Longitudinal intravital imaging nerve degeneration and sprouting in the toes of spared nerve injured mice.

Neuropathic pain is pain caused by damage to the somatosensory nervous system. Both degenerating injured nerves and neighboring sprouting nerves can contribute to neuropathic pain. However, the mesoscale changes in cutaneous nerve fibers over time after the loss of the parent nerve has not been investigated in detail. In this study, we followed the changes in nerve fibers longitudinally in the toe tips of mice that had undergone spared nerve injury (SNI). Nav1.8-tdTomato, Thy1-GFP and MrgD-GFP mice were used to observe the small and large cutaneous nerve fibers. We found that peripheral nerve plexuses degenerated within 3 days of nerve injury, and free nerve endings in the epidermis degenerated within 2 days. The timing of degeneration paralleled the initiation of mechanical hypersensitivity. We also found that some of the Nav1.8-positive nerve plexuses and free nerve endings in the fifth toe survived, and sprouting occurred mostly from 7 to 28 days. The timing of the sprouting of nerve fibers in the fifth toe paralleled the maintenance phase of mechanical hypersensitivity. Our results support the hypotheses that both injured and intact nerve fibers participate in neuropathic pain, and that, specifically, nerve degeneration is related to the initiation of evoked pain and nerve sprouting is related to the maintenance of evoked pain.

Whole-brain efferent and afferent connectivity of mouse ventral tegmental area melanocortin-3 receptor neurons.

The mesolimbic dopamine (DA) system is involved in the regulation of multiple behaviors, including feeding, and evidence demonstrates that the melanocortin system can act on the mesolimbic DA system to control feeding and other behaviors. The melanocortin-3 receptor (MC3R) is an important component of the melanocortin system, but its overall role is poorly understood. Because MC3Rs are highly expressed in the ventral tegmental area (VTA) and are likely to be the key interaction point between the melanocortin and mesolimbic DA systems, we set out to identify both the efferent projection patterns of VTA MC3R neurons and the location of the neurons providing afferent input to them. VTA MC3R neurons were broadly connected to neurons across the brain but were strongly connected to a discrete set of brain regions involved in the regulation of feeding, reward, and aversion. Surprisingly, experiments using monosynaptic rabies virus showed that proopiomelanocortin (POMC) and agouti-related protein (AgRP) neurons in the arcuate nucleus made few direct synapses onto VTA MC3R neurons or any of the other major neuronal subtypes in the VTA, despite being extensively labeled by general retrograde tracers injected into the VTA. These results greatly contribute to our understanding of the anatomical interactions between the melanocortin and mesolimbic systems and provide a foundation for future studies of VTA MC3R neurons and the circuits containing them in the control of feeding and other behaviors.

Identification of intrinsic primary afferent neurons in mouse jejunum.

BACKGROUND: The gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut-brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum. METHODS: Advillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin-expressing enteric neurons using confocal microscopy, calcium imaging, and whole-cell patch-clamp electrophysiology. KEY RESULTS: Advillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene-related peptide (CGRP), but not nNOS. CONCLUSIONS AND INFERENCES: Advillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease.

Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

TNF-α-converting enzyme, a member of the ADAM (A disintegrin and metalloproteinase) protease family and also known as ADAM17, regulates inflammation and regeneration in health and disease. ADAM17 targets are involved in pain development and hypersensitivity in animal models of inflammatory and neuropathic pain. However, the role of ADAM17 in the pain pathway is largely unknown. Therefore, we used the hypomorphic ADAM17 (ADAM17ex/ex) mouse model to investigate the importance of ADAM17 in nociceptive behavior, morphology, and function of primary afferent nociceptors. ADAM17ex/ex mice were hyposensitive to noxious stimulation, showing elevated mechanical thresholds as well as impaired heat and cold sensitivity. Despite these differences, skin thickness and innervation were comparable to controls. Although dorsal root ganglia of ADAM17ex/ex mice exhibited normal morphology of peptidergic and nonpeptidergic neurons, a small but significant reduction in the number of isolectin ß-4-positive neurons was observed. Functional electrical properties of unmyelinated nociceptors showed differences in resting membrane potential, afterhyperpolarization, and firing patterns in specific subpopulations of sensory neurons in ADAM17ex/ex mice. However, spinal cord morphology and microglia activity in ADAM17ex/ex mice were not altered. Our data suggest that ADAM17 contributes to the processing of painful stimuli, with a complex mode of action orchestrating the function of neurons along the pain pathway.-Quarta, S., Mitric, M., Kalpachidou, T., Mair, N., Schiefermeier-Mach, N., Andratsch, M., Qi, Y., Langeslag, M., Malsch, P., Rose-John, S., Kress, M. Impaired mechanical, heat, and cold nociception in a murine model of genetic TACE/ADAM17 knockdown.

PACAP and its role in primary headaches.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide implicated in a wide range of functions, such as nociception and in primary headaches. Regarding its localization, PACAP has been observed in the sensory trigeminal ganglion (TG), in the parasympathetic sphenopalatine (SPG) and otic ganglia (OTG), and in the brainstem trigeminocervical complex. Immunohistochemistry has shown PACAP-38 in numerous cell bodies of SPG/OTG, co-stored with vasoactive intestinal peptide (VIP), nitric oxide synthase (NOS) and, to a minor degree, with choline acetyltransferase. PACAP has in addition been found in a subpopulation of calcitonin gene-related peptide (CGRP)-immunoreactive cells in the trigeminal system. The PACAP/VIP receptors (PAC1, VPAC1, and VPAC2) are present in sensory neurons and in vascular smooth muscle related to the trigeminovascular system. It is postulated that PACAP is involved in nociception. In support, abolishment of PACAP synthesis or reception leads to diminished pain responses, whereas systemic PACAP-38 infusion triggers pain behavior in animals and delayed migraine-like attacks in migraine patients without marked vasodilatory effects. In addition, increased plasma levels have been documented in acute migraine attacks and in cluster headache, in accordance with findings in experimental models of trigeminal activation. This suggest that the activation of the trigeminal system may result in elevated venous levels of PACAP, a change that can be reduced when headache is treated. The data presented in this review indicate that PACAP and its receptors may be promising targets for migraine therapeutics.

Localization of zinc transporters in the spinal cord of cynomolgus monkey.

Zinc is abundant in the spinal cord, where it participates in several physiological and pathophysiological processes, including neurotransmission, spinal cord injury, and amyotrophic lateral sclerosis. However, the mechanisms underlying zinc homeostasis in the spinal cord are largely unknown. Zinc transporters (ZnTs) are responsible for transporting zinc from the cytoplasm to the extracellular space or to intracellular compartments. In the present study, we examined the distribution of ZnT1-10 proteins in the spinal cord of cynomolgus monkey. Immunohistochemical studies demonstrate that all detected ZnT family members are expressed in the gray matter. ZnT1-10 immunoreactivity can be seen in both motor and sensory neurons in the dorsal and ventral horn from the cervical to sacral segments. No obvious immunostaining was found in the glia cells. The present study demonstrates that ZnT proteins are functionally important for regulating zinc metabolism in both motor and sensory functions in monkey spinal cord.

5-HT3A -driven green fluorescent protein delineates gustatory fibers innervating sour-responsive taste cells: A labeled line for sour taste?

Taste buds contain multiple cell types with each type expressing receptors and transduction components for a subset of taste qualities. The sour sensing cells, Type III cells, release serotonin (5-HT) in response to the presence of sour (acidic) tastants and this released 5-HT activates 5-HT3 receptors on the gustatory nerves. We show here, using 5-HT3A GFP mice, that 5-HT3 -expressing nerve fibers preferentially contact and receive synaptic contact from Type III taste cells. Further, these 5-HT3 -expressing nerve fibers terminate in a restricted central-lateral portion of the nucleus of the solitary tract (nTS)-the same area that shows increased c-Fos expression upon presentation of a sour tastant (30 mM citric acid). This acid stimulation also evokes c-Fos in the laterally adjacent mediodorsal spinal trigeminal nucleus (DMSp5), but this trigeminal activation is not associated with the presence of 5-HT3 -expressing nerve fibers as it is in the nTS. Rather, the neuronal activation in the trigeminal complex likely is attributable to direct depolarization of acid-sensitive trigeminal nerve fibers, for example, polymodal nociceptors, rather than through taste buds. Taken together, these findings suggest that transmission of sour taste information involves communication between Type III taste cells and 5-HT3 -expressing afferent nerve fibers that project to a restricted portion of the nTS consistent with a crude mapping of taste quality information in the primary gustatory nucleus.

α-Conotoxin Vc1.1 inhibits human dorsal root ganglion neuroexcitability and mouse colonic nociception via GABAB receptors.

OBJECTIVE: α-Conotoxin Vc1.1 is a small disulfide-bonded peptide from the venom of the marine cone snail Conus victoriae. Vc1.1 has antinociceptive actions in animal models of neuropathic pain, but its applicability to inhibiting human dorsal root ganglion (DRG) neuroexcitability and reducing chronic visceral pain (CVP) is unknown. DESIGN: We determined the inhibitory actions of Vc1.1 on human DRG neurons and on mouse colonic sensory afferents in healthy and chronic visceral hypersensitivity (CVH) states. In mice, visceral nociception was assessed by neuronal activation within the spinal cord in response to noxious colorectal distension (CRD). Quantitative-reverse-transcription-PCR, single-cell-reverse-transcription-PCR and immunohistochemistry determined γ-aminobutyric acid receptor B (GABABR) and voltage-gated calcium channel (CaV2.2, CaV2.3) expression in human and mouse DRG neurons. RESULTS: Vc1.1 reduced the excitability of human DRG neurons, whereas a synthetic Vc1.1 analogue that is inactive at GABABR did not. Human DRG neurons expressed GABABR and its downstream effector channels CaV2.2 and CaV2.3. Mouse colonic DRG neurons exhibited high GABABR, CaV2.2 and CaV2.3 expression, with upregulation of the CaV2.2 exon-37a variant during CVH. Vc1.1 inhibited mouse colonic afferents ex vivo and nociceptive signalling of noxious CRD into the spinal cord in vivo, with greatest efficacy observed during CVH. A selective GABABR antagonist prevented Vc1.1-induced inhibition, whereas blocking both CaV2.2 and CaV2.3 caused inhibition comparable with Vc1.1 alone. CONCLUSIONS: Vc1.1-mediated activation of GABABR is a novel mechanism for reducing the excitability of human DRG neurons. Vc1.1-induced activation of GABABR on the peripheral endings of colonic afferents reduces nociceptive signalling. The enhanced antinociceptive actions of Vc1.1 during CVH suggest it is a novel candidate for the treatment for CVP.

Distribution and sub-types of afferent fibre in the mouse urinary bladder.

AIM: Increased afferent fibre activity contributes to pathological conditions such as the overactive bladder syndrome. Nerve fibres running near the urothelium are considered to be afferent as no efferent system has yet been described. The aim of this study was to identify sub-types of afferent nerve fibres in the mouse bladder wall based on morphological criteria and analyse regional differences. MATERIALS AND METHODS: 27 bladders of six month old C57BL/6 mice were removed and tissues were processed for immunohistochemistry. Cryostat sections were cut and stained for Protein Gene Product 9.5 (PGP), calcitonin gene related polypeptide (CGRP), neurofilament (NF), vesicular acetylcholine transporter (VAChT) and neuronal nitric oxide synthase (nNOS). RESULTS: In the sub-urothelium, different types of afferent nerve fibre were found, i.e. immunoreactive (IR) to; CGRP, NF, VAChT, and/or nNOS. At the bladder base, the sub-urothelium was more densely innervated by CGRP-IR and VAChT-IR nerve fibres, then at the lateral wall. NF- and nNOS nerves were sparsely distributed in the sub-urothelium throughout the bladder. At the lateral wall the inner muscle is densely innervated by CGRP-IR nerve fibres. NF, VAChT and nNOS nerves were evenly distributed in the different muscle layers throughout the bladder. Nerve fibre terminals expressing CGRP and NF were found within the extra-mural ganglia at the bladder base. CONCLUSIONS: Different types of afferent nerve fibres were identified in the sub-urothelium of the mouse bladder. At the bladder base the sub-urothelium is more densely innervated than the lateral wall by CGRP-IR and VAChT-IR afferent nerve fibres. CGRP and NF afferent nerve fibres in the muscle layer probably relay afferent input to external ganglia located near the bladder base. The identification of different afferent nerves in the sub-urothelium suggests a functional heterogeneity of the afferent nerve fibres in the urinary bladder.

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice.

Somatosensory nerves transduce thermal, mechanical, chemical, and noxious stimuli caused by both endogenous and environmental agents. The cell bodies of these afferent neurons are located within the sensory ganglia. Sensory ganglia innervate a specific organ or portion of the body. For instance, the dorsal root ganglia (DRG) are located in the vertebral column and extend processes throughout the body and limbs. The trigeminal ganglia are located in the skull and innervate the face, and upper airways. Vagal afferents of the nodose ganglia extend throughout the gut, heart, and lungs. The nodose neurons control a diverse array of functions such as: respiratory rate, airway irritation, and cough reflexes. Thus, to understand and manipulate their function, it is critical to identify and isolate airway specific neuronal sub-populations. In the mouse, the airways are exposed to a fluorescent tracer dye, Fast Blue, for retrograde tracing of airway-specific nodose neurons. The nodose ganglia are dissociated and fluorescence activated cell (FAC) sorting is used to collect dye positive cells. Next, high quality ribonucleic acid (RNA) is extracted from dye positive cells for next generation sequencing. Using this method airway specific neuronal gene expression is determined.

Distinct afferent innervation patterns within the human proximal and distal esophageal mucosa.

Little is known about the mucosal phenotype of the proximal human esophagus. There is evidence to suggest that the proximal esophagus is more sensitive to chemical and mechanical stimulation compared with the distal. This may have physiological relevance (e.g., in prevention of aspiration of gastroesophageal refluxate), but also pathological relevance (e.g., in reflux perception or dysphagia). Reasons for this increased sensitivity are unclear but may include impairment in mucosal barrier integrity or changes in sensory innervation. We assessed mucosal barrier integrity and afferent nerve distribution in the proximal and distal esophagus of healthy human volunteers. In 10 healthy volunteers baseline proximal and distal esophageal impedance was measured in vivo. Esophageal mucosal biopsies from the distal and proximal esophagus were taken, and baseline transepithelial electrical resistance (TER) was measured in Ussing chambers. Biopsies were examined immunohistochemically for presence and location of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. In a further four healthy volunteers we investigated for colocalization of CGRP and protein gene product (PGP) 9.5 immunoreactivity in nerve fibers. Baseline impedance was higher in the proximal than in the distal esophagus [2,936 Ω (SD578) vs. 2,229 Ω (SD821); P = 0.03], however, baseline TER was not significantly different between them. Mucosal CGRP-immunoreactive nerves were found in the epithelium of both proximal and distal esophagus, but were located more superficially in the proximal mucosa compared with the distal [11.5 (SD7) vs. 21.7 (SD5) cell layers from lumen, P = 0.002] 19% of proximal, and 10% of distal mucosal PGP-immunoreactive fibers colocalized with CGRP. PGP-immunoreactive fibers were also significantly closer to the luminal surface in the proximal compared with the distal esophagus (P < 0.001). We conclude that mucosal barrier integrity is similar in proximal and distal esophagus, but proximal mucosal afferent nerves are in a more superficial location. The enhanced sensitivity to reflux-evoked symptoms of the proximal esophagus most likely has an anatomical basis.

Reinnervation of renal afferent and efferent nerves at 5.5 and 11 months after catheter-based radiofrequency renal denervation in sheep.

Previous studies indicate that catheter-based renal denervation reduces blood pressure and renal norepinephrine spillover in human resistant hypertension. The effects of this procedure on afferent sensory and efferent sympathetic renal nerves, and the subsequent degree of reinnervation, have not been investigated. We therefore examined the level of functional and anatomic reinnervation at 5.5 and 11 months after renal denervation using the Symplicity Flex catheter. In normotensive anesthetized sheep (n=6), electric stimulation of intact renal nerves increased arterial pressure from 99±3 to 107±3 mm Hg (afferent response) and reduced renal blood flow from 198±16 to 85±20 mL/min (efferent response). In a further group (n=6), immediately after denervation, renal sympathetic nerve activity was absent and the responses to electric stimulation were abolished. At 11 months after denervation (n=5), renal sympathetic nerve activity and the responses to electric stimulation were at normal levels. Immunohistochemical staining for renal efferent (tyrosine hydroxylase) and renal afferent nerves (calcitonin gene-related peptide), as well as renal norepinephrine levels, was normal 11 months after denervation. Findings at 5.5 months after denervation were similar (n=5). In summary, catheter-based renal denervation effectively ablated the renal afferent and efferent nerves in normotensive sheep. By 11 months after denervation the functional afferent and efferent responses to electric stimulation were normal. Reinnervation at 11 months after denervation was supported by normal anatomic distribution of afferent and efferent renal nerves. In view of this evidence, the mechanisms underlying the prolonged hypotensive effect of catheter-based renal denervation in human resistant hypertension need to be reassessed.

Terminals of the major thalamic input to visual cortex are devoid of synapsin proteins.

Synapsins are nerve-terminal proteins that are linked to synaptic transmission and key factors in several forms of synaptic plasticity. While synapsins are generally assumed to be ubiquitous in synaptic terminals, whether they are excluded from certain types of terminals is of interest. In the visual pathway, synapsins are lacking in photoreceptor and bipolar cell terminals as well as in retinogeniculate synapses. These are the terminals of the first three feedforward synapses in the visual pathway, implying that lack of synapsins may be a common property of terminals that provide the primary driver activity onto their postsynaptic neurons. To further investigate this idea, we studied the fourth driver synapse, thalamocortical synapses in visual cortex, using glutamatergic terminal antibody markers anti-VGluT1 and VGluT2, anti-Synapsin I and II, and confocal microscopy to analyze co-localization of these proteins in terminals. We also used pre-embedding immunocytochemical labeling followed by electron microscopy to investigate morphological similarities or differences between terminals containing synapsins or VGluT2. In visual cortex, synapsin coincided extensively with non-TC-neuron marker, VGluT1, while thalamocortical terminal marker VGluT2 and synapsin overlap was sparse. Morphologically, synapsin-stained terminals were smaller than non-stained, while VGluT2-positive thalamocortical terminals constituted the largest terminals in cortex. The size discrepancy between synapsin- and VGluT2-positive terminals, together with the complementary staining patterns, indicates that thalamocortical synapses are devoid of synapsins, and support the hypothesis that afferent sensory information is consistently transmitted without the involvement of synapsins. Furthermore, VGluT2 and synapsins were colocalized in other brain structures, suggesting that lack of synapsins is not a property of VGluT2-containing terminals, but a property of primary driver terminals in the visual system.

VGLUT3-immunoreactive afferents of the lateral septum: ultrastructural evidence for a modulatory role of glutamate.

Through its extensive connections with various brain regions, the lateral septum (LS) participates in the processing of cognitive, emotional and autonomic information. It is decisively involved in the generation of behavioral responses according to environmental demands. Modulatory afferents reaching the LS from the brain stem (e.g. dopaminergic, serotonergic) play a role in the adjustment of these behavioral responses. Recently, a population of vesicular glutamate transporter 3-immunoreactive (VGLUT3-ir) fibers forming prominent pericellular basket-like structures (PBLS) was described in the rat LS. These VGLUT3-ir PBLS are distributed in a layer-like pattern, which is very typical for modulatory afferents of the LS. There is meanwhile broad evidence that glutamate can act as a modulatory or co-transmitter and that those neurons, which make use of this transmission mode, primarily express VGLUT3. Thus, the VGLUT3-ir fibers within the LS could also display features typical for non-canonical glutamatergic transmission. Employing pre-embedding electron microscopy for VGLUT3 in rats, we show now that the VGLUT3-ir fibers outlining LS neurons represent axonal terminals, which primarily form symmetric synapses with somata and proximal dendrites of their target neurons. Occasionally, we also found VGLUT3-ir terminals that make canonical asymmetric synapses on distal dendrites and spines. Thus, VGLUT3-ir boutons in the LS form two different, disproportionate, populations of synaptic contacts with their target neurons. The larger one of them is indicative of employing glutamate as a modulatory transmitter.

Recent advances in olfactory receptor-based biosensors.

The biological olfactory system can recognize and discriminate thousands of volatile organic compounds (VOCs) with extremely high sensitivity and specificity. The most fundamental elements are olfactory receptors (ORs) in the cilia of olfactory sensory neurons (OSNs), which contribute greatly to the high-performance olfactory system. The excellent properties of ORs are generally recognized in the development of biomimetic OR-based biosensors. Over the past two decades, much work has been done in developing OR-based biosensors due to their promising potential in many applications. In this article, we will outline the latest advances of OR-based biosensors. Two current crucial issues in this field will be discussed, namely, the production methods and immobilization techniques of ORs. We will also elaborate on various OR-based biosensors and their latest developments. Finally, current research trends and future challenges in this field will be discussed.

Degeneration of capsaicin sensitive sensory nerves enhances myocardial injury in acute myocardial infarction in rats.

BACKGROUND: Evidence indicated an involvement of afferent nerves in the pathology of acute myocardial infarction. This study was undertaken to clarify the role and mechanisms by which the sensory afferent degeneration exacerbates the myocardial injury in acute myocardial infarction in rats. METHODS: The myocardial injury was assessed by analysis of 1) the differences in the infarct size, myocyte apoptosis, the caspase activity in the myocardium and cardiac troponin I in serum between the denervated and non-denervated rats; 2) the differences in the size of infarctiom with and without antagonisms of endogenous neurokinin 1 receptor or calcitonin gene related peptide receptor in acute myocardial infarction. RESULTS: Degeneration of the afferent nerves resulted in marked increase in the pain threshold and decrease in substance P and calcitonin gene related peptide in dorsal root ganglia, spinal dorsal horn and myocardium. Increases of the infarction size (39% ± 4% vs. 26% ± 4%,), troponin-I (28.4 ± 8.89 ng/ml, vs. 14.6 ± 9.75 ng/ml), apoptosis of myocytes (by 1.8 ± 0.2 folds) and caspase-3 activity (1.6 ± 0.3 vs. 1.05 ± 0.18) were observed in the denervated animals at 6h of myocardial infarction, compared with the non-denervated rats. Antagonisms of the endogenous neurokinin 1 receptor or calcitonin gene related peptide receptor caused increase of the size of infarction in the animals. CONCLUSION: Degeneration of capsaicin sensitive afferent nerves enhances the myocardial injury of acute myocardial infarction, possibly due to reduction of endogenous calcitonin gene related peptide and substance P.

Ultrastructural analysis of the synaptic connectivity of TRPV1-expressing primary afferent terminals in the rat trigeminal caudal nucleus.

Trigeminal primary afferents that express the transient receptor potential vanilloid 1 (TRPV1) are important for the transmission of orofacial nociception. However, little is known about how the TRPV1-mediated nociceptive information is processed at the first relay nucleus in the central nervous system (CNS). To address this issue, we studied the synaptic connectivity of TRPV1-positive (+) terminals in the rat trigeminal caudal nucleus (Vc) by using electron microscopic immunohistochemistry and analysis of serial thin sections. Whereas the large majority of TRPV1+ terminals made synaptic contacts of an asymmetric type with one or two postsynaptic dendrites, a considerable fraction also participated in complex glomerular synaptic arrangements. A few TRPV1+ terminals received axoaxonic contacts from synaptic endings that contained pleomorphic synaptic vesicles and were immunolabeled for glutamic acid decarboxylase, the synthesizing enzyme for the inhibitory neurotransmitter γ-aminobutyric acid (GABA). We classified the TRPV1+ terminals into an S-type, containing less than five dense-core vesicles (DCVs), and a DCV-type, containing five or more DCVs. The number of postsynaptic dendrites was similar between the two types of terminals; however, whereas axoaxonic contacts were frequent on the S-type, the DCV-type did not receive axoaxonic contacts. In the sensory root of the trigeminal ganglion, TRPV1+ axons were mostly unmyelinated, and a small fraction was small myelinated. These results suggest that the TRPV1-mediated nociceptive information from the orofacial region is processed in a specific manner by two distinct types of synaptic arrangements in the Vc, and that the central input of a few TRPV1+ afferents is presynaptically modulated via a GABA-mediated mechanism.

[Afferent nerve connection among "Hegu" (LI4), "Neiguan" (PC6), "Futu" (LI18) and thyroid gland region: fluorescent double labelling method].

OBJECTIVE: To observe the peripheral and central neural connection of acupoint "Hegu" (LI4), "Neiguan" (PC6), "Futu" (LI18) and the thyroid gland (TG) region with fluorescence double-labelling method. METHODS: Thirty male Wistar rats were divided into LI4-LI18 group, PC6-LI18 group, TG-LI 18 group, LI 4-TG group, and PC 6-TG group, with 6 rats in each. Fluorescence dyes Propidium Iodide (PI, 10 microL) and Bisbenzimide (Bb, 10 microL) were, separately, injected into those acupoints mentioned above and the TG region. Sixty hours after PI-injection and 12 hours after Bb-injection, the rats under deep anesthesia were transcardiacally perfused with PBS containing 4% polyoxymethylene, followed by taking the spinal cord and dorsal root ganglia (DRGs) of the cervical segments (C1-C8) and thoracic 1 (T1) segment. Fluorescent single- and dual-labeled cells of the sliced DRGs and cervical spinal cord were observed by fluorescence microscope. RESULTS: (1) All PI- and Bb-labeled cells were found to distribute in DRGs from C1-T1 segments. PI single-labeled cells from LI4 and PC6 mainly distributed in DRGs from C4 to C8. Bb single-labeled cells from LI18 and TG region distributed in DRGs from C1-C6. (2) Dual-labeled cells in the LI 4-LI 188 group, PC6-LI18 group, LI4-TG group, and PC6-TG group distributed in DRGs from C3 to C7, suggesting bifurcate peripheral processes of the cervical DRG neurons to innervate LI8, LI4, PC6 and the TG region. And the dual-labeled cells of the TG-LI 18 group distributed mainly in DRGs from C1 to C4. (3) A small number of single-labeled neurons(about 8% of total labeled cells in DRGs) and only several dual-labeled neurons were found in the anterior horn of the cervical spinal cord. CONCLUSION: LI18, LI4 and PC6 and the thyroid gland have the same peripheral cells in DRGs of C3-C7 segments, suggesting that the bifurcate peripheral innervation may provide an anatomic evidence for the analgesic effect of acupuncture of LI18, LI4 and PC6 on thyroidectomy.

Altered expression of P2X3 in vagal and spinal afferents following esophagitis in rats.

Purinergic P2X(3) receptors are predominantly expressed in small diameter primary afferent neurons and activation of these receptors by adenosine triphosphate is reported to play an important role in nociceptive signaling. The objective of this study was to investigate the expression of P2X(3) receptors in spinal and vagal sensory neurons and esophageal tissues following esophagitis in rats. Two groups of rats were used including 7 days fundus-ligated (7D-ligated) esophagitis and sham-operated controls. Esophagitis was produced by ligating the fundus and partial obstruction of pylorus that initiated reflux of gastric contents. The sham-operated rats underwent midline incision without surgical manipulation of the stomach. Expressions of P2X(3) receptors in thoracic dorsal root ganglia (DRGs), nodose ganglia (NGs), and esophageal tissues were evaluated by RT-PCR, western blot and immunohistochemistry. Esophageal neurons were identified by retrograde transport of Fast Blue from the esophagus. There were no significant differences in P2X(3) mRNA expressions in DRGs (T1-T3) and NGs between 7D-ligated and sham-operated rats. However, there was an upregulation of P2X(3) mRNA in DRGs (T6-T12) and in the esophageal muscle. At protein level, P2X(3) exhibited significant upregulation both in DRGs and in NGs of rats having chronic esophagitis. Immunohistochemical analysis exhibited a significant increase in P2X(3) and TRPV1 co-expression in DRGs and NGs in 7D-ligated rats compared to sham-operated rats. The present findings suggest that chronic esophagitis results in upregulation of P2X(3) and its co-localization with TRPV1 receptor in vagal and spinal afferents. Changes in P2X(3) expression in vagal and spinal sensory neurons may contribute to esophageal hypersensitivity following acid reflux-induced esophagitis.

Peripheral nerve pathways of afferent fibers innervating the lumbar spine in rats.

UNLABELLED: We investigated the pathways of afferent fibers innervating the lumbar spine. The neurotracer DiI was applied to reference sites at the L5 level in rats. One of 4 surgeries was performed before DiI application: (1) transaction of the dorsal ramus of the L2 spinal nerve, (2) transaction of the ventral ramus of the L2 spinal nerve, (3) transaction of the psoas major muscle at L3-L4, or (4) removal of the paravertebral sympathetic trunks from L3-L5. The number of DiI-labeled neurons in the dorsal root ganglia after surgery was compared with neuron numbers in surgery-naïve rats. The number of DiI-labeled neurons decreased drastically with transection of the L2 ventral ramus or psoas major muscle for the ventral and lateral portions of the disc and vertebral body and after transection of the L2 dorsal ramus for the facet joint and spinous process. Removal of the sympathetic trunks did not reduce the number of DiI-labeled neurons significantly in the extra-spinal canal sites. In contrast, significant reductions occurred after the removal of the paravertebral sympathetic trunks in the intra-spinal canal sites. Extra-spinal canal sites received afferent fibers primarily through somatic routes, but intra-spinal canal sites received afferent fibers via the sympathetic trunks. PERSPECTIVE: Extra-spinal canal sites of the lumbar spine received afferent fibers from muscles originating in the site. Intra-spinal canal sites received a considerable number of afferent fibers via the paravertebral sympathetic trunks. These results may provide new insights for nerve block treatment of low back pain.