the hippocampus.

Developmental Disruption of Recurrent Inhibitory Feedback Results in Compensatory Adaptation in the Renshaw Cell-Motor Neuron Circuit.

When activating muscles, motor neurons in the spinal cord also activate Renshaw cells, which provide recurrent inhibitory feedback to the motor neurons. The tight coupling with motor neurons suggests that Renshaw cells have an integral role in movement, a role that is yet to be elucidated. Here we used the selective expression of the nicotinic cholinergic receptor α2 (Chrna2) in mice to genetically target the vesicular inhibitory amino acid transporter (VIAAT) in Renshaw cells. Loss of VIAAT from Chrna2Cre -expressing Renshaw cells did not impact any aspect of drug-induced fictive locomotion in the neonatal mouse or change gait, motor coordination, or grip strength in adult mice of both sexes. However, motor neurons from neonatal mice lacking VIAAT in Renshaw cells received spontaneous inhibitory synaptic input with a reduced frequency, showed lower input resistance, and had an increased number of proprioceptive glutamatergic and calbindin-labeled putative Renshaw cell synapses on their soma and proximal dendrites. Concomitantly, Renshaw cells developed with increased excitability and a normal number of cholinergic motor neuron synapses, indicating a compensatory mechanism within the recurrent inhibitory feedback circuit. Our data suggest an integral role for Renshaw cell signaling in shaping the excitability and synaptic input to motor neurons.SIGNIFICANCE STATEMENT We here provide a deeper understanding of spinal cord circuit formation and the repercussions for the possible role for Renshaw cells in speed and force control. Our results suggest that while Renshaw cells are not directly required as an integral part of the locomotor coordination machinery, the development of their electrophysiological character is dependent on vesicular inhibitory amino acid transporter-mediated signaling. Further, Renshaw cell signaling is closely associated with the molding of motor neuron character proposing the existence of a concerted maturation process, which seems to endow this particular spinal cord circuit with the plasticity to compensate for loss of the Renshaw cell in adult circuit function.

Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice.

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left-right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.

Differential Neuroprotective Effects of Interleukin-1 Receptor Antagonist on Spinal Cord Neurons after Excitotoxic Injury.

Secondary damage following spinal cord injury (SCI) induces neuronal damage through inflammatory and excitotoxic pathways. We hypothesized that the interleukin-1 receptor antagonist (IL1RA) protects neuronal populations and suppresses apoptosis and gliosis after injury. Spinal cord slice cultures (SCSCs) were subjected to excitotoxic injury with N-methyl-D-aspartate (NMDA) and treated with IL1RA. Immunohistochemistry for neuronal nuclei (NeuN), MacII, glial fibrillary acidic protein, and TdT-mediated dUTP nick end labelling stains were used to evaluate neuronal survival, glial activation, and apoptosis. Treatment with IL1RA significantly reduced the number of apoptotic cells in both NMDA-lesioned and unlesioned cultures. Experimental injury with NMDA reduced the number of NeuN-positive ventral horn neurons, and IL1RA treatment counteracted this loss 1 day after injury. However, IL1RA had no effect on the number of presumable Renshaw cells, identified by their selective expression of the cholinergic nicotinic α2-receptor subunit (Chrna2). Activated microglial cells were more numerous in NMDA-lesioned cultures 1 day after injury, and IL1RA significantly reduced their numbers. We conclude that IL1RA modulates neuronal apoptosis and microglial activation in excitotoxically injured SCSCs. Renshaw cells were more susceptible to excitotoxic injury than other neurons and were not rescued by IL1RA treatment. Modulation of IL-1-mediated pathways may thus be effective in reducing excitotoxically induced neuronal damage after SCI, however only in specific neuronal populations, such as ventral horn neurons. These findings motivate further investigations of the possibility to antagonize inflammatory pathways after SCI.

Multimodal Eph/Ephrin signaling controls several phases of urogenital development.

A substantial portion of the human population is affected by urogenital birth defects resulting from a failure in ureter development. Although recent research suggests roles for several genes in facilitating the ureter/bladder connection, the underlying molecular mechanisms remain poorly understood. Signaling via Eph receptor tyrosine kinases is involved in several developmental processes. Here we report that impaired Eph/Ephrin signaling in genetically modified mice results in severe hydronephrosis caused by defective ureteric bud induction, ureter maturation, and translocation. Our data imply that ureter translocation requires apoptosis in the urogenital sinus and inhibition of proliferation in the common nephric duct. These processes were disturbed in EphA4/EphB2 compound knockout mice and were accompanied by decreased ERK-2 phosphorylation. Using a set of Eph, Ephrin, and signaling-deficient mutants, we found that during urogenital development, different modes of Eph/Ephrin signaling occur at several sites with EphrinB2 and EphrinA5 acting in concert. Thus, Eph/Ephrin signaling should be considered in the etiology of congenital kidney and urinary tract anomalies.

Multimodal use of calcitonin gene-related peptide and substance P in itch and acute pain uncovered by the elimination of vesicular glutamate transporter 2 from transient receptor potential cation channel subfamily V member 1 neurons.

Primary afferents are known to use glutamate as their principal fast neurotransmitter. However, it has become increasingly clear that peptides have an influential role in both mediating and modulating sensory transmission. Here we describe the transmission accounting for different acute pain states and itch transmitted via the transient receptor potential cation channel subfamily V member 1 (TRPV1) population by either ablating Trpv1-Cre-expressing neurons or inducing vesicular glutamate transporter 2 (VGLUT2) deficiency in Trpv1-Cre-expressing neurons. Furthermore, by pharmacological inhibition of substance P or calcitonin gene-related peptide (CGRP) signaling in Vglut2-deficient mice, we evaluated the contribution of substance P or CGRP to these sensory modulations, with or without the presence of VGLUT2-mediated glutamatergic transmission in Trpv1-Cre neurons. This examination, together with c-Fos analyses, showed that glutamate via VGLUT2 in the Trpv1-Cre population together with substance P mediate acute cold pain, whereas glutamate together with CGRP mediate noxious heat. Moreover, we demonstrate that glutamate together with both substance P and CGRP mediate tissue-injury associated pain. We further show that itch, regulated by the VGLUT2-mediated transmission via the Trpv1-Cre population, depends on CGRP and gastrin-releasing peptide receptor (GRPR) transmission because pharmacological blockade of the CGRP or GRPR pathway, or genetic ablation of Grpr, led to a drastically attenuated itch. Our study reveals how different neurotransmitters combined can cooperate with each other to transmit or regulate various acute sensations, including itch.

EphA4-mediated ipsilateral corticospinal tract misprojections are necessary for bilateral voluntary movements but not bilateral stereotypic locomotion.

In this study, we took advantage of the reported role of EphA4 in determining the contralateral spinal projection of the corticospinal tract (CST) to investigate the effects of ipsilateral misprojections on voluntary movements and stereotypic locomotion. Null EphA4 mutations produce robust ipsilateral CST misprojections, resulting in bilateral corticospinal tracts. We hypothesize that a unilateral voluntary limb movement, not a stereotypic locomotor movement, will become a bilateral movement in EphA4 knock-out mice with a bilateral CST. However, in EphA4 full knock-outs, spinal interneurons also develop bilateral misprojections. Aberrant bilateral spinal circuits could thus transform unilateral corticospinal control signals into bilateral movements. We therefore studied mice with conditional forebrain deletion of the EphA4 gene under control by Emx1, a gene expressed in the forebrain that affects the developing CST but spares brainstem motor pathways and spinal motor circuits. We examined two conditional knock-outs targeting forebrain EphA4 during performance of stereotypic locomotion and voluntary movement: adaptive locomotion over obstacles and exploratory reaching. We found that the conditional knock-outs used alternate stepping, not hopping, during overground locomotion, suggesting normal central pattern generator function and supporting our hypothesis of minimal CST involvement in the moment-to-moment control of stereotypic locomotion. In contrast, the conditional knock-outs showed bilateral voluntary movements under conditions when single limb movements are normally produced and, as a basis for this aberrant control, developed a bilateral motor map in motor cortex that is driven by the aberrant ipsilateral CST misprojections. Therefore, a specific change in CST connectivity is associated with and explains a change in voluntary movement.

Firing properties of Renshaw cells defined by Chrna2 are modulated by hyperpolarizing and small conductance ion currents Ih and ISK.

Renshaw cells in the spinal cord ventral horn regulate motoneuron output through recurrent inhibition. Renshaw cells can be identified in vitro using anatomical and cellular criteria; however, their functional role in locomotion remains poorly defined because of the difficulty of functionally isolating Renshaw cells from surrounding motor circuits. Here we aimed to investigate whether the cholinergic nicotinic receptor alpha2 (Chrna2) can be used to identify Renshaw cells (RCs(α2)) in the mouse spinal cord. Immunohistochemistry and electrophysiological characterization of passive and active RCs(α2) properties confirmed that neurons genetically marked by the Chrna2-Cre mouse line together with a fluorescent reporter mouse line are Renshaw cells. Whole-cell patch-clamp recordings revealed that RCs(α2) constitute an electrophysiologically stereotyped population with a resting membrane potential of -50.5 ± 0.4 mV and an input resistance of 233.1 ± 11 MΩ. We identified a ZD7288-sensitive hyperpolarization-activated cation current (Ih) in all RCs(α2), contributing to membrane repolarization but not to the resting membrane potential in neonatal mice. Additionally, we found RCs(α2) to express small calcium-activated potassium currents (I(SK)) that, when blocked by apamin, resulted in a complete attenuation of the afterhyperpolarisation potential, increasing cellular firing frequency. We conclude that RCs(α2) can be genetically targeted through their selective Chrna2 expression and that they display currents known to modulate rebound excitation and firing frequency. The genetic identification of Renshaw cells and their electrophysiological profile is required for genetic and pharmacological manipulation as well as computational simulations with the aim to understand their functional role.

A role for solute carrier family 10 member 4, or vesicular aminergic-associated transporter, in structural remodelling and transmitter release at the mouse neuromuscular junction.

The solute carrier and presynaptic vesicle protein solute carrier family 10 member 4, or vesicular aminergic-associated transporter (VAAT), was recently proven to have a modulatory role in central cholinergic signalling. It is currently unknown whether VAAT also affects peripheral cholinergic synapses. Here we demonstrated a regulatory role for the presynaptic vesicle protein VAAT in neuromuscular junction (NMJ) development and function. NMJs lacking VAAT had fewer branch points, whereas endplates showed an increased number of islands. Whereas the amplitude of spontaneous miniature endplate potentials in VAAT-deficient NMJs was decreased, the amplitude of evoked endplate potentials and the size of the readily releasable pool of vesicles were both increased. Moreover, VAAT-deficient NMJs displayed aberrant short-term synaptic plasticity with enhanced synaptic depression in response to high-frequency stimulation. Finally, the transcript levels of cholinergic receptor subunits in VAAT-deficient muscles were increased, indicating a compensatory postsynaptic sensitization. Our results suggested that VAAT modulates NMJ transmission efficiency and, as such, may represent a novel target for treatment of disorders affecting motor neurons.

Glutamate, substance P, and calcitonin gene-related peptide cooperate in inflammation-induced heat hyperalgesia.

The transient receptor potential cation channel subfamily V member 1 (TRPV1) is known as a thermosensor and integrator of inflammation-induced hyperalgesia. TRPV1 is expressed in a subpopulation of primary afferent neurons that express several different neurotransmitters. The role of the TRPV1 channel in the development of hyperalgesia is established, but the role of the neurotransmitter glutamate, used partially by the same neuronal population and thus probably mediating the response, is still under investigation. We have used a Trpv1-Cre mouse line in which we either ablated Trpv1-Cre expressing neurons or induced vesicular glutamate transporter 2 (Vglut2) deficiency in Trpv1-Cre expressing neurons and investigated specific states of hyperalgesia after persistent inflammation. Furthermore, by pharmacologic inhibition of substance P (SP) or calcitonin gene-related peptide (CGRP) signaling in Vglut2-deficient mice, we also evaluated the contribution of SP or CGRP to inflammation-induced hyperalgesia, with or without the presence of vesicular glutamate transporter 2 (VGLUT2)-mediated glutamatergic transmission in Trpv1-Cre neurons. This examination, together with c-Fos analyses, showed that VGLUT2-mediated glutamatergic transmission in Trpv1-Cre afferents together with SP or CGRP is essential for the development of the heat hyperalgesia associated with persistent inflammation. Additionally, SP-, CGRP-, and VGLUT2-mediated transmission together were found to play a role in the development of mechanical hyperalgesia after persistent inflammation.

A sensory subpopulation depends on vesicular glutamate transporter 2 for mechanical pain, and together with substance P, inflammatory pain.

Ablating or functionally compromising sets of sensory neurons has provided important insights into peripheral modality-specific wiring in the somatosensory system. Inflammatory hyperalgesia, cold pain, and noxious mechanosensation have all been shown to depend upon Na(v)1.8-positive sensory neurons. The release of fast-acting neurotransmitters, such as glutamate, and more slowly released neuropeptides, such as substance P (SP), contribute to the diversified responses to external stimuli. Here we show that deleting Vglut2 in Na(v)1.8(Cre)-positive neurons compromised mechanical pain and NGF-induced thermal hyperalgesia, whereas tactile-evoked sensation, thermal, formalin-evoked, and chronic neuropathic pain were normal. However, when Vglut2(f/f);Na(v)1.8(Cre) mice were injected with a SP antagonist before the formalin test, the second phase pain response was nearly completely abolished, whereas in control mice, the pain response was unaffected. Our results suggest that VGLUT2-dependent signaling originating from Na(v)1.8-positive neurons is a principal sensing mechanism for mechanical pain and, together with SP, inflammatory pain. These data define sets of primary afferents associated with specific modalities and provide useful genetic tools with which to analyze the pathways that are activated by functionally distinct neuronal populations and transmitters.