Effects and distribution of vagal capsaicin-sensitive substance P neurons with special reference to the trachea and lungs

The origin of substance P (SP)-immunoreactive neurons in the lower respiratory tract, esophagus and heart of guinea-pigs was demonstrated by surgical denervation or capsaicin pretreatment with subsequent determination of the tissue levels of SP by radioimmunoassay. In other experiments the effect of vagal nerve stimulation on the SP levels in these tissues was studied. The effects of capsaicin-sensitive afferents in the respiratory tract mucosa and bronchial smooth muscle was also studied by analysis of vascular permeability to Evans blue and insufflation-pressure changes. Our present data indicate that all SP nerves in the trachea and lung are afferent and capsaicin-sensitive. The trachea and stem bronchi receive SP afferents mainly from the right vagus nerve with cell bodies located in both the nodose and jugular ganglia. The SP innervation of the lung seems to have a dual origin: 1. Afferents from both vagal nerves with a crossed type of innervation pattern. 2. A non-vagal source which consists of about 40% of the SP nerves in the lung. These nerves probably originate from thoracic spinal ganglia. The effects of ether and capsaicin on insufflation pressure and increase in vascular permeability were dependent on the integrity of capsaicin-sensitive afferents of both vagal and non-vagal origin. In the guinea pig, systemic capsaicin pretreatment to adult animals seemed to result in irreversible changes in the respiratory tract, while in the rat a successive recovery of the functional response of capsaicin-sensitive afferents occurred. Different regimes of systemic capsaicin pretreatment induced different effects on the cholinergic (atropine-sensitive) insufflation-pressure response. Capsaicin pretreatment, using multiple injections over two days, depressed the cholinergic insufflation-pressure increase, while the cholinergic vagal component was unaffected in animals which received a single dose of capsaicin or local pretreatment with capsaicin on the vagal nerves. The local treatment was more effective with regard to SP depletion in target areas when using alcohol as solvent than when capsaicin was dissolved in paraffin oil, while the functional deficits were similar. The SP nerves in the esophagus were mainly of vagal afferent origin, while the heart atrium seemed to have a dual innervation by both vagal and non-vagal SP nerves.     

Intrinsic and synaptic long-term depression of NTS relay of nociceptin- and capsaicin-sensitive cardiopulmonary afferents hyperactivity

The nucleus tractus solitarius (NTS) in the caudal medulla is a gateway for a variety of cardiopulmonary afferents important for homeostatic regulation and defense against airway and cardiovascular insults and is a key central target potentially mediating the response habituation to these inputs. Here, whole-cell and field population action potential recordings and infrared imaging in rat brainstem slices in vitro revealed a compartmental pain-pathway-like organization of capsaicin-facilitated vs. nocistatin-facilitated/nociceptin-suppressed neuronal clusters in an NTS region, which receives cardiopulmonary A- and C-fiber afferents with differing capsaicin sensitivities. All capsaicin-sensitive neurons and a fraction of nociceptin-sensitive neurons expressed N-methyl-d-aspartate (NMDA) receptor-dependent synaptic long-term depression (LTD) following afferent stimulation. All neurons also expressed activity-dependent decrease of excitability (intrinsic LTD), which converted to NMDA receptor-dependent intrinsic long-term potentiation after GABA_A receptor blockade. Thus, distinct intrinsic and synaptic LTD mechanisms in the NTS specific to the relay of A- or C-fiber afferents may underlie the response habituation to persistent afferents hyperactivity that are associated with varying physiologic challenges and cardiopulmonary derangements—including hypertension, chronic cough, asthmatic bronchoconstriction, sustained elevated lung volume in chronic obstructive pulmonary disease or in continuous positive-airway-pressure therapy for sleep apnea, metabolic acidosis, and prolonged exposure to hypoxia at high altitude.     

Neonatal anandamide treatment results in prolonged mitochondrial damage in the vanilloid receptor type 1-immunoreactive B-type neurons of the rat trigeminal ganglion

Capsaicin acting on the vanilloid type 1 receptor (VR1) excites a subset of primary sensory neurons. Systemic capsaicin treatment of adult or neonatal rats results in selective damage of the B-type neurons in the rat sensory ganglia by causing a long-lasting mitochondrial lesion that has been described in detail in previous studies.The endocannabinoid, anandamide, exhibits an agonist effect on VR1 receptors. The physiological role of anandamide as a VR1 agonist is still uncertain. This study addresses whether high doses of anandamide induce similar ultrastructural changes to those described for capsaicin. The effect of neonatally administered anandamide (1 mg/kg) on neurons of the trigeminal ganglia and the hippocampal formation was examined in the light and electron microscope from the first day after injections to the 20th week after treatment. Anandamide was found to cause mitochondrial damage of the B-type neurons of trigeminal ganglia similar to what has been described for capsaicin. The time course of damage was also comparable. In addition to the cells of the trigeminal ganglia, B-type cells of dorsal root ganglia were also damaged. A-type neurons and satellite glial cells were not affected either in the trigeminal or in the dorsal root ganglia. In the hippocampal formation, where a subpopulation of local circuit neurons is known to contain cannabinoid type 1 (CB1) but not VR1 receptors, anandamide did not cause morphological changes of mitochondria either in the dentate gyrus or in Ammon's horn. At 3 weeks of age, all VR1-immunoreactive neurons in the trigeminal ganglia of animals treated neonatally with anandamide displayed swollen mitochondria.The results suggest that anandamide, at pharmacologically relevant doses, acts on the VR1 receptor and causes prolonged and selective mitochondrial damage of B-type sensory neurons, as has previously been described for capsaicin.     

Responses of neurons in paraventricular nucleus to activation of cardiac afferents and acute myocardial ischaemia in rats

Stimulation of cardiac sympathetic afferents increases sympathetic outflow and blood pressure. Chemicals released during myocardial ischaemia activate cardiac afferents. This study was to determine the responses of neurons in paraventricular nucleus (PVN) to the cardiac afferent activation caused by exogenous chemicals or myocardial ischaemia using an extracellular single-unit recording method. Rats were anaesthetized and underwent bilateral cervical vagal denervation (VD) and carotid and aortic baroreceptor denervation (BD). In 196 spontaneously active neurons in parvicellular PVN, 60 (30.6%), 36 (18.4%) and 91 (46.4%) neurons were respectively sensitive, mildly sensitive and insensitive to capsaicin, while nine (4.6%) neurons showed inhibitory responses to capsaicin. Epicardial application of capsaicin activated capsaicin-sensitive neurons in the PVN and increased mean arterial pressure. These neurons were also sensitive to exogenous bradykinin, adenosine and H(2)O(2). The neuron response is not secondary to a capsaicin-induced increase in mean arterial pressure because a similar degree of pressor response induced by aortic coarctation did not increase the neuron activity. Compared with intact rats, VD or BD or combined VD and BD increased the response of capsaicin-sensitive neurons to epicardial application of capsaicin, while stimulation of vagal afferents inhibited the response. Myocardial ischaemia caused increases in the activity of capsaicin-sensitive neurons and renal sympathetic nerve activity. The results indicate that chemical stimulation of cardiac sympathetic afferents activates capsaicin-sensitive neurons in parvicellular PVN, which is inhibited by the afferent activities of vagi and arterial baroreceptors. Acute myocardial ischaemia activates capsaicin-sensitive neurons in PVN and enhances sympathetic outflow.     

Evidence for substance P-immunoreactive spinal afferents that mediate bronchoconstriction

The origin and functional role of capsaicin-sensitive substance P-(SP-) immunoreactive (IR) nerve fibres in the lower airways were studied in the guinea-pig. Stellatectomy caused a significant reduction of SP-IR in the lung and pulmonary artery. Immunohistochemical analysis, however, did not reveal any clear-cut change in the number and distribution of SP-IR fibres in the lung of these animals. After combined stellatectomy plus local capsaicin treatment of the vagal nerves, most SP-IR nerves disappeared in the lower airways. The bronchoconstriction induced by capsaicin was significantly reduced after stellatectomy and abolished after stellatectomy plus capsaicin pretreatment of the vagal nerves. Ether inhalation caused bronchoconstriction, which was not influenced by stellatectomy but markedly reduced by combined capsaicin treatment of vagal nerves and stellatectomy. Stellate ganglion stimulation in animals that had been chemically sympathectomized by 6-OH-dopamine caused bronchoconstriction, which was resistant to cholinergic or adrenergic receptor blockade. This response was absent after systemic capsaicin pretreatment, suggesting that it was due to antidromic stimulation of afferent fibres traversing the stellate ganglion. In conclusion, the present data suggest that the lower airways receive SP-IR capsaicin-sensitive C-fibre afferents of both vagal and spinal origin. These sensory fibres seem to have branches both within the bronchial smooth muscle and around blood vessels.     

Activation of 5-hydroxytryptamine type 3 receptor-expressing C-fiber vagal afferents inhibits retrotrapezoid nucleus chemoreceptors in rats

Retrotrapezoid nucleus (RTN) chemoreceptors are regulated by inputs from the carotid bodies (CB) and from pulmonary mechanoreceptors. Here we tested whether RTN neurons are influenced by 5-hydroxytryptamine type 3 receptor-expressing C-fiber vagal afferents. In urethan-anesthetized rats, selective activation of vagal C-fiber afferents by phenylbiguanide (PBG) eliminated the phrenic nerve discharge (PND) and inhibited RTN neurons (n = 24). PBG had no inhibitory effect in vagotomized rats. Muscimol injection into the solitary tract nucleus, commissural part, reduced inhibition of PND and RTN by PBG (73%), blocked activation of PND and RTN by CB stimulation (cyanide) but had no effect on inhibition of PND and RTN by lung inflation. Bilateral injections of muscimol into interstitial solitary tract nucleus (NTS) reduced the inhibition of PND and RTN by PBG (53%), blocked the inhibitory effects of lung inflation but did not change the activation of PND and RTN neurons by CB stimulation. PBG and lung inflation activated postinspiratory neurons located within the rostral ventral respiratory group (rVRG) and inhibited inspiratory and expiratory neurons. Bilateral injections of muscimol into rVRG eliminated PND and partially decreased RTN neuron inhibition by PBG (32%). In conclusion, activation of cardiopulmonary C-fiber afferents inhibits the activity of RTN chemoreceptors. The pathway relays within a broad medial region of the NTS and involves the rVRG to a limited degree. The apnea triggered by activation of cardiopulmonary C-fiber afferents may be due in part to a reduction of the activity of RTN chemoreceptors.     

Role of primary afferent nerves in allodynia caused by diabetic neuropathy in rats

Both myelinated and unmyelinated afferents are implicated in transmitting diabetic neuropathic pain. Although unmyelinated afferents are generally considered to play a significant role in diabetic neuropathic pain, pathological changes in diabetic neuropathy occur mostly in myelinated A-fibers. In the present study, we first examined the role of capsaicin-sensitive C-fibers in the development of allodynia induced by diabetic neuropathy. We then studied the functional changes of afferent nerves pertinent to diabetic neuropathic pain. Diabetes was induced in rats by i.p. streptozotocin. To deplete capsaicin-sensitive C-fibers, rats were treated with i.p. resiniferatoxin (300 microg/kg). Mechanical and thermal sensitivities were measured using von Frey filaments and a radiant heat stimulus. Single-unit activity of afferents was recorded from the tibial nerve. Tactile allodynia, but not thermal hyperalgesia, developed in diabetic rats. Resiniferatoxin treatment did not alter significantly the degree and time course of allodynia. Post-treatment with resiniferatoxin also failed to attenuate allodynia in diabetic rats. The electrophysiological recordings revealed ectopic discharges and a higher spontaneous activity mainly in Adelta- and Abeta-fiber afferents in diabetic rats regardless of resiniferatoxin treatment. Furthermore, these afferent fibers had a lower threshold for activation and augmented responses to mechanical stimuli. Thus, our study suggests that capsaicin-sensitive C-fiber afferents are not required in the development of allodynia in this rat model of diabetes. Our electrophysiological data provide substantial new evidence that the abnormal sensory input from Adelta- and Abeta-fiber afferents may play an important role in diabetic neuropathic pain.     

Vanilloid receptor VR1-positive primary afferents are glutamatergic and contact spinal neurons that co-express neurokinin receptor NK1 and glutamate receptors

The vanilloid receptor VR1 (TRPV1) is a temperature- and capsaicin-sensitive cation channel expressed by a class of primary afferents involved in nociception. To confirm the hypothesis that VR1-positive primary afferents are glutamatergic and contact spinal neurons that express the main classes of ionotropic glutamate receptors, we performed multiple immunofluorescent staining for VR1 and the glutamate transporter VGLUT2 (a specific marker for glutamatergic transmission) or AMPA and NMDA receptor subunits. VR1-positive cells in the dorsal root ganglion and boutons of their central afferent fibers in the dorsal horn expressed VGLUT2, and the latter contacted AMPA- or NMDA receptor-positive perikarya. Based on our previous observations of preferential targeting of VR1-positive primary afferents to spinal neurons that express the neurokinin receptor NK1 (Hwang et al., 2003), we further quantified the frequency of termination of VR1-positive afferents onto NK1-positive neurons co-expressing glutamate receptors. A larger fraction of NK1/NMDA receptors-positive than NK1/AMPA receptors-positive sites were contacted by VR1-positive boutons. We conclude that VR1-positive primary afferents in the rat use glutamate as neurotransmitter and contact postsynaptic sites that co-express NK1 and ionotropic glutamate receptors.     

Stimulation of pulmonary vagal C-fibres by anandamide in anaesthetized rats: role of vanilloid type 1 receptors

This study was carried out to determine the effect of intravenous injection of anandamide on pulmonary C-fibre afferents and the cardiorespiratory reflexes. In anaesthetized, spontaneously breathing rats, intravenous bolus injection of anandamide near the right atrium immediately elicited the pulmonary chemoreflex responses, characterized by apnoea, bradycardia and hypotension. After perineural treatment of both cervical vagi with capsaicin to block the conduction of C-fibres, anandamide no longer evoked these reflex responses. In open-chest, and artificially ventilated rats, anandamide injection evoked an abrupt and intense discharge in vagal pulmonary C-fibres in a dose-dependent manner. After injection of the high dose, the fibre discharge generally started within 1 s, reached a peak in ∼2 s, and returned to baseline within 7 s. The stimulation of C-fibres by anandamide was completely and reversibly blocked by pretreatment with capsazepine, a competitive antagonist of the vanilloid type 1 receptor. Anandamide (0.4 mg kg⁻¹) stimulated ∼93 % of pulmonary C-fibres that were activated by capsaicin at a much lower dose (0.6 μg kg⁻¹); the response to anandamide showed similar intensity, but had slightly longer latency and duration than that to capsaicin. In conclusion, intravenous bolus injection of anandamide evokes a consistent and distinct stimulatory effect on pulmonary C-fibre terminals, and this effect appears to be mediated through an activation of the vanilloid type 1 receptor.     

Prostaglandin E2 depresses solitary tract-mediated synaptic transmission in the nucleus tractus solitarius

Prostaglandin E(2) (PGE(2)) is a prototypical inflammatory mediator that excites and sensitizes cell bodies [Kwong K, Lee LY (2002) PGE(2) sensitizes cultured pulmonary vagal sensory neurons to chemical and electrical stimuli. J Appl Physiol 93:1419-1428; Kwong K, Lee LY (2005) Prostaglandin E(2) potentiates a tetrodotoxin (TTX)-resistant sodium current in rat capsaicin-sensitive vagal pulmonary sensory neurons. J Physiol 56:437-450] and peripheral nerve terminals [Ho CY, Gu Q, Hong JL, Lee LY (2000) Prostaglandin E (2) enhances chemical and mechanical sensitivities of pulmonary C fibers in the rat. Am J Respir Crit Care Med 162:528-533] of primary vagal sensory neurons. Nearly all central nerve terminals of vagal afferents are in the nucleus tractus solitarius (NTS), where they operate with a high probability of release [Doyle MW, Andresen MC (2001) Reliability of monosynaptic sensory transmission in brain stem neurons in vitro. J Neurophysiol 85:2213-2223]. We studied the effect of PGE(2) on synaptic transmission between tractus solitarius afferent nerve terminals and the second-order NTS neurons in brain stem slices of Sprague-Dawley rats. Whole-cell patch recording in voltage clamp mode was used to study evoked excitatory postsynaptic glutamatergic currents (evEPSCs) from NTS neurons elicited by electrical stimulation of the solitary tract (ST). In 34 neurons, bath-applied PGE(2) (200 nM) decreased the evEPSC amplitude by 49+/-5%. In 22 neurons, however, PGE(2) had no effect. We also tested 15 NTS neurons for capsaicin sensitivity. Seven neurons generated evEPSCs that were equally unaffected by PGE(2) and capsaicin. Conversely, evEPSCs of the other eight neurons, which were PGE(2)-responsive, were abolished by 200 nM capsaicin. Furthermore, the PGE(2-)induced depression of evEPSCs was associated with an increase in the paired pulse ratio and a decrease in both the frequency and amplitude of the spontaneous excitatory postsynaptic currents (sEPSCs) and TTX-independent spontaneous miniature excitatory postsynaptic currents (mEPSCs). These results suggest that PGE(2) acts both presynaptically on nerve terminals and postsynaptically on NTS neurons to reduce glutamatergic responses.     

The actions of anandamide on rat superficial medullary dorsal horn neurons in vitro

Whole-cell patch-clamp recordings were made from neurons in the trigeminal nucleus caudalis and trigeminal ganglion, in vitro , to investigate the cellular actions of the endogenous cannabinoid, anandamide. Anandamide has been shown to act through both the cannabinoid receptor 1 (CB1) and the vanilloid receptor 1 (VR1). Anandamide (30 μ m ) caused a 54 % increase in the rate of miniature excitatory post-synaptic currents (mEPSCs), without affecting their amplitude. The effect of anandamide was blocked by the VR1 antagonist capsazepine (20 μ m ), but not by the CB1-specific antagonist AM251 (3 μ m ). Application of the VR1 receptor agonist capsaicin (300 n m ) caused a 4200 % increase in the mEPSC rate. In dissociated trigeminal ganglion neurons, both anandamide and capsaicin caused an outward current in neurons that were voltage clamped at +40 mV. The maximal outward current produced by anandamide (EC₅₀, 10 μ m ) was 45 % of that produced by capsaicin (10 μ m ). Co-application of the VR1 antagonist capsazepine (30 μ m ) completely reversed the effects of both capsaicin and anandamide. The anandamide transport inhibitor, AM404 (30 μ m ) caused a 40 % increase in mEPSC rate in the slice preparation and an outward current in dissociated neurons. The latter current was reversed by the VR1 antagonist iodoresiniferatoxin (1 μ m ). The fatty acid amide hydrolase (FAAH) inhibitors phenylmethylsulfonyl fluoride (PMSF) (20 μ m ) and OL53 (1 μ m ) did not enhance the effect of anandamide in either the slice or dissociated neuron preparations. These results suggest that within the superficial medullary dorsal horn, anandamide (30 μ m ) acts presynaptically to enhance the release of glutamate via activation of the VR1 receptor.     

Thalamic neural activation in the cyclophosphamide-induced visceral pain model in mice

The afferent nociceptive information from the lower urinary tract terminates in the dorsal horn of the spinal cord, and then projects to the thalamus. In the present study, we examined the effects of visceral nociception from the lower urinary tract on the neural activity of thalamic neurons using cyclophosphamide (CP)-induced cystitis, a model of visceral nociception. The levels of c-fos mRNA as well as protein, a marker of neural activation, were investigated in the thalamus using in situ hybridization histochemistry and immunohistochemistry. The effects of pretreatment with capsaicin were also examined. In the CP-treated group, the c-fos mRNA as well as protein was significantly induced predominantly in the paraventricular area of the thalamus. The induction of c-fos mRNA exhibited a dose-dependency. The induction of c-fos mRNA of CP-treated mice was significantly inhibited by capsaicin pretreatment to deplete C-fibers. Our results indicate that visceral nociception from the lower urinary tract activates thalamic neurons and this activation is mediated in part through the activation of the capsaicin-sensitive C-fiber afferents. The present findings suggest that the levels of c-fos in the paraventricular area of the thalamus may be a useful marker for evaluating the afferent nerve activity from the lower urinary tract.     

Localization of TRPV1 and P2X3 in unmyelinated and myelinated vagal afferents in the rat

The vagus nerve is dominated by afferent fibers that convey sensory information from the viscera to the brain. Most vagal afferents are unmyelinated, slow-conducting C-fibers, while a smaller portion are myelinated, fast-conducting A-fibers. Vagal afferents terminate in the nucleus tractus solitarius (NTS) in the dorsal brainstem and regulate autonomic and respiratory reflexes, as well as ascending pathways throughout the brain. Vagal afferents form glutamatergic excitatory synapses with postsynaptic NTS neurons that are modulated by a variety of channels. The organization of vagal afferents with regard to fiber type and channels is not well understood. In the present study, we used tract tracing methods to identify distinct populations of vagal afferents to determine if key channels are selectively localized to specific groups of afferent fibers. Vagal afferents were labeled with isolectin B4 (IB4) or cholera toxin B (CTb) to detect unmyelinated and myelinated afferents, respectively. We find that TRPV1 channels are preferentially found in unmyelinated vagal afferents identified with IB4, with almost half of all IB4 fibers showing co-localization with TRPV1. These results agree with prior electrophysiological findings. In contrast, we found that the ATP-sensitive channel P2X3 is found in a subset of both myelinated and unmyelinated vagal afferent fibers. Specifically, 18% of IB4 and 23% of CTb afferents contained P2X3. The majority of CTb-ir vagal afferents contained neither channel. Since neither channel was found in all vagal afferents, there are likely further degrees of heterogeneity in the modulation of vagal afferent sensory input to the NTS beyond fiber type.     

Respiratory sensations evoked by activation of bronchopulmonary C-fibers

C-fibers represent the majority of vagal afferents innervating the airways and lung, and can be activated by inhaled chemical irritants and certain endogenous substances. Stimulation of bronchopulmonary C-fibers with selective chemical activators by either inhalation or intravenous injection evokes irritation, burning and choking sensations in the throat, neck and upper chest (mid-sternum region) in healthy human subjects. These irritating sensations are often accompanied by bouts of coughs either during inhalation challenge or when a higher dose of the chemical activator is administered by intravenous injection. Dyspnea and breathless sensation are not always evoked when these afferents are activated by different types of chemical stimulants. This variability probably reflects the chemical nature of the stimulants, as well as the possibility that different subtypes of C-fibers encoded by different receptor proteins are activated. These respiratory sensations and reflex responses (e.g., cough) are believed to play an important role in protecting the lung against inhaled irritants and preventing overexertion under unusual physiological stresses (e.g., during strenuous exercise) in healthy individuals. More importantly, recent studies have revealed that the sensitivity of bronchopulmonary C-fibers can be markedly elevated in acute and chronic airway inflammatory diseases, probably caused by a sensitizing effect of certain endogenously released inflammatory mediators (e.g., prostaglandin E(2)) that act directly or indirectly on specific ion channels expressed on the sensory terminals. Normal physiological actions such as an increase in tidal volume (e.g., during mild exercise) can then activate these C-fiber afferents, and consequently may contribute, in part, to the lingering respiratory discomforts and other debilitating symptoms in patients with lung diseases.     

Transganglionic degeneration of capsaicin-sensitive C-fiber primary afferent terminals

Local treatment of rat peripheral nerves with capsaicin induces permanent impairment of afferent C-fiber functions and changes in the response properties of spinal dorsal horn neurons. In this study a new experimental approach, the "capsaicin gap" technique, has been utilized in an attempt to unravel pathomorphological alterations which commence in the domain of primary sensory neurons as a consequence of perineural treatment with capsaicin. The technique relies on the facts that peripheral nerves in the spinal dorsal horn are represented in a strict somatotopic manner, and on the observation that in the adult rat systemic injection of appropriate doses of capsaicin results in a selective degeneration of primary afferent fibers within Rexed's laminae I and II of the spinal cord. We have assumed that, if treatment of a peripheral nerve with capsaicin results in an irreversible destruction of primary afferent terminals related to that nerve, then the corresponding spinal projection areas will be free of degeneration following a subsequent systemic injection of capsaicin. The topographical distribution of degenerating axon terminals, induced by a systemic injection of capsaicin, was studied within the lumbar spinal cord of rats whose sciatic nerves had been treated with capsaicin or its vehicle one to three months previously. In the dorsal horn ipsilateral to the vehicle-treated nerve, axon terminal degeneration was found in the entire mediolateral extent of Rexed's laminae I and II. In contrast, in the dorsal horn ipsilateral to the capsaicin-treated nerve, virtually no degeneration was observed in areas which correspond to the projection territories of sciatic afferents. It is concluded that the central terminals of capsaicin-sensitive sciatic afferents underwent transganglionic degeneration as a result of prior perineural treatment with capsaicin, and a subsequent systemic injection of this neurotoxin therefore failed to cause axon terminal degeneration in somatotopic areas served by the treated nerve. Comparative quantitative morphometric analysis of cell populations of dorsal root ganglia related to capsaicin- or vehicle-treated nerves disclosed (1) a marked reduction in the proportion of small-sized neurons, (2) a fall of about 80% in the percentage of neurons which undergo degeneration after the systemic injection of capsaicin, and (3) a marked decrease in the total number of neurons in ganglia ipsilateral to the capsaicin-treated nerves. Quantitative electron microscopic studies on saphenous nerves treated perineurally with capsaicin revealed a 32% reduction in the number of unmyelinated axons as compared with the controls, whereas the number of myelinated fibers was unchanged.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of bronchopulmonary vagal afferent nerves with bradykinin, acid and vanilloid receptor agonists in wild-type and TRPV1–/– mice

The vanilloid receptor TRPV1 (formerly VR1) has been implicated in the activation of nociceptive sensory nerves by capsaicin, noxious heat, protons, bradykinin, cannabinoids such as anandamide, and certain metabolites of arachidonic acid. Using TRPV1 knockout mouse (TRPV1–/–) we address the question of whether TRPV1 is obligatory for action potential discharge in vagal C-fibre terminals evoked by capsaicin, anandamide, acid and bradykinin. The response of a defined subtype of the vagal afferent bronchopulmonary C-fibres (conduction velocity < 0.7 ms⁻¹) to the putative TRPV1 activators was studied in vitro in the mouse isolated/perfused lung–nerve preparation. Capsaicin (1 μ m ) evoked action potential discharge of ∼90% (28/31) of C-fibres in the TRPV1+/+ mice, but failed to activate bronchopulmonary C-fibres in TRPV1–/– animals  ( n = 10)  . Anandamide (3–100 μ m ) induced concentration-dependent activation of capsaicin-sensitive TRPV1+/+ C-fibres with a threshold of 3–10 μ m , but failed to evoke substantive discharge in TRPV1–/– C-fibres. In the TRPV1+/+ mice, the B₂ receptor-mediated activation by bradykinin (1 μ m ) was restricted to the capsaicin-sensitive C-fibres. Bradykinin was effective in evoking B₂ receptor-mediated action potential discharge in TRPV1–/– C-fibres, but the response was significantly ( P < 0.05) less persistent than in TRPV1+/+ C-fibres. Exposing the tissue to acid (pH = 5) excited both TRPV1+/+ and TRPV1–/– C-fibres. We conclude that TRPV1 is obligatory for vagal C-fibre activation by capsaicin and anandamide. By contrast, whereas TRPV1 may have a modulatory role in bradykinin and acid-induced activation of bronchopulmonary C-fibres, it is not required for action potential discharge evoked by these stimuli.     

Plasticity of nodose ganglion neurons after capsaicin- and vagotomy-induced nerve damage in adult rats

Previous reports show that vagal afferent innervation of the stomach eventually regenerates from surviving nodose ganglion (NG) neurons after subdiaphragmatic vagotomy. Systemic capsaicin treatment destroys gastric vagal afferent neurons expressing vanilloid receptor 1 (VR1). However, it is not known whether gastric innervation lost after neuronal destruction can be restored. Here, we report that capsaicin-induced damage of NG neurons innervating the stomach in adult rats is followed by restoration of vagal afferent projections. Specifically, we compared measures of neuronal plasticity in NG and vagi after subdiaphragmatic vagotomy or capsaicin treatment. The numbers of VR1-immunoreactive neurons projecting to the stomach were significantly reduced 10 days after either capsaicin treatment or vagotomy. However, the VR1-immunoreactive afferent innervation of the stomach was restored to levels exceeding those of vagotomized rats by 37 days after capsaicin, whereas neither total afferent innervation nor VR1-immunoreactive innervation reached control levels, even by 67 days after vagotomy. Capsaicin treatment significantly increased NG neuronal nitric oxide synthase (nNOS) immunoreactivity at 10 days after capsaicin, and this increase was sustained for the duration of the study, indicating higher nNOS demand in restoration of vagal projections. Vagotomy was associated with a much smaller increase in the number of nNOS-immunoreactive NG neurons, detectable only at 10 days after surgery. The number of nNOS-immunopositive gastric-projecting neurons was dramatically reduced 10 days after either capsaicin treatment or vagotomy but returned to the control level in both groups at 67 days. We found a significantly higher number of growth cones in capsaicin-treated animals compared with controls. Capsaicin significantly increased the number of nNOS-immunopositive and nNOS-immunonegative growth cones in NG at all time points. Vagotomy did not increase the number of nNOS⁻ growth cones in NG. We conclude that capsaicin treatment may result in more significant restorative capacities than vagotomy, mainly because of sprouting of capsaicin-insensitive nerve fibers.     

Absence of mechanical hyperalgesia after exercise (delayed onset muscle soreness) in neonatally capsaicin-treated rats

Delayed onset muscle soreness (DOMS) appears with some delay after unaccustomed, strenuous exercise, especially after lengthening contraction (LC). It is characterized by tenderness and movement related pain, namely muscular mechanical hyperalgesia. To clarify the involvement of C-fibers in this mechanical hyperalgesia, we examined whether DOMS could be induced in rats treated neonatally with capsaicin. We confirmed that a large portion of unmyelinated afferent fibers were lost in capsaicin treated rats. In these animals, LC failed to induce muscular mechanical hyperalgesia. mRNA of nerve growth factor (NGF) in the muscle, which plays a pivotal role in maintaining mechanical hyperalgesia, was upregulated in the capsaicin treated animals similar to the vehicle treated animals. These results demonstrate that C-fiber afferents are essential in transmitting the nociceptive information from exercised muscle in DOMS.     

C-fiber depletion alters response properties of neurons in trigeminal nucleus principalis

The effects of C-fiber depletion induced by neonatal capsaicin treatment on the functional properties of vibrissa-sensitive low-threshold mechanoreceptive (LTM) neurons in the rat trigeminal nucleus principalis were examined in adult rats. Neonatal rats were injected either with capsaicin or its vehicle within 48 h of birth. The depletion of unmyelinated afferents was confirmed by the significant decrease in plasma extravasation of Evan's blue dye induced in the hindlimb skin of capsaicin-treated rats by cutaneous application of mustard oil and by the significant decrease of unmyelinated fibers in both the sciatic and infraorbital nerves. The mechanoreceptive field (RF) and response properties of 31 vibrissa-sensitive neurons in capsaicin-treated rats were compared with those of 32 vibrissa-sensitive neurons in control (untreated or vehicle-treated) rats. The use of electronically controlled mechanical stimuli allowed quantitative analysis of response properties of vibrissa-sensitive neurons; these included the number of center- and surround-RF vibrissae within the RF (i.e., those vibrissae which when stimulated elicited >/=1 and <1 action potential per stimulus, respectively), the response magnitude and latency, and the selectivity of responses to stimulation of vibrissae in different directions with emphasis on combining both the response magnitude and direction of vibrissal deflection in a vector analysis. Neonatal capsaicin treatment was associated with significant increases in the total number of vibrissae, in the number of center-RF vibrissae per neuronal RF, and in the percentage of vibrissa-sensitive neurons that also responded to stimulation of other types of orofacial tissues. Compared with control rats, capsaicin-treated rats showed significant increases in the response magnitude to stimulation of surround-RF vibrissae as well as in response latency variability to stimulation of both center- and surround-RF vibrissae. C-fiber depletion also significantly altered the directional selectivity of responses to stimulation of vibrissae. For neurons with multiple center-RF vibrissae, the proportion of center-RF vibrissae with net vector responses oriented toward the same quadrant was significantly less in capsaicin-treated compared with control rats. These changes in the functional properties of principalis vibrissa-sensitive neurons associated with marked depletion of C-fiber afferents are consistent with similarly induced alterations in LTM neurons studied at other levels of the rodent somatosensory system, and indeed may contribute to alterations previously described in the somatosensory cortex of adult rodents. Furthermore, these results provide additional support to the view that C fibers may have an important role in shaping the functional properties of LTM neurons in central somatosensory pathways.     

Role of capsaicin-sensitive primary afferent inputs from the masseter muscle in the C<Subscript>1</Subscript> spinal neurons responding to tooth-pulp stimulation in rats

The aim of the present study was to demonstrate the convergence of inputs from masseter muscle (MM) and tooth pulp (TP) onto C₁ spinal neurons and to determine whether the afferent fibers express the functional vanilloid receptor (VR1). Extracellular single-unit recordings were made from 61 C₁ units responding to TP electrical stimulation with a constant temporal relationship to a digastric electromyogram signal in pentobarbital anesthetized rats. Eighty-four percent of C₁ neurons responding to TP stimulation also responded to the ipsilateral MM stimulation. Of these neurons, 61% were considered to be afferent inputs from A-fibers and the remaining units (39%) were C-fibers, based on calculation of the nerve conduction velocity. Intramuscular injection of capsaicin (0.05 and 0.1%) produced a reduction in a MM-induced C₁ neuronal activity in a dose-dependent manner and this effect was antagonized by pretreatment with an antagonist of VR1, capsazepine. Some of these units were also excited by noxious heat stimulation (>43°C). The trigeminal root ganglion (TRG) neurons that innervated the MM were retrogradely labeled with Fluorogold (FG) and the small-diameter FG-labeled TRG neurons expressed the immunoreactivity for VR1. After intramuscular mustard oil injection (noxious chemical stimulation), the C₁ neuronal activity induced by both touch and pinch stimuli was enhanced and their receptive field sizes were significantly expanded. These changes were reversed within 15–20 min. These results suggest that there may be the convergence of noxious afferents inputs from the MM and TP afferents on the same C₁ neurons in rats, and that the afferent fibers expressing the functional VR1 may contribute to the hyperalgesia and/or referred pain associated with temporomandibular joint disorder.