Transcriptional profiling of dental sensory and proprioceptive trigeminal neurons using single-cell RNA sequencing

Dental primary afferent(DPA)neurons and proprioceptive mesencephalic trigeminal nucleus(MTN)neurons,located in the trigeminal ganglion and the brainstem,respectively,are essential for controlling masticatory functions.Despite extensive transcriptomic studies on various somatosensory neurons,there is still a lack of knowledge about the molecular identities of these populations due to technical challenges in their circuit-validated isolation.Here,we employed high-depth single-cell RNA sequencing(scRNA-seq)in combination with retrograde tracing in mice to identify intrinsic transcriptional features of DPA and MTN neurons.Our transcriptome analysis revealed five major types of DPA neurons with cell type-specific gene enrichment,some of which exhibit unique mechano-nociceptive properties capable of transmitting nociception in response to innocuous mechanical stimuli in the teeth.Furthermore,we discovered cellular heterogeneity within MTN neurons that potentially contribute to their responsiveness to mechanical stretch in the masseter muscle spindles.Additionally,DPA and MTN neurons represented sensory compartments with distinct molecular profiles characterized by various ion channels,receptors,neuropeptides,and mechanoreceptors.Together,our study provides new biological insights regarding the highly specialized mechanosensory functions of DPA and MTN neurons in pain and proprioception.

Whole-Brain Mapping of Direct Inputs to and Axonal Projections from GABAergic Neurons in the Parafacial Zone

The GABAergic neurons in the parafacial zone（PZ） play an important role in sleep-wake regulation and have been identified as part of a sleep-promoting center in the brainstem, but the long-range connections mediating this function remain poorly characterized. Here, we performed whole-brain mapping of both the inputs and outputs of the GABAergic neurons in the PZ of the mouse brain. We used the modified rabies virus Env A-DG-Ds Red combined with a Cre/lox P gene-expression strategy to map the direct monosynaptic inputs to the GABAergic neurons in the PZ, and found that they receive inputs mainly from the hypothalamic area, zona incerta, and parasubthalamic nucleus in the hypothalamus; the substantia nigra, pars reticulata and deep mesencephalic nucleus in the midbrain;and the intermediate reticular nucleus and medial vestibular nucleus（parvocellular part） in the pons and medulla.We also mapped the axonal projections of the PZ GABAergic neurons with adeno-associated virus, and defined the reciprocal connections of the PZ GABAergic neurons with their input and output nuclei. The newlyfound inputs and outputs of the PZ were also listed compared with the literature. This cell-type-specific neuronal whole-brain mapping of the PZ GABAergic neurons may reveal the circuits underlying various functions such as sleep-wake regulation.

Effects of Electroacupuncture on Metabolic Changes in Motor Cortex and Striatum of 6-Hydroxydopamine-Induced Parkinsonian Rats

Objective:To explore the possible underlying mechanism by investigating the effect of electroacupuncture(EA)treatment on the primary motor cortex and striatum in a unilateral 6-hydroxydopamine(6-0HDA)induced rat Parkinson's disease(PD)model.Methods:Male Sprague-Dawley rats were randomly divided into sham group(n=16),model group(n=14),and EA group(n=14).EA stimulation at Dazhui(GV 14)and Baihui(GV20)was applied to PD rats in the EA group for 4 weeks.Behavioral tests were conducted to evaluate the effectiveness of EA treatment.Metabolites were detected by 7.0 T proton nuclear magnetic resonance.Results:Following 4 weeks of EA treatment in PD model rats,the abnormal behavioral impairment induced by 6-0HDA was alleviated.In monitoring changes in metabolic activity,ratios of myoinositol/creatine(Cr)and N-acetyl aspartate(NAA)/Cr in the primary motor cortex were significantly lower at the injected side than the non-injected side in PD rats(P=0.024 and 0.020).The ratios of glutamate+glutamine(Glx)/Cr and NAA/Cr in the striatum were higher and lower,respectively,at the injected side than the non-injected side(P=0.046 and 0.008).EA treatment restored the balance of metabolic activity in the primary motor cortex and striatum.In addition,the taurine/Cr ratio and GIx/Cr ratio were elevated in the striatum of PD model rats compared to sham-lesioned rats(P=0.026 and 0.000).EA treatment alleviated the excessive glutamatergic transmission by down-regulating the striatal Glx/Cr ratio(P=0.001).The Glx/Cr ratio was negatively correlated with floor plane spontaneous locomotion in PD rats(P=0.027 and P=0.0007).Conclusions:EA treatment is able to normalize the metabolic balance in the primary motor cortex and striatum of PD rats,which may contribute to its therapeutic effect on motor deficits.The striatal GIx/Cr ratio may serve as a potential indicator of PD and a therapeutic target of EA treatment.

Mitochondrial Reactive Oxygen Species Elicit Acute and Chronic Itch via Transient Receptor Potential Canonical 3 Activation in Mice

Mitochondrial reactive oxygen species(mROS)that are overproduced by mitochondrial dysfunction are linked to pathological conditions including sensory abnormalities.Here,we explored whether mROS overproduction induces itch through transient receptor potential canonical 3(TRPC3),which is sensitive to ROS.Intradermal injection of antimycin A(AA),a selective inhibitor of mitochondrial electron transport chain complex III for mROS overproduction,produced robust scratching behavior in naïve mice,which was suppressed by MitoTEMPO,a mitochondria-selective ROS scavenger,and Pyr10,a TRPC3-specific blocker,but not by blockers of TRPA1 or TRPV1.AA activated subsets of trigeminal ganglion neurons and also induced inward currents,which were blocked by MitoTEMPO and Pyr10.Besides,dry skin-induced chronic scratching was relieved by MitoTEMPO and Pyr10,and also by resveratrol,an antioxidant.Taken together,our results suggest that mROS elicit itch through TRPC3,which may underlie chronic itch,representing a potential therapeutic target for chronic itch.