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Primary afferent neurons transduce distension of the bladder wall into action potentials that are relayed into the spinal cord and brain, where autonomic reflexes necessary for maintaining continence are coordinated with pathways involved in sensation. However, the relationship between spinal circuits involved with physiological and nociceptive signalling from the bladder has only been partially characterised. We used ex vivo bladder afferent recordings to characterise mechanosensitive afferent responses to graded distension (0-60 mm Hg) and retrograde tracing from the bladder wall to identify central axon projections within the dorsal horn of the lumbosacral (LS) spinal cord. Labelling of dorsal horn neurons with phosphorylated-MAP-kinase (pERK), combined with labelling for neurochemical markers (calbindin, calretinin, gamma aminobutyric acid, and parvalbumin) after in vivo bladder distension (20-60 mm Hg), was used to identify spinal cord circuits processing bladder afferent input. Ex vivo bladder distension evoked an increase in primary afferent output, and the recruitment of both low- and high-threshold mechanosensitive afferents. Retrograde tracing revealed bladder afferent projections that localised with pERK-immunoreactive dorsal horn neurons within the superficial laminae (superficial dorsal horn), dorsal gray commissure, and lateral collateral tracts of the LS spinal cord. Populations of pERK-immunoreactive neurons colabelled with calbindin, calretinin, or gamma aminobutyric acid, but not parvalbumin. Noxious bladder distension increased the percentage of pERK-immunoreactive neurons colabelled with calretinin. We identified LS spinal circuits supporting autonomic and nociceptive reflexes responsible for maintaining continence and bladder sensations. Our findings show for the first time that low- and high-threshold bladder afferents relay into similar dorsal horn circuits, with nociceptive signalling recruiting a larger number of neurons.

Activation of GABA A receptors on sensory axons produces a primary afferent depolarization (PAD) that modulates sensory transmission in the spinal cord. While axoaxonic synaptic contacts of GABAergic interneurons onto afferent terminals have been extensively studied, less is known about the function of extrasynaptic GABA receptors on afferents. Thus, we examined extrasynaptic α 5 GABA A receptors on low-threshold proprioceptive (group Ia) and cutaneous afferents. Afferents were impaled with intracellular electrodes and filled with neurobiotin in the sacrocaudal spinal cord of rats. Confocal microscopy was used to reconstruct the afferents and locate immunolabelled α 5 GABA A receptors. In all afferents α 5 GABA A receptors were found throughout the extensive central axon arbors. They were most densely located at branch points near sodium channel nodes, including in the dorsal horn. Unexpectedly, proprioceptive afferent terminals on motoneurons had a relative lack of α 5 GABA A receptors. When recording intracellularly from these afferents, blocking α 5 GABA A receptors (with L655708, gabazine, or bicuculline) hyperpolarized the afferents, as did blocking neuronal activity with tetrodotoxin, indicating a tonic GABA tone and tonic PAD. This tonic PAD was increased by repeatedly stimulating the dorsal root at low rates and remained elevated for many seconds after the stimulation. It is puzzling that tonic PAD arises from α 5 GABA A receptors located far from the afferent terminal where they can have relatively little effect on terminal presynaptic inhibition. However, consistent with the nodal location of α 5 GABA A receptors, we find tonic PAD helps produce sodium spikes that propagate antidromically out the dorsal roots, and we suggest that it may well be involved in assisting spike transmission in general. NEW & NOTEWORTHY GABAergic neurons are well known to form synaptic contacts on proprioceptive afferent terminals innervating motoneurons and to cause presynaptic inhibition. However, the particular GABA receptors involved are unknown. Here, we examined the distribution of extrasynaptic α 5 GABA A receptors on proprioceptive Ia afferents. Unexpectedly, these receptors were found preferentially near nodal sodium channels throughout the afferent and were largely absent from afferent terminals. These receptors produced a tonic afferent depolarization that modulated sodium spikes, consistent with their location.

Limited data are available regarding the spinal projections of afferent fibers in the phrenic nerve. We describe a method that robustly labels phrenic afferent spinal projections in adult rats. The proximal end of the cut phrenic nerve was secured in a microtube filled with a transganglionic tracer (cholera toxin β-subunit, CT-β, or Cascade Blue) and tissues harvested 96-h later. Robust CT-β labeling occurred in C3-C5 dorsal root ganglia cell bodies and phrenic afferent projections were identified in the mid-cervical dorsal horn (laminae I-III), intermediate grey matter (laminae IV, VII) and near the central canal (laminae X). Afferent fiber labeling was reduced or absent when CT-β was delivered to the intrapleural space or directly to the hemidiaphragm. Soaking the phrenic nerve with Cascade Blue also produced robust labeling of mid-cervical dorsal root ganglia cells bodies, and primary afferent fibers were observed in spinal grey matter and dorsal white matter. Our results show that the 'nerve soak' method effectively labels both phrenic motoneurons and phrenic afferent projections, and show that primary afferents project throughout the ipsilateral mid-cervical gray matter., (Copyright © 2016. Published by Elsevier B.V.)

During vertebrate development, centrally projecting sensory axons of the dorsal root ganglia neurons first reach the embryonic spinal cord at the dorsolateral margin. Instead of immediately projecting into the grey matter, they bifurcate and extend rostrally and caudally to establish the longitudinal dorsal funiculus during a stereotyped waiting period of approximately 48 h. Collateral fibres then extend concurrently across multiple spinal segments and project to their appropriate targets within the grey matter. This rostrocaudal extension of sensory afferents is crucial for the intersegmental processing of information throughout the spinal cord. However, the precise cues that prevent premature entry during the waiting period remain to be identified. Here, we show that semaphorin 5B (Sema5B), a member of the semaphorin family of guidance molecules, is expressed in the chick spinal cord during this waiting period and dorsal funiculus formation. Sema5B expression is dynamic, with a reduction of expression apparent in the spinal cord concomitant with collateral extension. We show that Sema5B inhibits the growth of NGF-dependent sensory axons and that this effect is mediated in part through the cell adhesion molecule TAG-1. Knockdown of Sema5B in the spinal cord using RNA interference leads to the premature extension of cutaneous nociceptive axons into the dorsal horn grey matter. These premature projections predominantly occur at the site of dorsal root entry. Our results suggest that Sema5B contributes to a repulsive barrier for centrally projecting primary sensory axons, forcing them to turn and establish the dorsal funiculus.

Gain control of primary afferent neurotransmission at their intraspinal terminals occurs by several mechanisms including primary afferent depolarization (PAD). PAD produces presynaptic inhibition via a reduction in transmitter release. While it is known that descending monoaminergic pathways complexly regulate sensory processing, the extent these actions include modulation of afferent-evoked PAD remains uncertain. We investigated the effects of serotonin (5HT), dopamine (DA) and noradrenaline (NA) on afferent transmission and PAD. Responses were evoked by stimulation of myelinated hindlimb cutaneous and muscle afferents in the isolated neonatal mouse spinal cord. Monosynaptic responses were examined in the deep dorsal horn either as population excitatory synaptic responses (recorded as extracellular field potentials; EFPs) or intracellular excitatory postsynaptic currents (EPSCs). The magnitude of PAD generated intraspinally was estimated from electrotonically back-propagating dorsal root potentials (DRPs) recorded on lumbar dorsal roots. 5HT depressed the DRP by 76%. Monosynaptic actions were similarly depressed by 5HT (EFPs 54%; EPSCs 75%) but with a slower time course. This suggests that depression of monosynaptic EFPs and DRPs occurs by independent mechanisms. DA and NA had similar depressant actions on DRPs but weaker effects on EFPs. IC50 values for DRP depression were 0.6, 0.8 and 1.0 µM for 5HT, DA and NA, respectively. Depression of DRPs by monoamines was nearly-identical in both muscle and cutaneous afferent-evoked responses, supporting a global modulation of the multimodal afferents stimulated. 5HT, DA and NA produced no change in the compound antidromic potentials evoked by intraspinal microstimulation indicating that depression of the DRP is unrelated to direct changes in the excitability of intraspinal afferent fibers, but due to metabotropic receptor activation. In summary, both myelinated afferent-evoked DRPs and monosynaptic transmission in the dorsal horn are broadly reduced by descending monoamine transmitters. These actions likely integrate with modulatory actions elsewhere to reconfigure spinal circuits during motor behaviors.

Afferent feedback alters muscle activity during locomotion and must be tightly controlled. As primary afferent depolarization-induced presynaptic inhibition (PAD-PSI) regulates afferent signaling, we investigated hindlimb PAD-PSI during locomotion in an in vitro rat spinal cord-hindlimb preparation. We compared the relation of PAD-PSI, measured as dorsal root potentials (DRPs), to observed ipsilateral and contralateral limb endpoint forces. Afferents activated during stance-phase force strongly and proportionately influenced DRP magnitude in the swinging limb. Responses increased with locomotor frequency. Electrical stimulation of contralateral afferents also preferentially evoked DRPs in the opposite limb during swing (flexion). Nerve lesioning, in conjunction with kinematic results, support a prominent contribution from toe Golgi tendon organ afferents. Thus, force-dependent afferent feedback during stance binds interlimb sensorimotor state to a proportional PAD-PSI in the swinging limb, presumably to optimize interlimb coordination. These results complement known actions of ipsilateral afferents on PAD-PSI during locomotion., (© 2013 New York Academy of Sciences.)

Background: Secretagogin (Scgn), a member of the EF-hand calcium-binding protein (CaBP) superfamily, has recently been found in subsets of developing and adult neurons. Here, we have analyzed the expression of Scgn in dorsal root ganglia (DRGs) and trigeminal ganglia (TGs), and in spinal cord of mouse at the mRNA and protein levels, and in comparison to the well-known CaBPs, calbindin D-28k, parvalbumin and calretinin. Rat DRGs, TGs and spinal cord, as well as human DRGs and spinal cord were used to reveal phylogenetic variations., Results: We found Scgn mRNA expressed in mouse and human DRGs and in mouse ventral spinal cord. Our immunohistochemical data showed a complementary distribution of Scgn and the three CaBPs in mouse DRG neurons and spinal cord. Scgn was expressed in ~7% of all mouse DRG neuron profiles, mainly small ones and almost exclusively co-localized with calcitonin gene-related peptide (CGRP). This co-localization was also seen in human, but not in rat DRGs. Scgn could be detected in the mouse sciatic nerve and accumulated proximal to its constriction. In mouse spinal cord, Scgn-positive neuronal cell bodies and fibers were found in gray matter, especially in the dorsal horn, with particularly high concentrations of fibers in the superficial laminae, as well as in cell bodies in inner lamina II and in some other laminae. A dense Scgn-positive fiber network and some small cell bodies were also found in the superficial dorsal horn of humans. In the ventral horn, a small number of neurons were Scgn-positive in mouse but not rat, confirming mRNA distribution. Both in mouse and rat, a subset of TG neurons contained Scgn. Dorsal rhizotomy strongly reduced Scgn fiber staining in the dorsal horn. Peripheral axotomy did not clearly affect Scgn expression in DRGs, dorsal horn or ventral horn neurons in mouse., Conclusions: Scgn is a CaBP expressed in a subpopulation of nociceptive DRG neurons and their processes in the dorsal horn of mouse, human and rat, the former two co-expressing CGRP, as well as in dorsal horn neurons in all three species. Functional implications of these findings include the cellular refinement of sensory information, in particular during the processing of pain.

Background: Pain-related (nociceptive) information is carried from the periphery to the dorsal horn of the spinal cord mostly by two populations of small diameter primary afferents, the peptidergic and the non-peptidergic. The peptidergic population expresses neuropeptides, such as substance P and calcitonin gene-related peptide, while the non-peptidergic fibers are devoid of neuropeptides, express the purinergic receptor P2X3, and bind the isolectin B4 (IB4). Although it has been known for some time that in rat the peptidergic afferents terminate mostly in lamina I and outer lamina II and non-peptidergic afferents in inner lamina II, the extent of the termination of the latter population in lamina I was never investigated as it was considered as very minor. Because our preliminary evidence suggested otherwise, we decided to re-examine the termination of non-peptidergic afferents in lamina I, in particular with regards to their innervation of projection neurons expressing substance P receptors (NK-1r). We used retrograde labeling of neurons from the parabrachial nucleus combined with lectin IB4 binding and immunocytochemistry. Samples were examined by confocal and electron microscopy., Results: By confocal microscopy, we studied the termination of non-peptidergic afferents in lamina I using IB4 binding and P2X3 immunoreactivity as markers, in relation to CGRP immunoreactivy, a marker of peptidergic afferents. The number of IB4 or P2X3-labeled fibers in lamina I was higher than previously thought, although they were less abundant than CGRP-labeled afferents. There were very few fibers double-labeled for CGRP and either P2X3 or IB4. We found a considerable number of IB4-positive fiber varicosities in close apposition to NK-1r-positive lamina I projection neurons, which were distinct from peptidergic varicosities. Furthermore, we confirmed at the ultrastructural level that there were bona fide synapses between P2X3-immunoreactive non-peptidergic boutons and neurokinin-1 receptor-positive lamina I dendrites., Conclusions: These results indicate the presence of direct innervation by non-peptidergic nociceptive afferents of lamina I projection neurons expressing NK-1r. Further investigations are needed to better understand the role of these connections in physiological conditions and chronic pain states.

Gamma motor neurons (MNs), the efferent component of the fusimotor system, regulate muscle spindle sensitivity. Muscle spindle sensory feedback is required for proprioception that includes sensing the relative position of neighboring body parts and appropriately adjust the employed strength in a movement. The lack of a single and specific genetic marker has long hampered functional and developmental studies of gamma MNs. Here we show that the serotonin receptor 1d (5-ht1d) is specifically expressed by gamma MNs and proprioceptive sensory neurons. Using mice expressing GFP driven by the 5-ht1d promotor, we performed whole-cell patch-clamp recordings of 5-ht1d::GFP⁺ and 5-ht1d::GFP⁻ motor neurons from young mice. Hierarchal clustering analysis revealed that gamma MNs have distinct electrophysiological properties intermediate to fast-like and slow-like alpha MNs. Moreover, mice lacking 5-ht1d displayed lower monosynaptic reflex amplitudes suggesting a reduced response to sensory stimulation in motor neurons. Interestingly, adult 5-ht1d knockout mice also displayed improved coordination skills on a beam-walking task, implying that reduced activation of MNs by Ia afferents during provoked movement tasks could reduce undesired exaggerated muscle output. In summary, we show that 5-ht1d is a novel marker for gamma MNs and that the 5-ht1d receptor is important for the ability of proprioceptive circuits to receive and relay accurate sensory information in developing and mature spinal cord motor circuits., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neurons in spinal dorsal horn lamina I play a pivotal role for nociception that critically depends on a proper balance between excitatory and inhibitory inputs. Any modification in synaptic strength may challenge this delicate balance. Long-term potentiation (LTP) at glutamatergic synapses between nociceptive C-fibers and lamina I neurons is an intensively studied cellular model of pain amplification. In contrast, nothing is presently known about long-term changes of synaptic strength at inhibitory synapses in the spinal dorsal horn. Using a spinal cord-dorsal root slice preparation from rats, we show that conditioning stimulation of primary afferent fibers with a stimulating protocol that induces LTP at C-fiber synapses also triggered LTP at GABAergic synapses (LTP(GABA)). This LTP(GABA) was heterosynaptic in nature and was mediated by activation of group I metabotropic glutamate receptors. Opening of ionotropic glutamate receptor channels of the AMPA/KA or NMDA subtype was not required for LTP(GABA). Paired-pulse ratio, coefficient of variation, and miniature IPSCs analysis revealed that LTP(GABA) was expressed presynaptically. Nitric oxide as a retrograde messenger signal mediated this increase of GABA release at spinal inhibitory synapses. This novel form of synaptic plasticity in spinal nociceptive circuits may be an essential mechanism to maintain the relative balance between excitation and inhibition and to improve the signal-to-noise ratio in nociceptive pathways.

Regeneration of a cut muscle nerve fails to restore the stretch reflex, and the companion paper to this article [Alvarez FJ, Titus-Mitchell HE, Bullinger KL, Kraszpulski M, Nardelli P, Cope TC. J Neurophysiol (August 10, 2011). doi:10.1152/jn.01095.2010] suggests an important central contribution from substantial and persistent disassembly of synapses between regenerated primary afferents and motoneurons. In the present study we tested for physiological correlates of synaptic disruption. Anesthetized adult rats were studied 6 mo or more after a muscle nerve was severed and surgically rejoined. We recorded action potentials (spikes) from individual muscle afferents classified as IA like (*IA) by several criteria and tested for their capacity to produce excitatory postsynaptic potentials (EPSPs) in homonymous motoneurons, using spike-triggered averaging (STA). Nearly every paired recording from a *IA afferent and homonymous motoneuron (93%) produced a STA EPSP in normal rats, but that percentage was only 17% in rats with regenerated nerves. In addition, the number of motoneurons that produced aggregate excitatory stretch synaptic potentials (eSSPs) in response to stretch of the reinnervated muscle was reduced from 100% normally to 60% after nerve regeneration. The decline in functional connectivity was not attributable to synaptic depression, which returned to its normally low level after regeneration. From these findings and those in the companion paper, we put forward a model in which synaptic excitation of motoneurons by muscle stretch is reduced not only by misguided axon regeneration that reconnects afferents to the wrong receptor type but also by retraction of synapses with motoneurons by spindle afferents that successfully reconnect with spindle receptors in the periphery.

The TRPA1 channel has been proposed to be a molecular transducer of cold and inflammatory nociceptive signals. It is expressed on a subset of small primary afferent neurons both in the peripheral terminals, where it serves as a sensor, and on the central nerve endings in the dorsal horn. The substantia gelatinosa (SG) of the spinal cord is a key site for integration of noxious inputs. The SG neurons are morphologically and functionally heterogeneous and the precise synaptic circuits of the SG are poorly understood. We examined how activation of TRPA1 channels affects synaptic transmission onto SG neurons using whole-cell patch-clamp recordings and morphological analyses in adult rat spinal cord slices. Cinnamaldehyde (TRPA1 agonist) elicited a barrage of excitatory postsynaptic currents (EPSCs) in a subset of the SG neurons that responded to allyl isothiocyanate (less specific TRPA1 agonist) and capsaicin (TRPV1 agonist). Cinnamaldehyde evoked EPSCs in vertical and radial but not islet or central SG cells. Notably, cinnamaldehyde produced no change in inhibitory postsynaptic currents and nor did it produce direct postsynaptic effects. In the presence of tetrodotoxin, cinnamaldehyde increased the frequency but not amplitude of miniature EPSCs. Intriguingly, cinnamaldehyde had a selective inhibitory action on monosynaptic C- (but not Adelta-) fiber-evoked EPSCs. These results indicate that activation of spinal TRPA1 presynaptically facilitates miniature excitatory synaptic transmission from primary afferents onto vertical and radial cells to initiate action potentials. The presence of TRPA1 channels on the central terminals raises the possibility of bidirectional modulatory action in morphologically identified subclasses of SG neurons.

Myocardial ischemia is a complex process leading to the simultaneous release of a number of mediators, including thromboxane A(2) (TxA(2)) and bradykinin (BK), that activate cardiac spinal afferents. The present study tested the hypothesis that TxA(2) and BK reciprocally interact to excite ischemically sensitive cardiac afferents. Nerve activity of single cardiac afferent units was recorded from the left sympathetic chain or rami communicantes (T(2)-T(5)) of anesthetized cats. Fifty-two ischemically sensitive afferents (conduction velocity = 0.27-3.35 m/s, 7 Adelta-fibers and 45 C-fibers) were identified. Repeated injections (1 microg) of BK into the left atrium (LA) 4 min after the administration of U-46619 (5 microg into the LA), a TxA(2) mimetic, induced a significantly larger cardiac afferent response than the first response to BK (0.61 +/- 0.14 to 1.95 +/- 0.29 vs. 0.66 +/- 0.09 to 2.75 +/- 0.34 impulses/s, first injection vs. second injection, n = 8). Conversely, blockade of TxA(2) receptors with BM-13,177 (30 mg/kg iv) attenuated the responses of eight other afferents to BK (1 microg into the LA) by 45%. In contrast, repeated BK (1 microg into the LA) induced consistent discharge activity in six separate afferents. We then observed that the coadministration of U-46619 (5 microg) and BK (1 microg into the LA) together caused a total response that was significantly higher than the predicted response by the simple addition of the individual responses. BK (1 microg) facilitated eight cardiac afferent responses to U-46619 (5 microg into the LA) by 64%. In contrast, repeated U-46619 (5 microg into the LA) without intervening BK stimulation evoked consistent responses in seven other ischemically sensitive afferents. Finally, inhibition of cyclooxygenase with indomethacin (5 mg/kg iv) eliminated the potentiating effects of BK on the cardiac afferent response to U-46619 (5 microg into the LA) but did not alter the afferent response to U-46619. These data suggest that BK and TxA(2) reciprocally interact to stimulate ischemically sensitive cardiac afferent endings leading to synergistic afferent responses and that the BK sensitization effect is mediated by cyclooxygenase products.

Task-dependent modulation of primary afferent depolarization (PAD) was studied in the cervical spinal cord of two monkeys performing a wrist flexion and extension task with an instructed delay period. We implanted two nerve cuff electrodes on proximal and distal parts of the superficial radial nerve (SR) and a recording chamber over a hemi-laminectomy in the lower cervical vertebrae. Antidromic volleys (ADVs) in the SR were evoked by intraspinal microstimuli (ISMS, 3-10 Hz, 3-30 microA) applied through a tungsten microelectrode, and the area of each ADV was measured. In total, 434 ADVs were evoked by ISMS in two monkeys, with onset latency consistently shorter in the proximal than distal cuffs. Estimated conduction velocity suggest that most ADVs were caused by action potentials in cutaneous fibers originating from low-threshold tactile receptors. Modulation of the size of ADVs as a function of the task was examined in 281 ADVs induced by ISMS applied at 78 different intraspinal sites. The ADVs were significantly facilitated during active movement in both flexion and extension (P<0.05), suggesting an epoch-dependent modulation of PAD. This facilitation started 400-900 ms before the onset of EMG activity. Such pre-EMG modulation is hard to explain by movement-induced reafference and probably is associated with descending motor commands.

Traumatic injury or inflammatory irritation of the peripheral nervous system often leads to persistent pathophysiological pain states. It has been well-documented that, after peripheral nerve injury or inflammation, functional and anatomical alterations sweep over the entire peripheral nervous system including the peripheral nerve endings, the injured or inflamed afferent fibers, the dorsal root ganglion (DRG), and the central afferent terminals in the spinal cord. Among all the changes, ectopic discharge or spontaneous activity of primary sensory neurons is of great clinical interest, as such discharges doubtless contribute to the development of pathological pain states such as neuropathic pain. Two key sources of abnormal spontaneous activity have been identified following peripheral nerve injury: the injured afferent fibers (neuroma) leading to the DRG, and the DRG somata. The purpose of this review is to provide a global account of the abnormal spontaneous activity in various animal models of pain. Particular attention is focused on the consequence of peripheral nerve injury and localized inflammation. Further, mechanisms involved in the generation of spontaneous activity are also reviewed; evidence of spontaneous activity in contributing to abnormal sympathetic sprouting in the axotomized DRG and to the initiation of neuropathic pain based on new findings from our research group are discussed. An improved understanding of the causes of spontaneous activity and the origins of neuropathic pain should facilitate the development of novel strategies for effective treatment of pathological pain.

The management of pancreatic pain is a significant clinical problem so understanding of how sensory signals are generated in pancreatic tissue is fundamental. We aimed to characterize mechanosensitive and chemosensitive properties of pancreatic spinal and vagal afferents in vitro. Spinal and vagal afferent preparations from Sprague-Dawley rats were established incorporating the left splanchnic nerve or vagus nerves respectively. The common bile duct was cannulated for distension of the pancreatic duct with fluid. Nerve discharge evoked by blunt probing, duct distension or electrical stimulation was obtained from teased nerve bundles using standard extra-cellular recording. Discharge from 197 spinal afferent bundles was recorded, of which 57% displayed spontaneous activity. Blunt probing revealed 61 mechanosensitive receptive fields which were associated primarily with arteries/blood vessels (33/61) and the parenchyma (22/61). All mechanosensitive responses were slowly adapting, with 33% continuing to discharge after termination of the stimulus and 60% displaying a response threshold <10 g. Application of chemical mediators (bradykinin, histamine, 5-hydroxytryptamine, cholecystokinin octapeptide) evoked a response from 31/57 units, with 33% excitatory and 23% inhibitory. Spontaneous discharge was recorded from 72% of 135 vagal bundles. Mechanosensitive receptive fields were not identified in the pancreas but were evident in adjacent organs. No spinal or vagal afferent response to duct distension was obtained. In conclusion, pancreatic mechanosensitive spinal afferents are common, in contrast to pancreatic mechanosensitive vagal afferents indicating that pancreatic sensory innervation is predominantly spinal. Chemosensitive spinal afferent nerve endings are present in the pancreas and respond to a variety of inflammatory and physiological mediators.

Despite increasing evidence for transcriptional control of neural connectivity, how transcription factors regulate discrete steps in axon guidance remains obscure. Projection neurons in the dorsal spinal cord relay sensory signals to higher brain centers. Some projection neurons send their axons ipsilaterally, whereas others, commissural neurons, send axons contralaterally. We show that two closely related LIM homeodomain proteins, Lhx2 and Lhx9, are expressed by a set of commissural relay neurons (dI1c neurons) and are required for the dI1c axon projection. Midline crossing by dI1c axons is lost in Lhx2/9 double mutants, a defect that results from loss of expression of Rig-1 from dI1c axons. Lhx2 binds to a conserved motif in the Rig-1 gene, suggesting that Lhx2/9 regulate directly the expression of Rig-1. Our findings reveal a link between the transcriptional programs that define neuronal subtype identity and the expression of receptors that guide distinctive aspects of their trajectory.

A better understanding of the mechanisms linked to chemokine pronociceptive effects is essential for the development of new strategies to better prevent and treat chronic pain. Among chemokines, MCP-1/CCL2 involvement in neuropathic pain processing is now established. However, the mechanisms by which MCP-1/CCL2 exerts its pronociceptive effects are still poorly understood. In the present study, we demonstrate that MCP-1/CCL2 can alter pain neurotransmission in healthy rats. Using immunohistochemical studies, we first show that CCL2 is constitutively expressed by primary afferent neurons and their processes in the dorsal horn of the spinal cord. We also observe that CCL2 is co-localized with pain-related peptides (SP and CGRP) and capsaicin receptor (VR1). Accordingly, using in vitro superfusion system of lumbar dorsal root ganglion and spinal cord explants of healthy rats, we show that potassium or capsaicin evoke calcium-dependent release of CCL2. In vivo, we demonstrate that intrathecal administration of CCL2 to healthy rats produces both thermal hyperalgesia and sustained mechanical allodynia (up to four consecutive days). These pronociceptive effects of CCL2 are completely prevented by the selective CCR2 antagonist (INCB3344), indicating that CCL2-induced pain facilitation is elicited via direct spinal activation of CCR2 receptor. Therefore, preventing the activation of CCR2 might provide a fruitful strategy for treating pain.

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas delta-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.

The c-kit receptor tyrosine kinase is expressed in a subpopulation of small- and medium-sized neurons of the dorsal root ganglia (DRG) and in the superficial layer of the spinal cord. Stem cell factor (SCF), a ligand of the c-kit receptor, induces neurite outgrowth from DRG and supports the survival of c-kit-expressing neurons. To clarify the possible function of the SCF/c-kit receptor system in the adult animal, we investigated the expression of c-kit receptor in the spinal cord and DRG in relation to pain by using H2C7, a newly developed anti-c-kit monoclonal antibody. S.c. and intrathecal injection of SCF markedly reduced the paw withdrawal threshold to mechanical stimuli and intrathecal SCF at 10 pg maximally induced mechanical allodynia in conscious mice. Intrathecal SCF also reduced the paw withdrawal latency to heat stimuli significantly but transiently. The c-kit receptor was co-expressed in 58.4% of calcitonin gene-related peptide (CGRP) -positive, but only 5.1% of isolectin B4-positive, DRG neurons. In the spinal cord, the c-kit receptor was detected in the superficial layer of the dorsal horn and co-localized there with CGRP in central terminals of DRG neurons. Selective elimination of unmyelinated C-fibers by neonatal capsaicin treatment resulted in marked reduction of the c-kit receptor and CGRP expression in the superficial layer of the spinal cord. Cell-size profiles showed that c-kit receptor expression was significantly up-regulated and down-regulated in medium-sized DRG neurons after neonatal capsaicin treatment and nerve injury, respectively. These results suggest that the c-kit receptor is mainly expressed in peptidergic small-sized DRG neurons and may be involved in pain regulation both peripherally and centrally.

Injury to the peripheral nerves often induces produces spontaneous pain, hyperalgesia (increased responsiveness to noxious stimuli), and allodynia (painful responses to normally innocuous stimuli). In contrast to inflammatory pain, the currently available therapeutics for neuropathic pain are either relatively ineffective or accompanied by considerable side effects. Numerous animal models of chronic pain following nerve injury have been introduced. All these neuropathic pain models are generated by partial nerve injury, where a few primary afferents are axotomized, while the others are spared. Among these models, the L5 spinal nerve ligation (SNL) model is unique because in this model, the L4 dorsal root ganglion (DRG) neurons are clearly separated from the axotomized L5 DRG neurons. Previous studies have focused considerable attention on the directly damaged primary afferents and their influence on the activity of the dorsal horn neurons. However, increasing evidence suggests that DRG neurons with intact axons also exhibit alterad excitability and gene expression, and these changes might play functional roles in the pathomechanisms of neuropathic pain. For example, L5 SNL increases the expression of substance P, calcitonin gene-related peptide, brain-derived neurotrophic factor, and the transient receptor potential ion channels TRPV1 and TRPA1 in the uninjured L4 DRG neurons. Furthermore, compelling evidence suggests that the glial cells in the spinal cord may also play a role in the pathogenesis of neuropathic pain. Recent studies have shown that peripheral nerve injury results in the activation of mitogen-activated protein kinases (MAPK) in spinal glial cells and that MAPK inhibitors diminish nerve injury-induced pain hypersensitivity. This review mainly focuses on the DRG neurons and spinal glial cells and will review the roles of MAPK in the nociceptive pathways for neuropathic pain.

To define the somatotopic arrangement of neurons in the trigeminal spinal subnucleus caudalis and upper cervical cord activated by acute noxious stimulation of various orofacial sites, pERK expression was analyzed in these neurons. After capsaicin injection into the tongue, lower gum, upper and lower lips, or mental region, pERK-like immunoreactive (pERK-LI) cells were distributed mainly in the dorsal half of the trigeminal spinal nucleus interporalis (Vi) and caudalis (Vc) transition zone (Vi/Vc zone), middle Vc, and Vc and upper cervical cord transition zone (Vc/C2 zone). pERK-LI cells were distributed throughout the dorsal to ventral portion of the Vi/Vc zone, middle Vc, and Vc/C2 zone following capsaicin injection into the anterior hard palate, upper gum, buccal mucosa, or vibrissal pad and in the ventral portion of the Vi/Vc zone, middle Vc, and Vc/C2 zone following snout, ophthalmic, or ocular injection of capsaicin. The rostrocaudal distribution area of pERK-LI cells was more extensive from the Vi/Vc zone to the Vc/C2 zone after intraoral injection than that after facial injection, and the rostrocaudal distribution of pERK-LI cells from the Vi/Vc zone to the Vc/C2 zone had a somatotopic arrangement, with the snout being represented most rostrally and ophthalmic, ocular, or mental regions represented most caudally. These findings suggest that the pERK-LI cells expressed from the Vi/Vc zone to the Vc/C2 zone following injection of capsaicin in facial and intraoral structures may be differentially involved in pain perception in facial and intraoral sites., (Copyright 2008 Wiley-Liss, Inc.)

To study the acute and long-term effects of local gut inflammation on the sensitivity of the spinal sensory neurons, the expressions of vanilloid receptor 1 (VR1) and calcitonin gene-related peptide (CGRP) in the colon-innervated primary sensory neurons in dorsal root ganglia (DRG) were examined in rats with trinitrobenzenesulfonic acid (TNBS)-induced experimental colitis. The neurons projecting to the distal colon were identified by DiI(3) retrograde labelling. Macroscopic examination, mean damage score and myeloperoxidase (MPO) activity were determined to assess the inflammatory status of the colon tissue. The number of CGRP and VR1 immunoreactive neurons at different stages of inflammation (on days 7, 21 and 42 after TNBS treatment) were compared. On day 7 after TNBS treatment, macroscopic damage of the mucosa could be easily detected and the percentage of colon-innervated DRG neurons expressing CGRP and VR1 increased nearly two folds respectively [(95.38±9.45)% vs (42.86±.02)% for CGRP, (89.23±8.21)% vs (32.54±4.58)% for VR1]. When the colon inflammatory reaction was resolved on days 21 and 42 after TNBS treatment, the percentage of colon-innervated DRG neurons expressing CGRP and VR1 were still higher than that in the control group [(86.25±8.21)%, (68.28±7.12)% vs (42.86±5.02)% for CGRP; (67.22±6.52)%, (56.25±4.86)% vs (32.54±4.58)% for VR1]. These results suggest that the local gut inflammation increases the expressions of CGRP and VR1 in gut-innervated DRG sensory neurons. More importantly, this abnormal status persists even after the gut inflammatory reaction has been resolved for certain time.

Environmental stimuli such as temperature and pressure are sensed by dorsal root ganglion (DRG) neurons. DRG neurons are heterogeneous, but molecular markers that identify unique functional subpopulations are mainly lacking. ThermoTRPs are members of the transient receptor potential family of ion channels and are gated by shifts in temperature. TRPM8 is activated by cooling, and TRPM8-deficient mice have severe deficits in cool thermosensation. The anatomical and functional properties of TRPM8-expressing fibers have not been not comprehensively investigated. We use mice engineered to express the farnesylated enhanced green fluorescent protein (EGFPf) from the TRPM8 locus (TRPM8(EGFPf)) to explore this issue. Virtually all EGFPf-positive cultured DRG neurons from hemizygous mice (TRPM8(EGFPf/+)) responded to cold and menthol. In contrast, EGFPf-positive DRGs from homozygous mice (TRPM8(EGFPf/EGFPf)) had drastically reduced cold responses and no menthol responses. In vivo, EGFPf-positive neurons marked a unique population of DRG neurons, a majority of which do not coexpress nociceptive markers. The fraction of DRG neurons expressing EGFPf was not altered under an inflammatory condition, although an increase in TRPV1-coexpressing neurons was observed. TRPM8(EGFPf) neurons project to the superficial layer I of the spinal cord, making distinct contacts when compared with peptidergic projections. At the periphery, TRPM8(EGFPf) projections mark unique endings in the most superficial layers of epidermis, including bush/cluster endings of the mystacial pads. We show that TRPM8 expression functionally associates with cold sensitivity in cultured DRGs, and provide the first glimpses of the unique anatomical architecture of cold fibers in vivo.

Peripheral sensory neurons detect diverse physical stimuli and transmit the information into the CNS. At present, the genetic tools for specifically studying the development, plasticity, and regeneration of the sensory axon projections are limited. We found that the gene encoding Advillin, an actin binding protein that belongs to the gelsolin superfamily, is expressed almost exclusively in peripheral sensory neurons. We next generated a line of knock-in mice in which the start codon of the Advillin is replaced by the gene encoding human placenta alkaline phosphatase (Avil-hPLAP mice). In heterozygous Avil-hPLAP mice, sensory axons, the exquisite sensory endings, as well as the fine central axonal collaterals can be clearly visualized with a simple alkaline phosphatase staining. Using this mouse line, we found that the development of peripheral target innervation and sensory ending formation is an ordered process with specific timing depending on sensory modalities. This is also true for the in-growth of central axonal collaterals into the brainstem and the spinal cord. Our results demonstrate that Avil-hPLAP mouse is a valuable tool for specifically studying peripheral sensory neurons. Functionally, we found that the regenerative axon growth of Advillin-null sensory neurons is significantly shortened and that deletion of Advillin reduces the plasticity of whisker-related barrelettes patterns in the hindbrain.

The hypocretin/orexin (HCRT/ORX) excitatory neuropeptides are expressed in a small population of lateral hypothalamic cells in mammals and fish. In humans, loss of these cells causes the sleep disorder narcolepsy. Identification of genes expressed in HCRT-producing cells may be revealing as to the regulation of sleep and the pathophysiology of narcolepsy. In this study, in situ hybridization analyses were performed to characterize the expression pattern of receptors and enzyme, which regulate ATP-mediated transmission in hypocretin cells of zebrafish larvae. The zebrafish cDNA encoding the ecto-nucleoside triphosphate diphosphohydrolase 3 (ENTPD3/NTPDase3) was isolated. This transcript was found to be expressed in zebrafish HCRT cells as previously reported in mammals. It was also expressed in the cranial nerves (gV, gVII, gIV and gX) and in primary sensory neurons (i.e., Rohon-Beard neurons) in the spinal cord. The expression of known zebrafish p2rx purinergic receptor family members was next studied and found to overlap with the entpd3 expression pattern. Specifically, p2rx2, p2rx3.1, p2rx3.2 and p2rx8 were expressed in the trigeminal ganglia and subsets of Rohon-Beard neurons. In contrast to mammals, p2rx2 was not expressed in HCRT cells; rather, p2rx8 was expressed with entpd3 in this hypothalamic region. The conservation of expression of these genes in HCRT cells and sensory neurons across vertebrates suggests an important role for ATP mediated transmission in the regulation of sleep and the processing of sensory inputs.

Introduction: Already low blood concentrations of ethanol acutely impair motor control and coordination. In vitro experiments have given evidence that spinal effects of ethanol contribute to this by reducing spinal excitability and enhancing presynaptic inhibition of Ia fibers. In this study, we investigated the influence of 0.7 g per kilogram of bodyweight ethanol on motoneuronal excitability and presynaptic inhibition in humans., Methods: The study was performed in 10 volunteers. Spinal excitability was measured by the maximal H-reflex of the soleus muscle normalized to the maximal muscular response (Hmax/Mmax). Presynaptic inhibition was measured by changes in heteronymous Ia-facilitation of the soleus H-reflex, which is achieved by stimulation of the femoral nerve. A decrease in facilitation can be ascribed to an increase in presynaptic inhibition. Changes of these parameters under the influence of 0.7 g per kilogram of bodyweight ethanol were assessed in comparison to control measurements before ethanol application., Results: Both parameters, Hmax/Mmax and Heteronymous facilitation, were significantly reduced under the influence of ethanol (Wilcoxon signed-rank test with Bonferroni correction for each, p<0.01)., Discussion: The increase in presynaptic inhibition by ethanol is probably caused by an increase in GABAA receptor-mediated Cl-conductance, which has been shown in spinal cord cultures. The role of presynaptic inhibition in movement is assumed to be there to control the afferent input of muscle spindles and tendon organs as a mechanism of specific input-selection. This study demonstrated that ethanol reduces spinal excitability and increases GABAergic presynaptic inhibition on Ia afferent fibers in humans.

Cocaine- and amphetamine-regulated transcript peptides (CART) have been implicated in the regulation of several physiological functions, including pain transmission. A dense plexus of CART-immunoreactive fibres has been described in the superficial laminae of the spinal cord, which are key areas in sensory information and pain processing. In this study, we used antibody against CART peptide, together with markers for various types of primary afferents, interneurons and descending systems to determine the origin of the CART-immunoreactive axons in the superficial laminae of the rat spinal cord. Calcitonin gene-related peptide (CGRP), a marker for peptidergic primary afferents in the dorsal horn, was present in 72.6% and 34.8% of CART-immunoreactive axons in lamina I and II, respectively. The majority of these fibres also contained substance P (SP), while a few were somatostatin (SOM)-positive. The other subpopulation of CART-immunoreactive boutons in lamina I and II also expressed SP and/or SOM without CGRP, but contained vesicular glutamate transporter 2, which is present mainly in excitatory interneuronal terminals. Our data demonstrate that the majority of CART-immunoreactive axons in the spinal dorsal horn originate from peptidergic nociceptive primary afferents, while the rest arise from excitatory interneurons that contain SP or SOM. This strongly suggests that CART peptide can affect glutamatergic neurotransmission as well as the release and effects of SP and SOM in nociception and other sensory processes.

The alpha(2)-adrenoceptors (alpha(2)-ARs) are located on primary afferent terminals and on neurons in the spinal cord dorsal horn. However, their relative contribution to the analgesic effect of the alpha(2)-AR agonists is not known. In this study, we determined the role of certain presynaptic alpha(2)-ARs in the antinociceptive effect produced by intrathecal administration of the alpha(2)-AR agonist clonidine. TRPV1-expressing sensory neurons were removed by resiniferatoxin (RTX). The effect of intrathecal injection of clonidine was measured by testing the paw withdrawal response to noxious mechanical or heat stimuli. In RTX-treated rats, the alpha(2A)-AR-immunoreactivity co-expressed with TRPV1-expressing terminals in the spinal cord was eliminated. However, the alpha(2C)-AR-immunoreactivity in the spinal cord was little changed. Surprisingly, intrathecal administration of clonidine produced a much greater increase in the mechanical withdrawal threshold in RTX- than in vehicle-treated rats. The duration of the clonidine effect was also significantly increased in RTX-treated rats. Furthermore, in the vehicle-treated group, although intrathecal injection of clonidine produced a large increase in the thermal withdrawal latency, it only had a small and short-lasting effect on the mechanical withdrawal threshold. This study provides new information that the antinociceptive effect of spinally administered alpha(2)-AR agonists is largely modality-specific. Loss of TRPV1-expressing sensory neurons leads to a reduction in presynaptic alpha(2A)-ARs but paradoxically potentiates the effect of clonidine on mechano-nociception.

Despite a large body of literature on the nociceptin (NC) opioid system in pain modulation, the mechanism of action of NC remains largely unexplored. Here, we investigated the role and mode of action of the spinal NC system in inflammatory pain. Preemptive intrathecal administration of NC attenuated thermal hyperalgesia and mechanical allodynia in rats with intraplantar complete Freund's adjuvant (CFA) injection. By using immunohistochemistry in L4 dorsal root ganglion (DRG) neurons, a marked increase of NC and ORL1 receptor immunoreactivity was detected following CFA. Intrathecal administration of NC attenuated the CFA-induced increases of calcitonin gene-related peptide, transient receptor potential vanilloid-1, and tumor necrosis factor-alpha in DRG neurons. Real-time reverse transcription-polymerase chain reaction showed that NC reduced the up-regulation of inducible nitric oxide synthase mRNA but not that of neuronal nitric oxide synthase mRNA in spinal cord segments after CFA. Furthermore, [Nphe1]NC(1-13)NH2, a selective opioid receptor-like 1 (ORL1) receptor antagonist, significantly antagonized the effects of NC on pain modulation and on the expression of inflammatory mediators, indicating a specific NC action through the ORL1 receptor. Together, these findings reveal novel mechanisms by which the NC system produces analgesia.

We examined the effects of a single injection of exogenous NT-3, administered at embryonic day (E) 13.5, on the survival of two populations of motor neurons and two populations of sensory neurons. Both wild-type and double knockout, Myf5-/-:MyoD-/-, mutant embryos were examined to determine the effects of the aforementioned neurotrophin on motor and sensory neuron survival in the presence and absence, respectively, of skeletal muscle. We found that, although NT-3 rescues select populations of motor neurons in the absence of muscles, there is a lack of increase in neuron survival when skeletal muscle is present. Additionally, NT-3 was found to rescue a select population of proprioceptive sensory neurons in the absence of target tissue, while, at times, exacerbating neuron cell death when target tissues are present. Lastly, we found that neurons in the spinal cord and brainstem show both a regional and functional specificity in their response to the administration of NT-3 in utero. Our results indicate the possibility that different pathways are involved in the survival of neurons during naturally occurring programmed cell death and during excessively occurring programmed cell death., ((c) 2007 Wiley-Liss, Inc.)

In lampreys, brain stem reticulospinal (RS) neurons constitute the main descending input to the spinal cord and activate the spinal locomotor central pattern generators. Cholinergic nicotinic inputs activate RS neurons, and consequently, induce locomotion. Cholinergic muscarinic agonists also induce locomotion when applied to the brain stem of birds. This study examined whether bath applications of muscarinic agonists could activate RS neurons and initiate motor output in lampreys. Bath applications of 25 microM muscarine elicited sustained, recurring depolarizations (mean duration of 5.0 +/- 0.5 s recurring with a mean period of 55.5 +/- 10.3 s) in intracellularly recorded rhombencephalic RS neurons. Calcium imaging experiments revealed that muscarine induced oscillations in calcium levels that occurred synchronously within the RS neuron population. Bath application of TTX abolished the muscarine effect, suggesting the sustained depolarizations in RS neurons are driven by other neurons. A series of lesion experiments suggested the caudal half of the rhombencephalon was necessary. Microinjections of muscarine (75 microM) or the muscarinic receptor (mAchR) antagonist atropine (1 mM) lateral to the rostral pole of the posterior rhombencephalic reticular nucleus induced or prevented, respectively, the muscarinic RS neuron response. Cells immunoreactive for muscarinic receptors were found in this region and could mediate this response. Bath application of glutamatergic antagonists (6-cyano-7-nitroquinoxaline-2,3-dione/D-2-amino-5-phosphonovaleric acid) abolished the muscarine effect, suggesting that glutamatergic transmission is needed for the effect. Ventral root recordings showed spinal motor output coincides with RS neuron sustained depolarizations. We propose that unilateral mAchR activation on specific cells in the caudal rhombencephalon activates a circuit that generates synchronous sustained, recurring depolarizations in bilateral populations of RS neurons.

The anatomy of the extrinsic innervation of the avian magnum has not been accurately demonstrated previously. In the present study, choleratoxin subunit B-conjugated horseradish peroxidase (CB-HRP) was used as a retrograde tracer to determine the sympathetic postganglionic and sensory innervation of the magnum of hens. With regard to the sympathetic postganglionic innervation, following CB-HRP injections under the serosa of the magnum, CB-HRP-positive neurons were found bilaterally in the C12-LS13 ganglia of the sympathetic chain, splanchnic ganglia and adrenal ganglia. The number of labelled neurons in the left ganglia of the sympathetic chain and splanchnic ganglia was approximately 2.1 times that in the right ganglia. This suggests that the unilateral magnum is bilaterally innervated with sympathetic postganglionic nerves, the left nerves being predominant. With regard to the sensory innervation, following tracer injections, CB-HRP-positive neurons were found bilaterally in the spinal ganglia C13-LS12, jugular ganglia and nodose ganglia. The number of positive cells in the left ganglia was about 2.2 times that in the right ganglia. In the spinal ganglia, 85.6% of the labelled neurons were in the T5-LS2 and LS8-LS11 ganglia. These results suggest that the sensory nerve fibres of the magnum reach the central nervous system principally via two groups of spinal ganglia and vagus nerves, and that the innervation is bilateral although the left-hand route predominates. Moreover, 45.7% of all the CB-HRP-labelled neurons were found in the rectal region of the intestinal nerve of Remak (INR), which suggests that the INR plays a very important role in the functional regulation of the magnum.

Gamma-aminobutyric acid type B receptors (GABAB) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABAB1 and GABAB2 subunits, a process that occurs within the endoplasmic reticulum. However, the mechanisms that regulate GABAB receptor oligomerization at the plasma membrane remain largely unknown. We first characterized the functional cytoarchitecture of an organotypic co-culture model of rat dorsal root ganglia and spinal cord. Subsequently, we studied the interactions between GABAB subunits after chronic stimulation of sensory fibres with capsaicin. Surface labelling of recombinant proteins showed a decrease in subunit co-localization and GABAB2 labelling, after capsaicin treatment. In these conditions, fluorescence lifetime imaging measurements further demonstrated a loss of interactions between green fluorescent protein-GABAB1b and t-dimer discosoma sp red fluorescent protein-GABAB2 subunits. Finally, we established that the GABAB receptor undergoes clathrin-dependent internalization and rapid recycling to the plasma membrane following activation with baclofen, a GABAB agonist. However, in cultures chronically stimulated with capsaicin, the agonist-induced endocytosis was decreased, reflecting changes in the dimeric state of the receptor. Taken together, our results indicate that the chronic stimulation of sensory fibres can dissociate the GABAB heterodimer and alters its responsiveness to the endogenous ligand. Chronic stimulation thus modulates receptor oligomerization, providing additional levels of control of signalling.

The occurrence of vagally mediated afferent signaling by lung irritants is well known. However, spinal visceral afferent pathways also might be relevant to pulmonary irritation. In the present study, responses and modulation of superficial and deep T3 spinal neurons were examined using inhaled ammonia, and the peripheral afferent fibers were also characterized in part. Extracellular potentials of single thoracic (T3) spinal neurons were recorded in pentobarbital anesthetized, paralyzed, and ventilated male rats. Ammonia vapor (0.5, 1.0, 2.0 ml) was injected into the inspiratory line of the ventilator for 20 s. Inhaled ammonia (IA, 1.0 ml) excited 5/6 neurons and inhibited one spinal neuron recorded in superficial laminae, whereas deeper neurons responded with excitatory (E, n = 20), inhibitory (I, n = 4) or biphasic patterns (6 E-I, 3 I-E). Electrical and chemical stimulation of C1-C2 spinal neurons primarily suppressed T3 neuronal responses to IA. Resiniferatoxin (2 microg/kg, i.v.), which desensitizes afferent fibers containing transient receptor potential vanilloid receptor-1 (TRPV-1), abolished excitatory responses of 8/8 neurons to IA. Bilateral cervical vagotomy did not affect IA responses in 5 superficial neurons while 7 deeper neurons showed variable responses. 82% (32/39) of the spinal neurons responding to IA also received convergent noxious inputs from somatic fields in the chest and back areas. These results suggested that superficial and deeper spinal neuronal activation by inhaled ammonia mainly depended upon pulmonary sympathetic afferent fibers expressing TRPV-1. Additionally, C1-C2 spinal neurons, supraspinal sites and vagal afferents modulated the thoracic spinal neuronal responses to lower airway irritation.

As different classes of sensory neurons project into the CNS, their axons segregate and establish distinct trajectories and target zones. One striking instance of axonal segregation is the projection of sensory neurons into the spinal cord, where proprioceptive axons avoid the superficial dorsal horn-the target zone of many cutaneous afferent fibers. PlexinA1 is a proprioceptive sensory axon-specific receptor for sema6C and sema6D, which are expressed in a dynamic pattern in the dorsal horn. The loss of plexinA1 signaling causes the shafts of proprioceptive axons to invade the superficial dorsal horn, disrupting the organization of cutaneous afferents. This disruptive influence appears to involve the intermediary action of oligodendrocytes, which accompany displaced proprioceptive axon shafts into the dorsal horn. Our findings reveal a dedicated program of axonal shaft positioning in the mammalian CNS and establish a role for plexinA1-mediated axonal exclusion in organizing the projection pattern of spinal sensory afferents.

The purpose of this study was to examine how upper thoracic spinal neurons responded to activation and desensitization of cardiac transient receptor potential vanilloid-1 (TRPV1)-containing afferent fibers. Extracellular potentials of single T3 spinal neurons were recorded in pentobarbital-anesthetized, paralyzed, and ventilated male rats. To activate cardiac nociceptive receptors, a catheter was placed in the pericardial sac to administer various chemicals: bradykinin (BK; 10 microg/ml, 0.2 ml), capsaicin (CAP, 10 microg/ml, 0.2 ml), or a mixture of algesic chemicals (AC; 0.2 ml) containing adenosine 10(-3) M, BK, serotonin, histamine, and PGE(2), 10(-5) M for each. Spinal neurons that responded to intrapericardial BK and/or CAP were used in this study. Results showed that 81% (35/43) of the neurons had excitatory responses to both intrapericardial BK and CAP, and the remainder responded to either BK or CAP. Intrapericardial resiniferatoxin (RTX) (0.2 microg/ml, 0.2 ml, 1 min), which desensitizes TRPV1-containing nerve endings, abolished excitatory responses to both BK (n = 8) and CAP (n = 7), and to AC (n = 5) but not to somatic stimuli. Intrapericardial capsazepine (1 mg/ml, 0.2 ml, 3 min), a specific antagonist of TRPV1, sharply attenuated excitatory responses to CAP in 5/5 neurons, but responses to BK in 5/5 neurons was maintained. Additionally, intrapericardial capsazepine had no significant effect on excitatory responses to AC in 3/3 neurons. These data indicated that intrapericardial BK-initiated spinal neuronal responses were linked to cardiac TRPV1-containing afferent fibers, but were not dependent on TRPV1. Intraspinal signaling for cardiac nociception was mediated through CAP-sensitive afferent fibers innervating the heart.

Microstimulation within the motor regions of the spinal cord is often assumed to activate motoneurons and propriospinal neurons close to the electrode tip. However, previous work has shown that intraspinal microstimulation (ISMS) in the gray matter activates sensory afferent axons as well as alpha-motoneurons (MNs). Here we report on the recruitment of sensory afferent axons and MNs as ISMS amplitudes increased. Intraspinal microstimulation was applied through microwires implanted in the dorsal horn, intermediate region and ventral horn of the L(5)-L(7) segments of the spinal cord in four acutely decerebrated cats, two of which had been chronically spinalized. Activation of sensory axons was detected with electroneurographic recordings from dorsal roots. Activation of MNs was detected with electromyographic (EMG) recordings from hindlimb muscles. Sensory axons were nearly always activated at lower stimulus levels than MNs irrespective of the stimulating electrode location. EMG response latencies decreased as ISMS stimulus intensities increased, suggesting that MNs were first activated transsynaptically and then directly as intensity increased. ISMS elicited antidromic activity in dorsal root filaments with entry zones up to 17 mm rostral and caudal to the stimulation sites. We posit that action potentials elicited in localized terminal branches of afferents spread antidromically to all terminal branches of the afferents and transsynaptically excite MNs and interneurons far removed from the stimulation site. This may help explain how focal ISMS can activate many MNs of a muscle even though they are distributed in long thin columns.

Previous results from our group and others showed that skin and muscle afferents are equipped with tetrodotoxin-resistant (TTX-r) channels. The great majority of the TTX-r fibres are unmyelinated (C or group IV) and are assumed to have nociceptive functions. Therefore, a block of the TTX-sensitive (TTX-s) fibres offers the possibility to study reactions of central nervous neurones to a purely nociceptive input. The present study compared spinal synaptic field potentials (SFPs) evoked by electrical stimulation of TTX-r afferent fibres from skin and muscle at various depths of the spinal segments L4 and L5 in the rat. Cutaneous input was produced by stimulation of the sural nerve (SU), input from muscle by stimulation of the gastrocnemius-soleus nerves (GS). To block the (non-nociceptive) TTX-s afferents, a pool containing TTX (concentration 1microM) was built around the dorsal roots L3-L6. As a measure of synaptic activity, the area of averaged SFPs was determined. After TTX application, the SFPs of fast conducting myelinated afferent fibres vanished completely. Simultaneously, the size of the potentials evoked by electrical stimulation of slowly conducting TTX-r skin and muscle afferents increased significantly. The field potentials of TTX-r GS afferents had a maximum in laminae IV-VI of the dorsal horn, whereas the SFPs induced by SU stimulation were more evenly distributed over all laminae. The results are a further indication that nociceptive input from skin and muscle is differently processed at the spinal level.

The visceral reflexes of the pelvic organs are mediated by connections between primary afferents innervating the pelvic organs and parasympathetic preganglionic neurons in the intermediolateral column of the sacral spinal cord. The present immunohistochemical study revealed many varicosities expressing transient receptor potential vanilloid 1 (TRPV1) that were closely apposed to the preganglionic neuronal perikarya at embryonic day 16 in mice. Many, but not all, varicosities expressing TRPV1 in the intermediolateral column were also immunopositive for calcitonin gene-related peptide. In contrast, no nerve fibers expressing TRPV1 projected to the sympathetic preganglionic cell column in the lumbar spinal cord in prenatal stages. The results of the present study raised the possibility that the primary afferents transmit signals elicited by the activation of TRPV1 receptors to the sacral parasympathetic preganglionic neurons. Thus, the functional circuit for pelvic spinal reflexes, such as micturition induced by urine influx, might develop in the prenatal stages in mice.

Tetanic stimulation of the sciatic nerve produces spinal long-term potentiation (LTP) of C-fiber evoked field potentials, which is NMDA dependent and may be the substrate of inflammation- or nerve injury-produced central sensitization. Glial glutamate transporter GLT-1 has been considered as an important regulator of excitatory synaptic transmission and nociception. In the present study, we investigated the effects of GLT-1 on the spinal LTP and Fos expression induced by tetanically sciatic stimulation. Intrathecal administration of dihydrokainate (DHK), a GLT-1 selective inhibitor, partially inhibited (0.1 mM) or completely blocked (3.0 mM) the spinal LTP, which may be related to an accumulation of extracellular glutamate. Intrathecal DHK (3.0 mM) also suppressed tetanic stimulation-induced spinal Fos expression. Double immunofluorescence showed no Fos expression in glial fibrillary acidic protein (GFAP)-positive cells, and the cell DNA fragment study failed to detect a significant apoptosis of spinal neurons. These results suggest that disruption of GLT-1 may be associated with the inhibition of functional activation of spinal neurons expressing Fos, but not with glutamate excitotoxicity. In conclusion, glial GLT-1 may play an important role in tetanically sciatic stimulation-induced LTP of spinal nociceptive neurons via the regulation of extracellular levels of glutamate to an appropriate concentration.

The finding that sensory neuron-specific G-protein-coupled receptor mRNA is solely expressed in small primary sensory neurons suggests involvement of the receptor in nociceptive modulation. The present study was designed to assess effects of intrathecal administration of bovine adrenal medulla 8-22 and (Tyr6)-gamma2-MSH-6-12, selective sensory neuron-specific receptor agonists, on nocifensive behaviors and expression of spinal c-Fos-like immunoreactivity evoked by intraplantar injection of 2.5% formalin in rats. The agonists were administered 10 min before (pretreatment) and/or after (post-treatment) injection of formalin. Pretreatment with bovine adrenal medulla 8-22 dose-dependently (3, 10 and 30 nmol) decreased time lifting and licking the paw mainly in the second phase. Intrathecal bovine adrenal medulla 8-22 (30 nmol) remarkably suppressed nocifensive behaviors in the first and second phases and the expression of formalin-evoked c-Fos-like immunoreactivity in laminae I-II and V-VI of the spinal dorsal horn at L4-5. Moreover, naloxone (20 microg, intrathecal) failed to antagonize the inhibitory effects of bovine adrenal medulla 8-22. Post-treatment with bovine adrenal medulla 8-22 also exerted inhibition on the second phase behaviors in a dose-dependent manner with a similar efficacy observed in pretreatment groups. Furthermore, post-treatment with (Tyr6)-gamma2-MSH-6-12 (0.5, 1.5 and 5 nmol) also suppressed formalin-evoked nocifensive behaviors in the second phase and c-Fos-like immunoreactivity in the spinal dorsal horn similar with bovine adrenal medulla 8-22. Our results suggest that sensory neuron-specific receptor may play an important role in modulation of spinal nociceptive transmission. This is the first to demonstrate that activation of sensory neuron-specific receptor produces analgesia in the persistent pain model.

The aim of our study was to assess the ability of brain-derived neurotrophic factor (BDNF) to rescue motor and sensory neurons from programmed cell death. It is clearly demonstrated that the administration of a single injection of a putative neurotrophic factor to mouse embryos in utero on embryonic day (E) 14.5 is sufficient to significantly reduce the death of motor neurons when assessed on E18.5. However, the trophic requirements of somatic neurons have not been unequivocally determined in a mammalian species in vivo. Indeed, the unexpectedly high numbers of surviving neurons observed in neurotrophin and tyrosine kinase receptor knockout mice are probably the consequence of functional redundancy between the neurotrophins and their receptors. We studied spinal cord and facial motor nucleus neurons and proprioceptive neurons in the dorsal root ganglion and mesencephalic nucleus. The action of BDNF was assessed in wild-type fetuses to gain insight into its ability to rescue neurons from naturally occurring programmed cell death. In addition, we used Myf5(-/-):MyoD(-/-) embryos, which completely lack skeletal musculature, to assess the ability of BDNF to rescue neurons from excessively occurring programmed cell death. We found that BDNF differentially rescued neurons from naturally vs. excessively occurring cell death and that its ability to do so varied among neuronal subpopulations.

In view of the Lack of theoretical information, end-to-side neurorrhaphy is a frequent object of experimental interest. End-to-side neurorrhaphy is based on collateral sprouting of an intact axon. The quantitative assessment of collateral sprouts sent by an intact motor and sensory axon was the goal of the present study. End-to-side neurorrhaphy of the distal stump of transected musculocutaneous nerve (MCN) with intact ulnar nerve (UN) was performed in a rat model. Collateral sprouts were quantitatively evaluated by counting of motoneurons and DRG neurons following their retrograde labeling by Fluoro-Ruby and Fluoro-Emerald applied to the UN and MCN, respectively. The results suggest a comparable capacity of both intact sensory and motor axons to send collateral sprouts into a denervated nerve stump. The ratio of sensory/motor neurons, the axons of which reinnervated distal MCN stumps, was very similar to that of intact UN (6.500 and 6.747, respectively), but different from intact MCN (5.029). This suggests that the pruning process occurred to balance the collateral sprouts at a ratio of sensory/motor neurons for the donor UN, but not according to the number of sensory and motor bands of Bungner available in the distal stump of the MCN. The present experimental study confirms end-to-side neurorrhaphy as a suitable method of nerve reconstruction.

This article provides a critical analysis of the concepts of the sensory innervation of the brain and spinal cord presented in Sotnikov's articles in issues 2 and 3 of this volume. Criteria for the identification of intracerebral primary sensory neurons and the possible roles of neuronal cilia are discussed. The significance of asynaptic dendrites and bipolar cells is considered. The importance of the correct use of a variety of terms is emphasized, and the need for using combined morphological and electrophysiological analysis in investigating sensory intracerebral exteroceptors is noted.

Published data and our own results relating to exteroceptor and a variety of interceptor neurons in the brain and spinal cord, such as intraspinal Hesse ocelli and light-sensitive epiphyseal and ependymal neurons, are presented. Light-sensitive ganglion neurons in invertebrates are also described, along with intrinsic spinal cord bipolar sensory neurons within the spinal cord, primary chemo-and thermosensitive neurons, and sensory unipolar neurons associated with the three fine "central nerves" of Motavkin, which perforate the sheath of the spinal cord and ending with bush-like receptors close to vessels or near the ependyma of the central canal. Data on all known intracortical interoceptors in vertebrates are generalized into a single scheme. It is hypothesized that the brains of animals and humans have an intrinsic sensory innervation comparable with the innervation of other organs and containing local primary sensory neurons and their asynaptic dendrites, which can be divided into two groups: interceptor and exteroceptor.

Natriuretic peptides (NP) and the corresponding receptors are present in the rodent spinal cord. We have studied the structures which respond to atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide with an increased synthesis of cGMP. NP-responsive cGMP-producing structures were observed in laminae I-III, and X, and in addition in ependymal cells, astrocytes and a subpopulation of dorsal root ganglion cells. As the cGMP concentration is controlled by the rate of synthesis and the rate of breakdown by phosphodiesterases, we studied NP-responsive structures in spinal cord slices incubated in the presence of different phosphodiesterase inhibitors. We studied EHNA and BAY 60-7550 as selective PDE2 inhibitors, sildenafil as a selective PDE5 inhibitors, dipyridamole as a mixed type PDE5 and PDE10 inhibitor, rolipram as a PDE4 inhibitor, and SCH 81566 as a selective PDE9 inhibitor. Double immunostainings showed that cGMP-IR colocalized partial with the vesicular acetylcholine transporter molecule in lamina X, with Substance P in a subpopulation of neuronal fibers situated dorsolateral, and with a subpopulation of CGRP-IR dorsal root ganglion neurons. Colocalization of cGMP-IR was absent with parvalbumin, synaptophysin, and the vesicular transporter molecules for GABA and glutamate. It is concluded that NPs in the spinal cord are probably involved in integrating intersegmental sensory processing in the spinal cord although the greater part of the NP-responsive cGMP-producing fibers could not be characterized. PDE2, 5, and 9 are involved in regulating NP-stimulated cGMP levels in the spinal cord. NPs may have a role in regulating cerebrospinal fluid homeostasis.

Spinal marginal zone (MZ) neurons play a crucial role in the transmission of nociceptive and thermoreceptive information to the brain. The precise areas to which physiologically characterized MZ neurons project in the ventral posterior lateral (VPL) nucleus of the thalamus have not been clearly established. Here, we examine this projection in rats using the method of antidromic activation to map the axon terminals of neurons recorded from the MZ. Thirty-three neurons were antidromically activated using pulses of < or =30 microA in the contralateral VPL. In every case, the most rostral point from which the MZ neuron could be antidromically activated was surrounded by stimulating tracks in which large-amplitude current pulses failed to activate the examined neuron, indicating the termination of the spinothalamic tract (STT) axon. Each of 30 examined neurons responded to noxious but not innocuous mechanical stimuli applied to their cutaneous receptive fields, which ranged in size from two digits to the entire limb. Of 17 thermally tested neurons, 16 responded to innocuous or noxious thermal stimuli. Among STT neurons that responded to thermal stimuli, 50% responded to innocuous cooling as well as noxious heat and cold, 31% responded to noxious heat and cold, and 19% responded only to noxious heat. Axons from cells responsive to innocuous cooling terminated in the core region of VPL, significantly dorsal and medial relative to other thermally responsive subgroups. In rats, thermally responsive subgroups of MZ neurons project directly to distinct regions of VPL.

Systemic administration of resiniferatoxin (RTX), an ultrapotent capsaicin analogue, removes transient receptor potential vanilloid type 1 (TRPV1)-expressing afferent neurons and impairs thermal but not mechanical nociception in adult animals. In this study, we determined how loss of TRPV1-expressing sensory neurons alters the antinociceptive effect of mu opioids and mu opioid receptors in the spinal cord. The effect of morphine and (D-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO) was measured by testing the paw mechanical withdrawal threshold in rats treated with RTX or vehicle. RTX treatment deleted TRPV1-immunoreactive dorsal root ganglion neurons and nerve terminals in the spinal dorsal horn. Also the mu opioid receptor immunoreactivity was markedly reduced in the superficial dorsal horn of RTX-treated rats. However, RTX treatment did not affect the dorsal horn neurons labeled with both TRPV1- and mu opioid receptor-immunoreactivity. Surprisingly, intrathecal morphine or DAMGO produced a greater increase in the withdrawal threshold in RTX- than in vehicle-treated rats. The duration of the effect of intrathecal morphine and DAMGO in RTX-treated rats was also profoundly increased. Furthermore, the antinociceptive effect of systemic morphine was significantly potentiated in RTX-treated rats. The B(MAX) (but not K(D)) of [3H]-DAMGO binding and DAMGO-stimulated [35S]GTPgammaS activity in the dorsal spinal cord were significantly reduced in the RTX group. This study provides novel information that loss of TRPV1 afferent neurons eliminates presynaptic mu opioid receptors present on TRPV1-expressing afferent neurons but paradoxically potentiates the analgesic effect of mu opioid agonists. Mechano-nociception, transmitted through non-TRPV1 sensory neurons, is subject to potent modulation by mu opioid agonists.

Dorsal root ganglion (DRG) neurons extend axons to specific targets in the gray matter of the spinal cord. During development, DRG axons grow into the dorsolateral margin of the spinal cord and projection into the dorsal mantle layer occurs after a ;waiting period' of a few days. Netrin 1 is a long-range diffusible factor expressed in the ventral midline of the developing neural tube, and has chemoattractive and chemorepulsive effects on growing axons. Netrin 1 is also expressed in the dorsal spinal cord. However, the roles of dorsally derived netrin 1 remain totally unknown. Here, we show that dorsal netrin 1 controls the correct guidance of primary sensory axons. During the waiting period, netrin 1 is transiently expressed or upregulated in the dorsal spinal cord, and the absence of netrin 1 results in the aberrant projection of sensory axons, including both cutaneous and proprioceptive afferents, into the dorsal mantle layer. Netrin 1 derived from the dorsal spinal cord, but not the floor plate, is involved in the correct projection of DRG axons. Furthermore, netrin 1 suppresses axon outgrowth from DRG in vitro. Unc5c(rcm) mutant shows abnormal invasion of DRG axons as observed in netrin 1 mutants. These results are the first direct evidence that netrin 1 in the dorsal spinal cord acts as an inhibitory cue for primary sensory axons and is a crucial signal for the formation of sensory afferent neural networks.