Agouti-related peptide (AgRP) neurons of the arcuate nucleus of the hypothalamus (ARC) promote homeostatic feeding at times of caloric insufficiency, yet they are rapidly suppressed by food-related sensory cues before ingestion. Here we identify a highly selective inhibitory afferent to AgRP neurons that serves as a neural determinant of this rapid modulation. Specifically, GABAergic projections arising from the ventral compartment of the dorsomedial nucleus of the hypothalamus (vDMH) contribute to the preconsummatory modulation of ARCAgRP neurons. In a manner reciprocal to ARCAgRP neurons, ARC-projecting leptin receptor-expressing GABAergic vDMH neurons exhibit rapid activation upon availability of food that additionally reflects the relative value of the food. Thus, leptin receptor-expressing GABAergic vDMH neurons form part of the sensory network that relays real-time information about the nature and availability of food to dynamically modulate ARCAgRP neuron activity and feeding behavior.

The long belief that dental primary afferent (DPA) neurons are entirely composed of nociceptive neurons has been challenged by several anatomical and functional investigations. In order to characterize non-nociceptivepopulation among DPA neurons, retrograde transport fluorescent dye was placed in upper molars of rats and immunohistochemical detection of peripherin and neurofilament 200 in the labeled trigeminal ganglia was performed. As the results, majority ofDPA neurons were peripherin-expressing small-sized neurons, showing characteristic ofnociceptive C-fibers. However, 25.7% of DPA were stained with antibody against neurofilament 200, indicating significant portion of DPA neurons are related to large myelinated Aβ fibers. There were a small number of neurons thatexpressed both peripherin and neurofilament 200, suggestive of Aδ fibers. The possible transition of neurochemical properties by neuronal injury induced by retrograde labeling technique was ruled out by detection of minimal expression of neuronal injury marker, ATF-3. These results suggest that in addition to the large population of C-fiber-related nociceptive neurons, a subset of DPA neurons is myelinated large neurons, which is related to low-threshold mechanosensitive Aβ fibers. We suggest that these Aβ fiber-related neurons might play a role as mechanotransducers of fluid movement within dentinal tubules.

Exposure to intense sound can cause damage to the delicate sensory and neuronal components of the cochlea leading to hearing loss. Such damage often causes the dendrites of the spiral ganglion neurons (SGN), the neurons that provide the afferent innervation of the hair cells, to swell and degenerate thus damaging the synapse. In models of neuropathic pain, axotomy, another form of afferent nerve damage, is accompanied by altered voltage-gated sodium channel (VGSC) expression, leading to neuronal hyperactivity. In this study, adult Wistar rats were exposed to noise which produced a mild, 20 dB hearing threshold elevation and their VGSC expression was investigated. Quantitative PCR showed decreased Na(V)1.1 and Na(V)1.6 mRNA expression in the SGN following noise exposure (29% and 56% decrease respectively) while Na(V)1.7 mRNA expression increased by approximately 20% when compared to control rats. Immunohistochemistry extended these findings, revealing increased staining for Na(V)1.1 along the SGN dendrites and Na(V)1.7 in the cell bodies after noise. These results provide the first evidence for selective changes in VGSC expression following moderate noise-induced hearing loss and could contribute to elevated hearing thresholds and to the generation of perceptual anomalies commonly associated with cochlear damage, such as tinnitus and hyperacusis.

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.

Although nociceptive afferents innervating the body have been heavily studied form many years, much less attention has been paid to trigeminal afferent biology. In particular, very little is known concerning trigeminal nociceptor responses to heat, and almost nothing in the rat. This study uses a highly controlled and reproducible diode laser stimulator to investigate the activation of trigeminal afferents to noxious skin heating.

The gut-brain axis embodies the bi-directional communication between the gastrointestinal tract and the central nervous system (CNS), where vagal afferent neurons (VANs) serve as sensors for a variety of gut-derived signals. The gut is colonized by a large and diverse population of microorganisms that communicate via small (effector) molecules, which also act on the VAN terminals situated in the gut viscera and consequently influence many CNS processes. However, the convoluted in vivo environment makes it difficult to study the causative impact of the effector molecules on VAN activation or desensitization. Here, we report on a VAN culture and its proof-of-principle demonstration as a cell-based sensor to monitor the influence of gastrointestinal effector molecules on neuronal behavior. We initially compared the effect of surface coatings (poly-L-lysine vs. Matrigel) and culture media composition (serum vs. growth factor supplement) on neurite growth as a surrogate of VAN regeneration following tissue harvesting, where the Matrigel coating, but not the media composition, played a significant role in the increased neurite growth. We then used both live-cell calcium imaging and extracellular electrophysiological recordings to show that the VANs responded to classical effector molecules of endogenous and exogenous origin (cholecystokinin serotonin and capsaicin) in a complex fashion. We expect this study to enable platforms for screening various effector molecules and their influence on VAN activity, assessed by their information-rich electrophysiological fingerprints.

Cell bodies of afferent neurons located in lumbosacral dorsal root ganglia (DRG) provide Adelta- and C-fibres to the urinary bladder, reporting bladder wall tension, volume and noxious stimuli. Recent studies suggested an involvement of muscarinic acetylcholine receptors (mAChRs) not only in detrusor contractility but also in modulating afferent function, and this has been linked to the beneficial effects of muscarinic antagonists in the treatment of overactive bladder. Here, we aimed to determine the inventory of mAChR subtypes expressed by bladder afferent neurons in the mouse. Bladder afferent neurons were identified by retrograde neuronal tracing using Fast Blue (FB) or 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorhydrate (DiI) injection into the detrusor muscle. DRG L6-S1 were recognized as the major location of bladder afferent perikarya with an additional smaller peak at L1/L2. Retrogradely labelled bladder afferents located in DRG L4-S2 were subjected to immunohistochemistry or to laser-assisted microdissection with subsequent RT-PCR to study expression of mAChRs subtypes M1R-M5R. Immunolabelling for mAChR subtype M2R, validated on DRG from M2R gene-deficient mice, demonstrated this subtype on 35% of FB-labelled bladder afferents. RT-PCR demonstrated expression of subtypes M2R, M3R and M4R, but not of M1R and M5R, in pooled samples (30 section profiles each) of laser microdissected DiI-labelled bladder afferent cell bodies. In conclusion, bladder afferent neurons express different subtypes of mAChRs (M2R, M3R and M4R). Thus, processing of sensory information from the bladder appears to be under direct cholinergic control.

Aβ-afferents in maxillary or V2 trigeminal ganglion (TG) neurons are somatosensory neurons that may be involved in both non-nociceptive and nociceptive functions in orofacial regions. However, electrophysiological properties of these V2 trigeminal Aβ-afferent neurons have not been well characterized so far. Here, we used rat ex vivo trigeminal nerve preparations and applied patch-clamp recordings to large-sized V2 TG neurons to characterize their electrophysiological properties. All the cells recorded had afferent conduction velocities in the range of Aβ-afferent conduction speeds. However, these V2 trigeminal Aβ-afferent neurons displayed different action potential (AP) properties. APs showed fast kinetics in some cells but slow kinetics with shoulders in repolarization phases in other cells. Based on the derivatives of voltages in AP repolarization with time (dV/dt), we classified V2 trigeminal Aβ-afferent neurons into four types: type I, type II, type IIIa and type IIIb. Type I V2 trigeminal Aβ-afferent neurons had the largest dV/dt of repolarization, the fastest AP conduction velocities, the shortest AP and afterhyperpolarization (AHP) durations, and the highest AP success rates. In contrast, type IIIb V2 trigeminal Aβ-afferent neurons had the smallest dV/dt of AP repolarization, the slowest AP conduction velocities, the longest AP and AHP durations, and the lowest AP success rates. The type IIIb cells also had significantly lower voltage-activated K+ currents. For type II and type IIIa V2 trigeminal Aβ-afferent neurons, AP parameters were in the range between those of type I and type IIIb V2 trigeminal Aβ-afferent neurons. Our electrophysiological classification of V2 trigeminal Aβ-afferent neurons may be useful in future to study their non-nociceptive and nociceptive functions in orofacial regions.

Sensory neurons are able to detect tissue ischaemia and both transmit information to the brainstem as well as release local vasoactive mediators. Their ability to sense tissue ischaemia is assumed to be primarily mediated through proton sensing ion channels, lack of oxygen however may also affect sensory neuron function. In this study we investigated the effects of anoxia on isolated capsaicin sensitive neurons from rat nodose ganglion. Acute anoxia triggered a reversible increase in [Ca2+]i that was mainly due to Ca2+-efflux from FCCP sensitive stores and from caffeine and CPA sensitive ER stores. Prolonged anoxia resulted in complete depletion of ER Ca2+-stores. Mitochondria were partially depolarised by acute anoxia but mitochondrial Ca2+-uptake/buffering during voltage-gated Ca2+-influx was unaffected. The process of Ca2+-release from mitochondria and cytosolic Ca2+-clearance following Ca2+ influx was however significantly slowed. Anoxia was also found to inhibit SERCA activity and, to a lesser extent, PMCA activity. Hence, anoxia has multiple influences on [Ca2+]i homeostasis in vagal afferent neurons, including depression of ATP-driven Ca2+-pumps, modulation of the kinetics of mitochondrial Ca2+ buffering/release and Ca2+-release from, and depletion of, internal Ca2+-stores. These effects are likely to influence sensory neuronal function during ischaemia.

Investigation of neural circuits underlying visceral pain is hampered by the difficulty in achieving selective manipulations of individual circuit components. In this study, we adapted a dual AAV approach, used for projection-specific transgene expression in the CNS, to explore the potential for targeted delivery of transgenes to primary afferent neurons innervating visceral organs. Focusing on the extrinsic sensory innervation of the mouse colon, we first characterized the extent of dual transduction following intrathecal delivery of one AAV9 vector and intracolonic delivery of a second AAV9 vector. We found that if the two AAV9 vectors were delivered one week apart, dorsal root ganglion (DRG) neuron transduction by the second vector was greatly diminished. Following delivery of the two viruses on the same day, we observed colocalization of the transgenes in DRG neurons, indicating dual transduction. Next, we delivered intrathecally an AAV9 vector encoding the inhibitory chemogenetic actuator hM4D(Gi) in a Cre-recombinase dependent manner, and on the same day injected an AAV9 vector carrying Cre-recombinase in the colon. DRG expression of hM4D(Gi) was demonstrated at the mRNA and protein level. However, we were unable to demonstrate selective inhibition of visceral nociception following hM4D(Gi) activation. Taken together, these results establish a foundation for development of strategies for targeted transduction of primary afferent neurons for neuromodulation of peripheral neural circuits.

Determining the number and placement of synaptic inputs along the distinct plasma membrane domains of neurons is essential for explaining the basis of neuronal activity and function. We detail a strategy that combines juxtacellular labeling, neuronal reconstructions and stereological sampling of inputs at the ultrastructural level to define key elements of the afferent 'synaptome' of a given neuron. This approach provides unbiased estimates of the total number and somato-dendritic distribution of synapses made with individual neurons. These organizational properties can be related to the activity of the same neurons previously recorded in vivo, for direct structure-function correlations at the single-cell level. The approach also provides the quantitative data required to develop biologically realistic models that simulate and predict neuronal activity and function.

Corticotropin-releasing hormone (CRH) is a critical neuropeptide modulating the mammalian stress response. It is involved in many functional activities within various brain regions, among which there is a subset of CRH neurons occupying a considerable proportion of the cortical GABAergic interneurons. Here, we utilized rabies virus-based monosynaptic retrograde tracing system to map the whole-brain afferent presynaptic partners of the CRH neurons in the anterior cingulate cortex (ACC). We find that the ACC CRH neurons integrate information from the cortex, thalamus, hippocampal formation, amygdala, and also several other midbrain and hindbrain nuclei. Furthermore, our results reveal that ACC CRH neurons receive direct inputs from two neuromodulatory systems, the basal forebrain cholinergic neurons and raphe serotoninergic neurons. These findings together expand our knowledge about the connectivity of the cortical GABAergic neurons and also provide a basis for further investigation of the circuit function of cortical CRH neurons.

Intestinal glucagon-like peptide-1 (GLP-1) and pancreatic insulin, released postprandially, commonly regulate glucose metabolism. Recent clinical experience indicates that the GLP-1R agonist and insulin in combination, compared to insulin alone, results in better glycemic and weight controls in type 2 diabetic patients. These observations suggest possible interactive effect of these hormones. These hormones, in addition to peripherally controlling glycemia, exert central regulation of food intake and glucose metabolism, the effect at least partly mediated by signaling to the brain via the vagal afferents. However, whether the vagal afferents are involved in the interactive effects of GLP-1 and insulin remains unknown. The present study explored possible cooperative effect of GLP-1 and insulin on vagal afferent neurons isolated from nodose ganglion (NG) of mice, while monitoring the neuronal activity by measuring cytosolic Ca2+ concentration ([Ca2+]i) with fura-2. GLP-1 at 10-8M increased [Ca2+]i in 8-11% of single NG neurons. GLP-1-induced [Ca2+]i increases were inhibited by GLP-1 receptor antagonist exendin (9-39). Majority (92%) of GLP-1-responseive NG neurons also responded to 10-7M insulin with [Ca2+]i increases. Both GLP-1 and insulin at lower concentration of 10-9M induced [Ca2+]i increases with smaller amplitude in lesser NG neuron population (4-7%). These hormones at 10-9M in combination recruited the unresponsive neurons to [Ca2+]i increases, and induced [Ca2+]i increases with greater amplitude in the responsive neurons. The results demonstrate that GLP-1 and insulin synergistically and additively activate vagal afferent neurons. This interaction may be linked to the postprandial functions mediated commonly by GLP-1 and insulin and in the beneficial outcome of the therapy with GLP-1 receptor agonist and insulin in combination.

Peripherally-restricted opiate compounds attenuate hyperalgesia in experimental models of inflammatory pain, but have little discernable effect on nociceptive behavior in normal animals. This suggests that activation of opioid receptors on peripheral sensory axons contributes to decreased afferent activity after injury. Previously, we reported that direct application of morphine to cutaneous receptive fields decreased mechanical and heat-evoked responses in a population of C-fiber nociceptors in inflamed skin. Consistent with reported behavioral studies, direct application of morphine had no effect on fiber activity in control skin. The aim of the present study was to determine whether mechanical responsiveness of nociceptors innervating inflamed skin was attenuated by direct activation of delta opioid receptors (DORs) on peripheral terminals. An ex vivo preparation of rat plantar skin and tibial nerve was used to examine effects of a selective DOR agonist, deltorphin II, on responsiveness of single fibers innervating inflamed skin. Electrical recordings were made eighteen hours after injection of complete Freund's adjuvant into the hindpaw. Deltorphin II produced an inhibition of the mechanical responsiveness of single fibers innervating inflamed skin; an effect blocked by the DOR-selective antagonist, naltrindole. The population of units responsive to deltorphin II was identified as consisting of C fiber mechanical nociceptors.

Propranolol, a representative adrenergic β-receptor antagonist, is widely used to prevent migraine attacks. Although propranolol is well known to inhibit tetrodotoxin-resistant (TTX-R) Na+ channels in cardiac myocytes, it is unclear whether the drug modulates these channels expressed in dural afferent neurons. In this study, we examined the effects of propranolol on TTX-R Na+ channels in medium-sized dural afferent neurons identified by the fluorescent dye DiI. The TTX-R Na+ currents (INa) were recorded from acutely isolated DiI-positive neurons using a whole-cell patch clamp technique under voltage-clamp conditions. Propranolol inhibited the noninactivating steady-state component more potently than the peak component of transient TTX-R INa. Propranolol also potently inhibited the slow voltage ramp-induced TTX-R INa in a concentration-dependent manner, suggesting that it preferentially inhibited the noninactivating or persistent INa in DiI-positive neurons. Propranolol had little effect on voltage dependence, but it increased the extent of the use-dependent inhibition of TTX-R Na+ channels. Propranolol also accelerated the onset of inactivation and retarded recovery from inactivation in these channels. Under current-clamp conditions, propranolol decreased the number of action potentials elicited by depolarizing current stimuli. In conclusion, the propranolol-mediated preferential inhibition of persistent INa and modulation of the inactivation kinetics of TTX-R Na+ channels might represent additional mechanisms for migraine prophylaxis.

Nicotinic acetylcholine receptors (nAChR) influence bladder afferent activity and reflex sensitivity, and have been suggested as potential targets for treating detrusor overactivity. Mechanisms may include indirect effects, e.g. involving the urothelium, and direct action on nAChR expressed by afferent neurons. Here we determined the nAChR repertoire of bladder afferent neurons by retrograde neuronal tracing and laser-assisted microdissection/reverse transcriptase polymerase chain reaction (RT-PCR), and quantified retrogradely labelled nAChRα3-subunit-expressing neurons by immunohistochemistry in nAChR α3β4α5 cluster enhanced green fluorescent protein (eGFP) reporter mice. Bladder afferents distinctly expressed mRNAs encoding for nAChR-subunits α3, α6, α7, β2-4, and weakly α4. Based upon known combinatorial patterns of subunits, this predicts the expression of at least three basically different subunits of nAChR - α3(∗), α6(∗) and α7(∗) - and of additional combinations with β-subunits and α5. Bladder afferents were of all sizes, and their majority (69%; n=1367) were eGFP-nAChRα3 positive. Immunofluorescence revealed immunoreactivities to neurofilament 68 (NF68), transient receptor potential cation channel vanilloid 1 (TRPV1), substance P (SP) and calcitonin gene-related peptide (CGRP) in eGFP-nAChRα3-positive and -negative neurons. For each antigen, all possible combinations of colocalisation with eGFP-nAChRα3 were observed, with eGFP-nAChRα3-positive bladder neurons without additional immunoreactivity being most numerous, followed by triple-labelled neurons. In conclusion, more than one population of bladder afferent neurons expresses nAChR, indicating that peripheral nicotinic initiation and modulation of bladder reflexes might result, in addition to indirect effects, from the direct activation of sensory terminals. The expression of multiple nAChR subunits offers the potential of selectively addressing functional aspects and/or sensory neuron subpopulations.

The vestibular system relays information about head position via afferent nerve fibers to the brain in the form of action potentials. Voltage-gated Na+ channels in vestibular afferents drive the initiation and propagation of action potentials, but their expression during postnatal development and their contributions to firing in diverse mature afferent populations are unknown. Electrophysiological techniques were used to determine Na+ channel subunit types in vestibular calyx-bearing afferents at different stages of postnatal development. We used whole cell patch clamp recordings in thin slices of gerbil crista neuroepithelium to investigate Na+ channels and firing patterns in central zone (CZ) and peripheral zone (PZ) afferents. PZ afferents are exclusively dimorphic, innervating type I and type II hair cells, whereas CZ afferents can form dimorphs or calyx-only terminals which innervate type I hair cells alone. All afferents expressed tetrodotoxin (TTX)-sensitive Na+ currents, but TTX-sensitivity varied with age. During the fourth postnatal week, 200-300 nM TTX completely blocked sodium currents in PZ and CZ calyces. By contrast, in immature calyces [postnatal day (P) 5-11], a small component of peak sodium current remained in 200 nM TTX. Application of 1 μM TTX, or Jingzhaotoxin-III plus 200 nM TTX, abolished sodium current in immature calyces, suggesting the transient expression of voltage-gated sodium channel 1.5 (Nav1.5) during development. A similar TTX-insensitive current was found in early postnatal crista hair cells (P5-9) and constituted approximately one third of the total sodium current. The Nav1.6 channel blocker, 4,9-anhydrotetrodotoxin, reduced a component of sodium current in immature and mature calyces. At 100 nM 4,9-anhydrotetrodotoxin, peak sodium current was reduced on average by 20% in P5-14 calyces, by 37% in mature dimorphic PZ calyces, but by less than 15% in mature CZ calyx-only terminals. In mature PZ calyces, action potentials became shorter and broader in the presence of 4,9-anhydrotetrodotoxin implicating a role for Nav1.6 channels in firing in dimorphic afferents.

Vagal afferent neurons represent the key neurosensory branch of the gut-brain axis, which describes the bidirectional communication between the gastrointestinal system and the brain. These neurons are important for detecting and relaying sensory information from the periphery to the central nervous system to modulate feeding behavior, metabolism, and inflammation. Confounding variables complicate the process of isolating the role of the vagal afferents in mediating these physiological processes. Therefore, we developed a microfluidic model of the sensory branch of the gut-brain axis. We show that this microfluidic model successfully compartmentalizes the cell body and neurite terminals of the neurons, thereby simulates the anatomical layout of these neurons to more accurately study physiologically-relevant processes.

Pain is normally mediated by nociceptive Aδ and C fibers, while Aβ fibers signal touch. However, after nerve injury, Aβ fibers may signal pain. Using a genetic model, we tested the hypothesis that phenotypic switching in neurotransmitters expressed by Aβ afferents might account for heritable differences in neuropathic pain behavior. The study examined selection-line rats in which one line, high autotomy (HA), shows higher levels of spontaneous pain in the neuroma neuropathy model, and of tactile allodynia in the spinal nerve ligation (SNL) model, than the companion low autotomy (LA) line. Changes in calcitonin gene-related peptide (CGRP) and Substance P expression were evaluated immunohistochemically in L4 and L5 dorsal root ganglia 7 days after SNL surgery. Expression of CGRP was decreased in axotomized small- and medium-diameter neurons in both rat lines. However, in HA but not in LA rats, there was a tenfold increase in CGRP immunoreactivity (CGRP-IR) in large-diameter neurons. Corresponding changes in CGRP-IR in axon terminals in the nucleus gracilis were also seen. Finally, there were indications of enhanced CGRP neurotransmission in deep laminae of the dorsal horn. Substance P immunoreactivity was also upregulated in large-diameter neurons, but this change was similar in the 2 lines. Our findings suggest that phenotypic switching contributes to the heritable difference in pain behavior in HA vs LA rats. Specifically, we propose that in HA rats, but less so in LA rats, injured, spontaneously active Aβ afferents both directly drive CGRP-sensitive central nervous system pain-signaling neurons and also trigger and maintain central sensitization, hence generating spontaneous pain and tactile allodynia.

Migraine is characterized by recurrent and disabling headaches; therefore, several drugs have been widely prescribed to prevent acute migraine attacks. Amitriptyline, a tricyclic antidepressant, is among the most commonly administered. It is poorly known, however, whether amitriptyline modulates the excitability of dural afferent neurons that transmit pain signals from the dura mater. In this study, the effects of amitriptyline on tetrodotoxin-resistant (TTX-R) Na+ channels were examined in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na+ currents (INa) were recorded from medium-sized DiI-positive neurons using a whole-cell patch clamp technique. Amitriptyline (3 μM) slightly reduced the peak component of transient INa and induced a marked decrease in the steady-state component of transient TTX-R INa, as well as in the slow ramp-induced TTX-R INa. Our findings suggest that amitriptyline specifically inhibits persistent Na+ currents mediated by TTX-R Na+ channels. While amitriptyline had minor effects on voltage-activation/inactivation, it increased the extent of the use-dependent inhibition of TTX-R Na+ channels. Amitriptyline also affected the inactivation kinetics of TTX-R Na+ channels by significantly accelerating the inactivation of TTX-R Na+ channels and slowing the subsequent recovery. Amitriptyline decreased the number of action potentials by increasing the threshold for their generation. In conclusion, the amitriptyline-mediated diverse modulation of TTX-R Na+ channels would be, at least in part, responsible for its prophylactic efficacy for migraine attacks.