Responses of neurons in rostral ventrolateral medulla to activation of cardiac receptors in rats

Ischemic stimulation of cardiac receptors reflexly excites the cardiovascular system. However, the supraspinal mechanisms involved in this reflex are not well defined. This study examined the responses of barosensitive neurons in the rostral ventrolateral medulla (RVLM) to stimulation of cardiac receptors and the afferent pathways involved in these responses. Single-unit activity of RVLM neurons was recorded in alpha-chloralose-anesthetized rats. Cardiac receptors were stimulated by epicardial application of 10 microg/ml of bradykinin (BK). Barosensitive neurons were silenced by stimulation of baroreceptors. Application of BK increased the mean arterial pressure from 65.2 +/- 1.9 to 89.3 +/- 2.9 mmHg and excited RVLM barosensitive neurons from 6.2 +/- 0.7 to 10.7 +/- 0.9 impulses/s (P < 0.05, n = 40). BK had no effect on 21 nonbarosensitive neurons. Blockade of stellate ganglia abolished the response of barosensitive neurons to BK. Cervical vagotomy significantly increased the baseline discharges of RVLM barosensitive neurons but had no effect on their responses to BK. Thus this study indicates that stimulation of cardiac receptors selectively activates RVLM barosensitive neurons through sympathetic afferent pathways. This information suggests that the RVLM barosensitive neurons are likely involved in the sympathetic control of circulation during myocardial ischemia.     

Responses of spinohypothalamic tract neurons in the thoracic spinal cord of rats to somatic stimuli and to graded distention of the bile duct

Anatomical studies indicate that a relatively large percentage of spinohypothalamic tract (SHT) neurons are located within thoracic spinal segments. The aim of this study was to characterize the responses of SHT neurons in these segments of rats to innocuous and noxious stimulation of the skin and of a visceral structure, the bile duct. In addition, we attempted to determine the trajectories of the axons of the examined neurons within the diencephalon and brainstem. Fifty-three SHT neurons were recorded within segments T8-T13 in urethane anesthetized rats. Each cell was antidromically activated using current pulses < or = 30 microA delivered from the tip of an electrode located within the contralateral hypothalamus. The recording points were located in the superficial dorsal horn (9) and deep dorsal horn (44). All examined SHT neurons had receptive fields on the posterior thorax and anterior and ventral abdomen of the ipsilateral side. Ninety percent of the 41 SHT neurons responded exclusively (13) or preferentially (24) to noxious cutaneous stimuli. Thirteen of 27 (48%) examined units were activated by forceful distention of the bile duct. Response thresholds ranged from 30 to 40 mmHg. Responses incremented as pressures were increased to 50-80 mmHg. The axons of 22 of 28 (79%) examined SHT neurons appeared to cross the midline within the hypothalamus and terminate in the ipsilateral hypothalamus, thalamus or midbrain. The results indicate that SHT neurons in thoracic spinal cord of rats are capable of conveying somatic and visceral nociceptive information from the bile duct directly to targets at various levels of the brain bilaterally.     

Chemical activation of C(1)-C(2) spinal neurons modulates activity of thoracic respiratory interneurons in rats

Discharge patterns of thoracic dorsal horn neurons are influenced by chemical activation of cell bodies in cervical spinal segments C(1)-C(2). The present aim was to examine whether such activation would specifically affect thoracic respiratory interneurons (TRINs) of the deep dorsal horn and intermediate zone in pentobarbital sodium-anesthetized, paralyzed, artificially ventilated rats. We also characterized discharge patterns and pathways of TRIN activation in rats. A total of 77 cells were classified as TRINs by location, continued burst activity related to phrenic discharge when the respirator was stopped, and lack of antidromic response from selected pathways. A variety of respiration-phased discharge patterns was documented whose pathways were interrupted by ipsilateral C(1) transection. Glutamate pledgets (1 M, 1 min) on the dorsal surface of the spinal cord inhibited 22/49, excited 15/49, or excited/inhibited 3/49 tested cells. Incidence of responses did not depend on whether the phase of TRIN discharge was inspiratory, expiratory, or biphasic. Phrenic nerve activity was unaffected by chemical activation of C(1)-C(2) in this preparation. Besides supraspinal input, TRIN activity may be influenced by upper cervical modulatory pathways.     

The glutamatergic nature of TRPV1-expressing neurons in the spinal dorsal horn

The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary afferent terminals and spinal dorsal horn neurons. However, the neurochemical phenotypes and functions of TRPV1-expressing post-synaptic neurons in the spinal cord are not clear. In this study, we tested the hypothesis that TRPV1-expressing dorsal horn neurons are glutamatergic. Immunocytochemical labeling revealed that TRPV1 and vesicular glutamate transporter-2 were colocalized in dorsal horn neurons and their terminals in the rat spinal cord. Resiniferatoxin (RTX) treatment or dorsal rhizotomy ablated TRPV1-expressing primary afferents but did not affect TRPV1- and vesicular glutamate transporter-2-expressing dorsal horn neurons. Capsaicin significantly increased the frequency of glutamatergic spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in almost all the lamina II neurons tested in control rats. In RTX-treated or dorsal rhizotomized rats, capsaicin still increased the frequency of spontaneous excitatory post-synaptic currents and miniature excitatory post-synaptic currents in the majority of neurons examined, and this effect was abolished by a TRPV1 blocker or by non-NMDA receptor antagonist. In RTX-treated or in dorsal rhizotomized rats, capsaicin also produced an inward current in a subpopulation of lamina II neurons. However, capsaicin had no effect on GABAergic and glycinergic spontaneous inhibitory post-synaptic currents of lamina II neurons in RTX-treated or dorsal rhizotomized rats. Collectively, our study provides new histological and functional evidence that TRPV1-expressing dorsal horn neurons in the spinal cord are glutamatergic and that they mediate excitatory synaptic transmission. This finding is important to our understanding of the circuitry and phenotypes of intrinsic dorsal horn neurons in the spinal cord.     

Intraesophageal chemicals enhance responsiveness of upper thoracic spinal neurons to mechanical stimulation of esophagus in rats

Esophageal hypersensitivity is one of the most common causes of noncardiac chest pain in patients. In this study, we investigated whether exposure of the esophagus to acid and other chemical irritants affected activity of thoracic spinal neurons responding to esophageal distension (ED) in rats. Extracellular potentials of single thoracic (T3) spinal neurons were recorded in pentobarbital sodium-anesthetized, -paralyzed, and -ventilated male rats. ED (0.2 or 0.4 ml, 20 s) was produced by water inflation of a latex balloon placed orally into the middle thoracic region of the esophagus. The chemicals were administered via a tube that was passed through the stomach and placed in the thoracic esophagus. To irritate the esophagus, 0.2 ml of HCl (0.01 N), bradykinin (10 microg/ml), or capsaicin (10 microg/ml) were injected for 1-2 min. Only neurons excited by ED were included in this study. Results showed that intraesophageal instillation of HCl, bradykinin, and capsaicin increased activity in 3/20 (15%), 7/25 (28%), and 9/20 (45%) neurons but enhanced excitatory responses to ED in 9/17 (53%), 8/15 (53%), and 7/11 (64%) of the remaining spinal neurons, respectively. Furthermore, intraesophageal chemicals were more likely to enhance the responsiveness of low-threshold neurons than high-threshold neurons to the esophageal mechanical stimulus. Normal saline (pH 7.4, 0.2 ml) or vehicle instilled in the esophagus did not significantly affect activity or ED responses of neurons. We conclude that enhanced responses of thoracic spinal neurons to ED by the chemically challenged esophagus may provide a possible pathophysiological basis for visceral hypersensitivity in patients with gastroesophageal reflux and/or esophagitis.     

Chemical activation of C1-C2 spinal neurons modulates intercostal and phrenic nerve activity in rats

Chemical activation of upper cervical spinal neurons modulates activity of thoracic respiratory interneurons in rats. The aim of the present study was to examine the effects of chemical activation of C(1)-C(2) spinal neurons on thoracic spinal respiratory motor outflows. Electroneurograms of left phrenic (n = 23) and intercostal nerves (ICNs, n = 93) between T(3) and T(8) spinal segments were recorded from 36 decerebrated, vagotomized, paralyzed, and ventilated male rats. To activate upper cervical spinal neurons, glutamate pledgets (1 M, 1 min) were placed on the dorsal surface of the C(1)-C(2) spinal cord. Glutamate on C(1)-C(2) increased ICN tonic activity in 56/59 (95%) ICNs. The average maximal tonic activity of ICN was increased by 174% (n = 59). After spinal transection at rostral C(1), glutamate on C(1)-C(2) still increased ICN tonic activity in 33/35 ICNs. However, the effects of C(1)-C(2) glutamate on ICN phasic activity were highly variable, with observations of augmentation or suppression of both inspiratory and expiratory discharge. C(1)-C(2) glutamate augmented the average amplitude of phrenic burst by 20%, whereas the increases in amplitude of ICN inspiratory activity, when they occurred, averaged 120%. The burst rate of phrenic nerve discharge was decreased from 34.2 +/- 1.6 to 26.3 +/- 2.0 (mean +/- SE) breaths/min during C(1)-C(2) glutamate. These data suggested that upper cervical propriospinal neurons might play a role in descending modulation of thoracic respiratory and nonrespiratory motor activity.     

Induction of TRPV1 desensitization by a biased receptor agonist

Selective suppression of hyperactive sensory neurons is an attractive strategy for managing pathological pain. Blocking Na⁺ channels to eliminate action potentials and desensitizing transduction channels can both reduce sensory neuron excitability. The novel synthetic vanilloid ligand cap-ET preserves agonist activation of intracellular Ca²⁺ signals and large organic cation transport but loses effective electric current induction. Cap-ET can therefore be used to deliver the membrane impermeable Na⁺ channel blocker QX-314 to substantially inhibit voltage-activated Na⁺ currents. We explored, besides facilitating entry of organic cationic therapeutics, whether cap-ET can also produce receptor desensitization similar to the natural agonist capsaicin. Using the YO-PRO-1 based fluorescent dye uptake assay, we found that cap-ET effectively triggered Ca²⁺ dependent desensitization of TRPV1 when the receptor was pre-sensitized with the surrogate oxidative chemical phenylarsine oxide (PAO), suggesting an alternative use of permanently charged cationic capsaicinoids in differential neuronal silencing.     

Induction of TRPV1 desensitization by a biased receptor agonist

Selective suppression of hyperactive sensory neurons is an attractive strategy for managing pathological pain. Blocking Na(+) channels to eliminate action potentials and desensitizing transduction channels can both reduce sensory neuron excitability. The novel synthetic vanilloid ligand cap-ET preserves agonist activation of intracellular Ca(2+) signals and large organic cation transport but loses effective electric current induction. Cap-ET can therefore be used to deliver the membrane impermeable Na(+) channel blocker QX-314 to substantially inhibit voltage-activated Na(+) currents. We explored, besides facilitating entry of organic cationic therapeutics, whether cap-ET can also produce receptor desensitization similar to the natural agonist capsaicin. Using the YO-PRO-1 based fluorescent dye uptake assay, we found that cap-ET effectively triggered Ca(2+) dependent desensitization of TRPV1 when the receptor was pre-sensitized with the surrogate oxidative chemical phenylarsine oxide (PAO), suggesting an alternative use of permanently charged cationic capsaicinoids in differential neuronal silencing.     

Non-peptidergic primary afferents are presynaptic to neurokinin-1 receptor immunoreactive lamina I projection neurons in rat spinal cord

ABSTRACT: 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.     

Role of TRPV1 and intracellular Ca2+ in excitation of cardiac sensory neurons by bradykinin

Bradykinin is an important mediator produced during myocardial ischemia and infarction that can activate and/or sensitize cardiac spinal (sympathetic) sensory neurons to trigger chest pain. Because a long-onset latency is associated with the bradykinin effect on cardiac spinal afferents, a cascade of intracellular signaling events is likely involved in the action of bradykinin on cardiac nociceptors. In this study, we determined the signal transduction mechanisms involved in bradykinin stimulation of cardiac nociceptors. Cardiac dorsal root ganglion (DRG) neurons in rats were labeled by intracardiac injection of a fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI). Whole cell current-clamp recordings were performed in acutely isolated DRG neurons. In DiI-labeled DRG neurons, 1 microM bradykinin significantly increased the firing frequency and lowered the membrane potential. Iodoresiniferatoxin, a highly specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, significantly reduced the excitatory effect of bradykinin. Furthermore, the stimulating effect of bradykinin on DiI-labeled DRG neurons was significantly attenuated by baicalein (a selective inhibitor of 12-lipoxygenase) or 2-aminoethyl diphenylborinate [an inositol 1,4,5-trisphosphate (IP(3)) antagonist]. In addition, the effect of bradykinin on cardiac DRG neurons was abolished after the neurons were treated with BAPTA-AM or thapsigargin (to deplete intracellular Ca(2+) stores) but not in the Ca(2+)-free extracellular solution. Collectively, these findings provide new evidence that 12-lipoxygenase products, IP(3), and TRPV1 channels contribute importantly to excitation of cardiac nociceptors by bradykinin. Activation of TRPV1 and the increase in the intracellular Ca(2+) are critically involved in activation/sensitization of cardiac nociceptors by bradykinin.     

N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.     

Myocardial ischemia induces the release of substance P from cardiac afferent neurons in rat thoracic spinal cord

Antibody-coated microprobes were inserted into the thoracic (T3-4) spinal cord in urethane-anesthetized Sprague-Dawley rats to detect the differences in the release of immunoreactive substance P-like (irSP) substances in response to differential activation of cardiac nociceptive sensory neurons (CNAN). CNAN were stimulated either by intrapericardial infusion of an inflammatory ischemic exudate solution (IES) containing algogenic substances (i.e., 10 mM each of adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine), or by transient occlusion of the left anterior descending coronary artery (CoAO). There was widespread basal release of irSP from the thoracic spinal cord. Stimulation of the CNAN by IES did not alter the pattern of release of irSP. Conversely, CoAO augmented the release of irSP from T3-4 spinal segments from laminae I-VII. This CoAO-induced irSP release was eliminated after thoracic dorsal rhizotomy. These results indicate that heterogeneous activation of cardiac afferents, as with focal coronary artery occlusion, represents an optimum input for activation of the cardiac neuronal hierarchy and for the resultant perception of angina. Excessive stimulation of cardiac nociceptive afferent neurons elicited during regional coronary artery occlusion involves the release of SP in the thoracic spinal cord and suggests that local spinal cord release of SP may be involved in the neural signaling of angina.     

c-Fos expression in rat brain stem and spinal cord in response to activation of cardiac ischemia-sensitive afferent neurons and electrostimulatory modulation

The purpose of this study was to identify central neuronal sites activated by stimulation of cardiac ischemia-sensitive afferent neurons and determine whether electrical stimulation of left vagal afferent fibers modified the pattern of neuronal activation. Fos-like immunoreactivity (Fos-LI) was used as an index of neuronal activation in selected levels of cervical and thoracic spinal cord and brain stem. Adult Sprague-Dawley rats were anesthetized with urethane and underwent intrapericardial infusion of an "inflammatory exudate solution" (IES) containing algogenic substances that are released during ischemia (10 mM adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine) or occlusion of the left anterior descending coronary artery (CoAO) to activate cardiac ischemia-sensitive (nociceptive) afferent fibers. IES and CoAO increased Fos-LI above resting levels in dorsal horns in laminae I-V at C2 and T4 and in the caudal nucleus tractus solitarius. Dorsal rhizotomy virtually eliminated Fos-LI in the spinal cord as well as the brain stem. Neuromodulation of the ischemic signal by electrical stimulation of the central end of the left thoracic vagus excited neurons at the cervical and brain stem level but inhibited neurons at the thoracic spinal cord during IES or CoAO. These results suggest that stimulation of the left thoracic vagus excites descending inhibitory pathways. Inhibition at the thoracic spinal level that suppresses the ischemic (nociceptive) input signal may occur by a short-loop descending pathway via signals from cervical propriospinal circuits and/or a longer-loop descending pathway via signals from the nucleus tractus solitarius.     

Responses of rat spinal ganglion neurons mediated by activation of serotonin receptors of the third type

Application of serotonin (5-hydroxytryptamine; 5-HT) to rat dorsal root ganglion neurons under conditions in which potassium conductance was blocked by cesium ions elicited depolarizing responses followed by an increase in membrane conductance. The responses did not exhibit desensitization and were due to activation of 5-HT receptors of the third type (5-HT₃Rs), since they were insensitive to methysergide, the 5-HT₂R antagonist, but were inhibited by tropicetrone (ISC 205–930) and metoclopramide, the 5-HT₃R antagonists. The reversal potential of the 5-HT-induced depolarizing responses was –11.9 mV; their amplitude decreased following a decrease in extracellular Na⁺ concentration but remained constant after intracellular injection of GTP. The amplitude of the responses increased following elevation of intracellular cAMP concentration caused by theophylline or sodium fluoride whose potentiating effect was reduced by butamide, a protein kinase A inhibitor. Potentiation of the 5-HT-induced responses was also produced by increased intracellular Ca²⁺ concentration following either direct intracellular injections or a burst of action potentials. The potentiation could be prevented by trifluoroperazine, the calmodulin inhibitor. The 5-HT effects were also potentiated by methylfurmetide, an activator of muscarinic acetylcholine receptors. The effect of methylfurmetide was slightly decreased by trifluoroperazine and was markedly decreased by polymixin B, a protein kinase C inhibitor. The effects of 5-HT were also enhanced by ethanol.Neirofiziologiya/Neurophysiology, Vol. 25, pp. 258–263, July–August, 1993.     

TRPV1 acts as proton channel to induce acidification in nociceptive neurons

The low extracellular pH of inflamed or ischemic tissues enhances painful sensations by sensitizing and activating the vanilloid receptor 1 (TRPV1). We report here that activation of TRPV1 results in a marked intracellular acidification in nociceptive dorsal root ganglion neurons and in a heterologous expression system. A characterization of the underlying mechanisms revealed a Ca(2+)-dependent intracellular acidification operating at neutral pH and an additional as yet unrecognized direct proton conductance through the poorly selective TRPV1 pore operating in acidic extracellular media. Large organic cations permeate through the activated TRPV1 pore even in the presence of physiological concentrations of Na(+), Mg(2+), and Ca(2+). The wide pore and the unexpectedly high proton permeability of TRPV1 point to a proton hopping permeation mechanism along the water-filled channel pore. In acidic media, the high relative proton permeability through TRPV1 defines a novel proton entry mechanism in nociceptive neurons.     

Responses of spontaneously active spinal neurons to temperature stimulation of the skin

Changes in spontaneous unit activity in the dorsal zones of the spinal cord in response to temperature stimulation of the skin of the thigh and leg were investigated in acute experiments on cats lightly anesthetized with pentobarbital. Two groups of neurons were distinguished by the character of their response. The firing rate of the first group of neurons was changed only in response to cold or warmth. The neurons of the second group responded in opposite ways to the two types of stimulation. Frequency characteristics of spontaneous activity of temperature-sensitive units located in both white and gray matter were determined. In all the lumbar segments their depth was not below the 5th layer of gray matter. Mechanisms of the changes in spontaneous activity and the possible pathways of conduction of temperature sensation are discussed.     

Fibronectin stimulates TRPV1 translocation in primary sensory neurons

Extracellular matrix (ECM) molecules are highly variable in their composition and receptor recognition. Their ubiquitous expression profile has been linked to roles in cell growth, differentiation, and survival. Recent work has identified certain ECM molecules that serve as dynamic signal modulators, versus the more-recognized role of chronic modulation of signal transduction. In this study, we investigated the role that fibronectin (FN) plays in the dynamic modulation of transient receptor potential family V type 1 receptor (TRPV1) translocation to the plasma membrane in trigeminal ganglia (TG) sensory neurons. Confocal immunofluorescence analyses identify co-expression of the TRPV1 receptor with integrin subunits that bind FN. TG neurons cultured upon or treated with FN experienced a leftward shift in the EC₅₀ of capsaicin-stimulated neuropeptide release. This FN-induced increase in TRPV1 sensitivity to activation is coupled by an increase in plasma membrane expression of TRPV1, as well as an increase in tyrosine phosphorylation of TRPV1 in TG neurons. Furthermore, TG neurons cultured on FN demonstrated an increase in capsaicin-mediated Ca²⁺ accumulation relative to neurons cultured on poly- d -lysine. Data presented from these studies indicate that FN stimulates tyrosine-phosphorylation-dependent translocation of the TRPV1 receptor to the plasma membrane, identifying FN as a critical component of the ECM capable of sensory neuron sensitization.