Probes for VGLUT2

The pedunculopontine tegmental nucleus (PPT) is implicated in many brain functions, ranging from sleep/wake control and locomotion, to reward mechanisms and learning. The PPT contains cholinergic, GABAergic and glutamatergic neurons with extensive ascending and descending axonal projections. Glutamatergic PPT (PPT vGlut2) neurons are thought to promote wakefulness, but the mechanisms through which this occurs are unknown. In addition, some researchers propose that PPT vGlut2 neurons promote locomotion, yet even though the PPT is a target for deep brain stimulation in Parkinson's disease, the role of the PPT in locomotion is debated. We hypothesized that PPT vGluT2 neurons drive arousal and specific waking behaviors via certain projections and modulate locomotion via others.We mapped the axonal projections of PPT vGlut2 neurons using conditional anterograde tracing and then photostimulated PPT vGlut2 soma or their axon terminal fields across sleep/wake states and analyzed sleep/wake behavior, muscle activity, and locomotion in transgenic mice.We found that stimulation of PPT vGlut2 soma and their axon terminals rapidly triggered arousals from NREM sleep, especially with activation of terminals in the basal forebrain (BF) and lateral hypothalamus (LH). With photoactivation of PPT vGlut2 terminals in the BF and LH, this wakefulness was accompanied by locomotion and other active behaviors, but stimulation of PPT vGlut2 soma and terminals in the substantia nigra triggered only quiet wakefulness without locomotion.These findings demonstrate the importance of the PPT vGluT2 neurons in driving various aspects of arousal and show that heterogeneous brain nuclei, such as the PPT, can promote a variety of behaviors via distinct axonal projections.

Mechanical allodynia (MA) represents one prevalent symptom of chronic pain. Previously we and others have identified spinal and brain circuits that transmit or modulate the initial establishment of MA. However, brain-derived descending pathways that control the laterality and duration of MA are still poorly understood. Here we report that the contralateral brain-to-spinal circuits, from Oprm1 neurons in the lateral parabrachial nucleus (lPBNOprm1), via Pdyn neurons in the dorsal medial regions of hypothalamus (dmHPdyn), to the spinal dorsal horn (SDH), act to prevent nerve injury from inducing contralateral MA and reduce the duration of bilateral MA induced by capsaicin. Ablating/silencing dmH-projecting lPBNOprm1 neurons or SDH-projecting dmHPdyn neurons, deleting Dyn peptide from dmH, or blocking spinal κ-opioid receptors all led to long-lasting bilateral MA. Conversely, activation of dmHPdyn neurons or their axonal terminals in SDH can suppress sustained bilateral MA induced by lPBN lesion.

Vocalization is an essential medium for social signaling in birds and mammals. Periaqueductal gray (PAG) a conserved midbrain structure is believed to be responsible for innate vocalizations, but its molecular regulation remains largely unknown. Here, through a mouse forward genetic screening we identified one of the key Wnt/β-catenin effectors TCF7L2/TCF4 controls ultrasonic vocalization (USV) production and syllable complexity during maternal deprivation and sexual encounter. Early developmental expression of TCF7L2 in PAG excitatory neurons is necessary for the complex trait, while TCF7L2 loss reduces neuronal gene expressions and synaptic transmission in PAG. TCF7L2-mediated vocal control is independent of its β-catenin-binding domain but dependent of its DNA binding ability. Patient mutations associated with developmental disorders, including autism spectrum disorders, disrupt the transcriptional repression effect of TCF7L2, while mice carrying those mutations display severe USV impairments. Therefore, we conclude that TCF7L2 orchestrates gene expression in midbrain to control vocal production through its DNA binding but not transcription activation domain.

Green light exposure has been shown to reduce pain in animal models. Here, we report a vision-associated enkephalinergic neural circuit responsible for green light-mediated analgesia. Full-field green light exposure at an intensity of 10 lux produced analgesic effects in healthy mice and in a model of arthrosis. Ablation of cone photoreceptors completely inhibited the analgesic effect, whereas rod ablation only partially reduced pain relief. The analgesic effect was not modulated by the ablation of intrinsically photosensitive retinal ganglion cells (ipRGCs), which are atypical photoreceptors that control various nonvisual effects of light. Inhibition of the retino-ventrolateral geniculate nucleus (vLGN) pathway completely abolished the analgesic effects. Activation of this pathway reduced nociceptive behavioral responses; such activation was blocked by the inhibition of proenkephalin (Penk)-positive neurons in the vLGN (vLGNPenk). Moreover, green light analgesia was prevented by knockdown of Penk in the vLGN or by ablation of vLGNPenk neurons. In addition, activation of the projections from vLGNPenk neurons to the dorsal raphe nucleus (DRN) was sufficient to suppress nociceptive behaviors, whereas its inhibition abolished the green light analgesia. Our findings indicate that cone-dominated retinal inputs mediated green light analgesia through the vLGNPenk-DRN pathway and suggest that this signaling pathway could be exploited for reducing pain.

Infections induce a set of pleiotropic responses in animals, including anorexia, adipsia, lethargy and changes in temperature, collectively termed sickness behaviours1. Although these responses have been shown to be adaptive, the underlying neural mechanisms have not been elucidated2-4. Here we use of a set of unbiased methodologies to show that a specific subpopulation of neurons in the brainstem can control the diverse responses to a bacterial endotoxin (lipopolysaccharide (LPS)) that potently induces sickness behaviour. Whole-brain activity mapping revealed that subsets of neurons in the nucleus of the solitary tract (NTS) and the area postrema (AP) acutely express FOS after LPS treatment, and we found that subsequent reactivation of these specific neurons in FOS2A-iCreERT2 (also known as TRAP2) mice replicates the behavioural and thermal component of sickness. In addition, inhibition of LPS-activated neurons diminished all of the behavioural responses to LPS. Single-nucleus RNA sequencing of the NTS-AP was used to identify LPS-activated neural populations, and we found that activation of ADCYAP1+ neurons in the NTS-AP fully recapitulates the responses elicited by LPS. Furthermore, inhibition of these neurons significantly diminished the anorexia, adipsia and locomotor cessation seen after LPS injection. Together these studies map the pleiotropic effects of LPS to a neural population that is both necessary and sufficient for canonical elements of the sickness response, thus establishing a critical link between the brain and the response to infection.

Human pluripotent stem cell (hPSC)-based replacement therapy holds great promise in treating Parkinson's disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we depicted the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including ventral midbrain, isthmus, and ventral hindbrain, resulting in heterogenous donor cell population. We reconstructed the differentiation trajectory of mDA lineage and identified CLSTN2 and PTPRO as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate highly enriched mDA neurons (up to 80%) following progenitor sorting and transplantation. Marker sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation, and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.

Discovered just over 20 years ago, dopamine neurons have the ability to cotransmit both dopamine and glutamate. Yet, the functional roles of dopamine neuron glutamate cotransmission and their implications for therapeutic use are just emerging. This review article encompasses the current body of evidence investigating the functions of dopamine neurons of the ventral midbrain that cotransmit glutamate. Since its discovery in dopamine neuron cultures, further work in vivo confirmed dopamine neuron glutamate cotransmission across species. From there, growing interest has led to research related to neural functioning including roles in synaptic signaling, development, and behavior. Functional connectome mapping reveals robust connections in multiple forebrain regions to various cell types, most notably to cholinergic interneurons in both the medial shell of the nucleus accumbens and the lateral dorsal striatum. Glutamate markers in dopamine neurons reach peak levels during embryonic development and increase in response to various toxins, suggesting dopamine neuron glutamate cotransmission may serve neuroprotective roles. Findings from behavioral analyses reveal prominent roles for dopamine neuron glutamate cotransmission in responses to psychostimulants, in positive valence and cognitive systems and for subtle roles in negative valence systems. Insight into dopamine neuron glutamate cotransmission informs the pathophysiology of neuropsychiatric disorders such as addiction, schizophrenia and Parkinson Disease, with therapeutic implications.

The nucleus tractus solitarii (NTS) is the primary central station that integrates visceral afferent information and regulates respiratory, gastrointestinal, cardiovascular, and other physiological functions. Leptin receptor b (LepRb)-expressing neurons of the NTS (NTSLepRb neurons) are implicated in central respiration regulation, respiratory facilitation, and respiratory drive enhancement. Furthermore, LepRb dysfunction is involved in obesity, insulin resistance, and sleep-disordered breathing. However, the monosynaptic inputs and outputs of NTSLepRb neurons in whole-brain mapping remain to be elucidated. Therefore, the exploration of its whole-brain connection system may provide strong support for comprehensively understanding the physiological and pathological functions of NTSLepRb neurons. In the present study, we used a cell type-specific, modified rabies virus and adeno-associated virus with the Cre-loxp system to map monosynaptic inputs and outputs of NTSLepRb neurons in LepRb-Cre mice. The results showed that NTSLepRb neurons received inputs from 48 nuclei in the whole brain from five brain regions, including especially the medulla. We found that NTSLepRb neurons received inputs from nuclei associated with respiration, such as the pre-Bötzinger complex, ambiguus nucleus, and parabrachial nucleus. Interestingly, some brain areas related to cardiovascular regulation-i.e., the ventrolateral periaqueductal gray and locus coeruleus-also sent a small number of inputs to NTSLepRb neurons. In addition, anterograde tracing results demonstrated that NTSLepRb neurons sent efferent projections to 15 nuclei, including the dorsomedial hypothalamic nucleus and arcuate hypothalamic nucleus, which are involved in regulation of energy metabolism and feeding behaviors. Quantitative statistical analysis revealed that the inputs of the whole brain to NTSLepRb neurons were significantly greater than the outputs. Our study comprehensively revealed neuronal connections of NTSLepRb neurons in the whole brain and provided a neuroanatomical basis for further research on physiological and pathological functions of NTSLepRb neurons.

Cannabinoids produce a number of central nervous system effects via the CB2 receptor (CB2R), including analgesia, antianxiety, anti-reward, hypoactivity and attenuation of opioid-induced respiratory depression. However, the cellular distributions of the CB2Rs in the brain remain unclear. We have reported that CB2Rs are expressed in midbrain dopamine (DA) neurons and functionally regulate DA-mediated behavior(s). Unexpectedly, high densities of CB2-like signaling were also found in a neighboring motor structure - the red nucleus (RN) of the midbrain. In the present study, we systematically explored CB2R expression and function in the RN. Immunohistochemistry and in situ hybridization assays showed high densities of CB2R-immunostaining and mRNA signal in RN magnocellular glutamate neurons in wildtype and CB1-knockout, but not CB2-knockout, mice. Ex vivo electrophysiological recordings in midbrain slices demonstrated that CB2R activation by JWH133 dose-dependently inhibited firing rates of RN magnocellular neurons in wildtype, but not CB2-knockout, mice, while having no effect on RN GABA neurons in transgenic GAD67-GFP reporter mice, suggesting CB2-mediated effects on glutamatergic neurons. In addition, microinjection of JWH133 into the RN produced robust ipsilateral rotations in wildtype, but not CB2-knockout mice, which was blocked by pretreatment with either a CB2 or DA D1 or D2 receptor antagonist, suggesting a DA-dependent effect. Finally, fluorescent tract tracing revealed glutamatergic projections from the RN to multiple brain areas including the ventral tegmental area, nucleus accumbens, thalamus, and cerebellum. These findings suggest that CB2Rs in RN glutamate neurons functionally modulate motor activity, and therefore, constitute a new target in cannabis-based medication development for motor disorders.

Sensorimotor gating is a fundamental pre-attentive process that is defined as the inhibition of a motor response by a sensory event. Sensorimotor gating, commonly measured using the prepulse inhibition (PPI) of the auditory startle reflex task, is impaired in patients suffering from various neurological and psychiatric disorders. PPI deficits are a hallmark of schizophrenia, and they are often associated with attention and other cognitive impairments. Although the reversal of PPI deficits in animal models is widely used in pre-clinical research for antipsychotic drug screening, the neurotransmitter systems and synaptic mechanisms underlying PPI are still not resolved, even under physiological conditions. Recent evidence ruled out the longstanding hypothesis that PPI is mediated by midbrain cholinergic inputs to the caudal pontine reticular nucleus (PnC). Instead, glutamatergic, glycinergic, and GABAergic inhibitory mechanisms are now suggested to be crucial for PPI, at the PnC level. Since amygdalar dysfunctions alter PPI and are common to pathologies displaying sensorimotor gating deficits, the present study was designed to test that direct projections to the PnC originating from the amygdala contribute to PPI.Using wild type and transgenic mice expressing eGFP under the control of the glycine transporter type 2 promoter (GlyT2-eGFP mice), we first employed tract-tracing, morphological reconstructions, and immunohistochemical analyses to demonstrate that the central nucleus of the amygdala (CeA) sends glutamatergic inputs lateroventrally to PnC neurons, including GlyT2+ cells. Then, we showed the contribution of the CeA-PnC excitatory synapses to PPI in vivo by demonstrating that optogenetic inhibition of this connection decreases PPI, and optogenetic activation induces partial PPI. Finally, in GlyT2-Cre mice, whole-cell recordings of GlyT2+ PnC neurons in vitro paired with optogenetic stimulation of CeA fibers, as well as photo-inhibition of GlyT2+ PnC neurons in vivo, allowed us to implicate GlyT2+ neurons in the PPI pathway.Our results uncover a feedforward inhibitory mechanism within the brainstem startle circuit by which amygdalar glutamatergic inputs and GlyT2+ PnC neurons contribute to PPI. We are providing new insights to the clinically relevant theoretical construct of PPI, which is disrupted in various neuropsychiatric and neurological diseases.