Taurine-activated chloride currents in the rat sacral dorsal commissural neurons

The electrophysiological and pharmacological properties of taurine (Tau)-activated Cl⁻ currents (I_Tau) were investigated in the dissociated rat sacral dorsal commissural nucleus (SDCN) neurons using the nystatin perforated patch recording configuration under voltage-clamp conditions. The reversal potential of I_Tau was close to the Cl⁻ equilibrium potential. The I_Tau was not affected by a preceding GABA response but cross-desensitized by a preceding glycine (Gly) response. Strychnine (STR), picrotoxin (PIC), bicuculline (BIC) and Zn²⁺ suppressed the I_Tau in a concentration-dependent manner. The pharmacology of the I_Tau and Gly-induced response (I_Gly) was similar, though Zn²⁺ inhibition on I_Tau differed from that on I_Gly in being much slower in recovery. Serotonin potentiated the I_Tau via protein kinase C. The results indicate that both Tau and Gly act on a strychnine-sensitive site to open the same Cl⁻ channels in the SDCN neurons, and suggest that Tau may act as a functional neurotransmitter in the mammalian SDCN.     

依托咪酯激活大鼠骶髓后连合核神经元GABAA门控氯通道电流

目的　 研究依托咪酯 (etomidate ,ET)在急性分离的大鼠骶髓后连合核 (sacraldorsalcommissuralnucleus,SDCN)神经元的药理学特性。方法　采用制霉菌素穿孔膜片钳全细胞记录。结果　在大鼠SDCN神经元 ,ET(10～ 30 0 0 μmol/L)在钳制电位为 - 40mV时 ,可引起内向电流 (IET) ,EC50 为 (33.35± 3.0 7) μmol/L ,该电流可被GABAA受体拮抗剂荷包牡丹碱 (bicuculline)及氯通道阻滞剂印防己毒素 (picrotoxin)以浓度依赖方式所阻断。结论　ET在大鼠SDCN神经元可通过作用于GABAA受体而诱发氯通道电流 ,此作用可能和全麻状态下脊髓水平的麻醉效应有关     

Adenosine suppresses GABAA receptor-mediated responses in rat sacral dorsal commissural neurons

The modulatory effect of adenosine on gamma-aminobutyric acid (GABA)-activated whole-cell currents were investigated in the neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under the voltage-clamp conditions. The results showed that: (1) GABA acted on GABAA receptor and elicited inward Cl- currents (IGABA) at a holding potential (VH) of -40 mV; (2) adenosine suppressed GABA-induced Cl- current without affecting the reversal potential of IGABA and the apparent affinity of GABA to its receptor; (3) N6-cyclohexyladenosine mimicked the suppression effect of adenosine on IGABA, whereas 8-cyclopentyl-1,3-dipropylxanthine blocked the suppression effect of adenosine; (4) adenosine fails to suppress IGABA on the neurons that were pretreated with bisindolylmaleimide I (BIM), while after pretreatment with H-89, the inhibitory effect of adenosine on IGABA were not affected; (5) the suppression effect of adenosine on IGABA remained in the presence of BAPTA-AM. The present results indicate that the suppression of adenosine on IGABA is mediated by adenosine A1 receptor and through a Ca2+-independent protein kinase C transduction pathway, and that the interactions between adenosine and GABA might participate in the modulation of nociceptive information transmission at the SDCN.     

Long-range oscillatory Ca2+ waves in rat spinal dorsal horn

Synchronous activity of large populations of neurons shapes neuronal networks during development. However, re-emergence of such activity at later stages of development could severely disrupt the orderly processing of sensory information, e.g. in the spinal dorsal horn. We used Ca2+ imaging in spinal cord slices of neonatal and young rats to assess under which conditions synchronous activity occurs in dorsal horn. No spontaneous synchronous Ca2+ transients were detected. However, increasing neuronal excitability by application of 4-aminopyridine after pretreatment of the slice with blockers of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, gamma-aminobutyric acid (GABA)(A) and glycine receptors evoked repetitive Ca2+ waves in dorsal horn. These waves spread mediolaterally with a speed of 1.0 +/- 0.1 mm/s and affected virtually every dorsal horn neuron. The Ca2+ waves were associated with large depolarizing shifts of the membrane potential of participating neurons and were most likely synaptically mediated because they were abolished by blockade of action potentials or N-methyl-D-aspartate (NMDA) receptors. They were most pronounced in the superficial dorsal horn and absent from the ventral horn. A significant proportion of the Ca2+ waves spread to the contralateral dorsal horn. This seemed to be enabled by disinhibition as primary afferent-induced dorsal horn excitation crossed the midline only when GABA(A) and glycine receptors were blocked. Interestingly, the Ca2+ waves occurred under conditions where AMPA/kainate receptors were blocked. Thus, superficial dorsal horn NMDA receptors are able to sustain synchronous neuronal excitation in the absence of functional AMPA/kainate receptors.     

大鼠脑干SP能神经元向腰骶髓后连合核的投射

采用荧光金逆行标记和免疫荧光组织化学技术,观察了大鼠脑干ＳＰ能神经元向腰骶髓后连合核（ＤＣＮ）的投射状况。将ＦＧ压力注入ＤＣＮ后,脑干中缝苍白核、中缝隐核、中缝大核和巨细胞网状核α部腹外侧部的一些ＦＧ逆标神经元呈现ＳＰ样免疫反应阳性。结果表明,脑干的ＳＰ能下行纤维可终止于脊髓ＤＣＮ区。     

Beta-alanine acts on glycine receptors in the rat sacral dorsal commissural neurons

Whole-cell current responses to bath application of beta-alanine (beta-ALA) were investigated in neurons acutely dissociated from the rat sacra! dorsal commissural nucleus (SDCN) using the nystatin perforated patch recording configuration under voltage-clamp conditions. beta-ALA activated inward currents in a concentration-dependent manner over the range of 10-3000 microM with an EC50 of 80.8 microM. The reversal potential of beta-ALA-activated currents (I beta-ALA) was close to the Cl- equilibrium potential. Strychnine and the chloride channel blocker picrotoxin suppressed the I beta-ALA in a concentration-dependent manner with the IC50 values of 0.19 microM and 343.6 microM, respectively. At the concentration of 3 microM, strychnine was sufficient to completely block 100 microM beta-ALA response, whereas it did not show a suppression of GABA response. In contrast, bicuculline, a potent GABAA receptor antagonist, at concentrations up to 10 microM, a dose that could block the GABA response completely, had little or no effect on I beta-ALA. Furthermore, the I beta-ALA was not affected by a preceding GABA response, but rather cross-desensitized with that evoked by glycine. The results indicate that beta-ALA activates the strychnine-sensitive glycine receptors in the SDCN neurons, and suggest that beta-ALA may act as a functional neurotransmitter in the mammalian SDCN.     

Potentiation of glycine responses by dideoxyforskolin and tamoxifen in rat spinal neurons

Dideoxyforskolin, a forskolin analogue unable to stimulate adenylate cyclase, and tamoxifen, an antioestrogen widely used against breast cancer, are both known to block some Cl- channels. Their effects on Cl- responses to glycine or GABA have been tested here by using whole-cell recording from cultured spinal neurons. Dideoxyforskolin (4 or 16 microm) and tamoxifen (0.2-5 microm) both potentiate responses to low glycine concentrations. They also induce blocking effects, predominant at high glycine concentrations. At 5 microm, tamoxifen increased responses to 15 microm glycine by a factor >4.5, reaching 20 in some neurons. Potentiation by extracellular dideoxyforskolin or tamoxifen persisted after intracellular application of the modulator and was not due to Zn2+ contamination. Potentiation by tamoxifen also persisted in a Ca2+-free extracellular solution, after intracellular Ca2+ buffering and protein kinase C blockade. Thus, the critical sites of action are not intracellular. The EC50 for glycine was lowered 6.6-fold by 5 microm tamoxifen. The kinetics and voltage-dependence of the effects of tamoxifen on glycine responses support the idea that this hydrophobic drug may act from a site located within the membrane. Tamoxifen (5 micro m) also increased responses to 2 micro m GABA by a factor of 3.5, but barely affected peak responses to 20 microm GABA. The demonstration that tamoxifen affects some of the main inhibitory receptors should be useful for better evaluating its neurological effects. Furthermore, the results identify a new class of molecules that potentiate glycine receptor function.     

Plasticity of synaptic inhibition in mouse spinal cord lamina II neurons during early postnatal development and after inactivation of the glycine receptor α3 subunit gene

Synaptic inhibition mediated by GABA_A receptors and glycine receptors (GlyRs) in the outer laminae of the spinal cord dorsal horn efficiently filters ascending nociceptive messages, controlling pathological pain symptoms. However, although many studies have utilized transgenic models to study spinal nociceptive processing, very little is known about the development of functional inhibitory synapses onto these interneurons in mice. Here we report that most interneurons in lamina II are placed under phasic control by both GABAergic and glycinergic synapses, a number of which exhibit dual GABA/glycine co‐release. A developmental switch is also apparent: a subpopulation of lamina II interneurons controlled exclusively by either GABAergic or glycinergic synapses becomes detectable only after postnatal days 15 and 21, respectively. Using mice older than postnatal day 21, we also characterized the plastic changes in glycinergic transmission resulting from the inactivation of the GlyR α3 subunit gene, a key player in inflammatory pain pathways. This allowed us to demonstrate that synapses containing GlyR α3 contribute in large part to synaptic inhibition in lamina II. In Glra3 knockout mice, we found that synaptic currents at the remaining glycinergic synapses, containing GlyR α1, showed faster decay kinetics with unchanged amplitudes but increased frequency. These findings explain the absence of any basal nociceptive hypersensitivity in Glra3 knockout mice, as GlyR α1 is still available for mediating synaptic inhibition at lamina II synapses, but cannot be modulated by the prostaglandin–E‐prostanoid type 2 (EP2) receptor–protein kinase A signalling cascade. Our results clearly demonstrate that presynaptic GABA/glycine release properties are influenced by the nature and complexity of postsynaptic inhibitory receptor subtypes.     

Proximal colon distention increases Fos expression in the lumbosacral spinal cord and activates sacral parasympathetic NADPHd-positive neurons in rats

Fos expression induced by nociceptive mechanical distention of the proximal colon was examined in the lumbosacral spinal cord in freely moving rats equipped with a chronic balloon in the proximal colon. Fos protein in lumbosacral neurons was detected immunocytochemically, and colocalization with nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) activity was determined histochemically at 1 hour after distention. Distention of the proximal colon (10 ml, 30 seconds on/off for 10 minutes, about 90 mm Hg) increased the number of Fos-positive cells in the lumbar 6 (L6) and sacral 1 and 2 (S1, S2) segments, whereas no change was observed in the L1–L5 and S3 segments compared with the sham distended group or with animals that received no treatment. In L6–S2 segments, Fos-positive neurons were increased by two-fold in laminae I-VII (mainly in laminae I and outer II) and area X (surrounding the central canal) and by nine-fold in the sacral parasympathetic nucleus. Results of time course studies indicate that the maximal increase in Fos expression observed at 1 hour after distention returns to basal levels within 4 hours. In the S1 segment, distention of the proximal colon increased the percentage of NADPHd/Fos-positive neurons selectively in the parasympathetic nucleus by 40% compared with less than 4% in the sham distention group; the number and pattern of NADPHd-stained cells were not modified. These results indicate that noxious distention of the proximal colon for a short duration in awake rats selectively activates neurons in the L6-S2 segments of the dorsal horn mainly in laminae involved in nociceptive and autonomic processing. The marked activation of NADPHd-positive neurons in the sacral parasympathetic nucleus suggests a possible role of nitric oxide in the visceroautonomic reflexes induced by distention of the proximal colon. J. Comp. Neurol. 390:311–321, 1998. © 1998 Wiley-Liss, Inc.     

Primary afferent projections from dorsal and ventral roots to autonomic preganglionic neurons in the cat sacral spinal cord: light and electron microscopic observations

HRP applied to cut dorsal and ventral roots of the cat sacral spinal cord labeled afferent axons with swellings in close apposition to labeled preganglionic neurons (PGNs) in the sacral parasympathetic nucleus. Electron microscopy allowed characterization of synaptic contacts between afferents and PGNs. The results suggest that both the dorsal and ventral root afferents can directly activate autonomic preganglionic neurons.     

Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neurons.

Although glycine receptors (GlyRs) are responsible for the main spinal inhibitory responses in adult vertebrates, in the embryo they have been reported to mediate depolarizing responses, which can sometimes activate dihydropyridine-sensitive L-type calcium channels. However, these channels are not the only targets of dihydropyridines (DHPs), and we questioned whether GlyRs might be directly modulated by DHPs. By whole-cell recording of cultured spinal neurons, we investigated modulation of glycine responses by the calcium channel antagonists, nifedipine, nitrendipine, nicardipine and (R)-Bay K 8644, and by the calcium channel, agonist (S)-Bay K 8644. At concentrations between 1 and 10 microM, all these DHPs could block glycine responses, even in the absence of extracellular Ca2+. The block was stronger at higher glycine concentrations, and increased with time during each glycine application. Nicardipine blocked GABAA responses from the same neurons in a similar manner. In addition to their blocking effects, nitrendipine and nicardipine potentiated the peak responses to low glycine concentrations. Both effects of extracellular nitrendipine on glycine responses persisted when the drug was present in the intracellular solution. Thus, these modulations are related neither to calcium channel modulation nor to possible intracellular effects of DHPs. Another type of calcium antagonist, verapamil (10-50 microM), also blocked glycine responses. Our results suggest that some of the effects of calcium antagonists, including the neuroprotective and anticonvulsant effects of DHPs, might result partly from their interactions with ligand-gated chloride channels.     

Characterization of the single-channel properties of NMDA receptors in laminae I and II of the dorsal horn of neonatal rat spinal cord

The single-channel properties of native NMDA receptors in laminae I and II of the dorsal horn of the neonatal rat spinal cord were studied using outside-out patch-clamp techniques. These receptors were found to have several features that distinguish them from native NMDA receptors elsewhere in the CNS. Single-channel currents activated by NMDA (100 nm) and glycine (10 microm) exhibited five distinct amplitude components with slope-conductance values of 19.9 +/- 0.8, 32.9 +/- 0.6, 42.2 +/- 1.1, 53.0 +/- 1.0 and 68.7 +/- 1.5 pS. Direct transitions were observed between all conductance levels but transitions between 69-pS openings and 20-, 33- and 42-pS openings were rare. There was no significant difference in the frequency of direct transitions from 42- to 20-pS compared to 20- to 42-pS transitions. The Kb (0 mV) for Mg2+ was 89 microm. The Mg2+ unblocking rate constant was similar to other reported values. However, the Mg2+ blocking rate constant was larger than other reported values, suggesting an unusually high sensitivity to Mg2+. The NR2B subunit-selective antagonist, ifenprodil, had no significant effect on overall channel activity but significantly decreased the mean open time of 53-pS openings. These results suggest neonatal laminae I and II NMDA receptors are not simply composed of NR1 and NR2B subunits or NR1 and NR2D subunits. It is possible that these properties are due to an as yet uninvestigated combination of two NR2 subunits with the NR1 subunit or a combination of NR3A, NR2 and NR1 subunits.     

Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ‐aminobutyric acid type A receptor (GABA_AR) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABA_AR antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N‐methyl‐D‐aspartate (NMDA)‐subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA‐subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN‐93, NMDA receptor antagonist D‐APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization. © 2013 Wiley Periodicals, Inc.     

BDNF sensitizes the response of lamina II neurons to high threshold primary afferent inputs

Brain-derived neurotrophic factor (BDNF) is up-regulated and released in the dorsal horn following peripheral inflammation and has therefore been implicated in spinal mechanisms of sensitization. Despite these observations, the mechanisms associated with such a role for BDNF are not yet fully determined. Here, we investigate the effect of BDNF on dorsal root-evoked synaptic transmission in lamina II neurons. In a transverse spinal cord slice preparation from neonatal rats (P1-15), the whole cell patch-clamp technique was used to record from these neurons. Brief application of BDNF (50-200 ng/mL) facilitated the evoked synaptic currents; they remained enhanced even after BDNF was washed out. A significant minority of cells was minimally affected by BDNF and consistent with this, not all neurons in lamina II were immunoreactive for the tyrosine kinase (trk) B receptor. No facilitation was elicited when N-methyl-d-aspartate (NMDA) receptors were blocked with D-APV, when the postsynaptic NMDA receptors were selectively blocked with intracellular MK-801, or when postsynaptic neurons were loaded with BAPTA. Additionally, inhibiting phospholipase C (PLC) or protein kinase C (PKC) prior to BDNF application completely blocked facilitation. However, once synaptic current underwent BDNF-induced facilitation, the PKC inhibitors failed to reverse the effect, suggesting that PKC is needed for initiation, but not maintenance of BDNF-induced facilitation. These results demonstrate that BDNF functions at the spinal level to enhance synaptic efficacy in an NMDA receptor-dependent manner and requires the action of the PLC/PKC pathway. This action of BDNF may contribute to central sensitization and exaggerated pain states.     

Identification of neuropeptides in pelvic and pudendal nerve afferent pathways to the sacral spinal cord of the cat

The distribution of several neuropeptides, including vasoactive intestinal polypeptide (VIP), substance P, somatostatin, leucine enkephalin, methionine enkephalin, and cholecystokinin, in sacral afferent pathways of the cat was examined by immunohistochemical techniques. Certain peptides (substance P, somatostatin, and leucine enkephalin) could be demonstrated in normal dorsal root ganglion cells; however, topical administration or injections of colchicine solution into ganglia 36-56 hours prior to removal markedly increased the number of cells labeled and the intensity of staining. Other peptides (VIP, cholecystokinin, and methionine enkephalin) were only detected in significant numbers of cells following intraganglionic injections of colchicine. The distribution of peptides in dorsal root ganglion cells projecting to the pelvic nerve (visceral) and the pudendal nerve (somatic) was examined by retrograde dye labeling combined with immunohistochemistry. Fluorescent dyes were applied to the cut ends of the nerves 2 weeks prior to removal. A considerably higher percentage of pelvic nerve afferent neurons than pudendal nerve afferent neurons exhibited peptide immunoreactivity; e.g., VIP (42% vs. 10%), cholecystokinin (29% vs. 12%), substance P (24% vs. 21%), leucine enkephalin (30% vs. 24%), and methionine enkephalin (10% vs. 3%). Somatostatin was present in only a small percentage of either type of afferent neuron (0.3-2%). The total percentage of peptide-containing pelvic afferent neurons exceeded 100% (137%), suggesting that more than one peptide is present in some visceral afferent neurons. This has been confirmed in preliminary experiments. The peptide-containing cells were in general less than 40 micron in average diameter; however, a significant percentage of substance P and cholecystokinin neurons ranged from 40 to 60 micron in average diameter. VIP cells had the smallest average diameter (30 micron) whereas somatostatin cells had the largest average diameter (36 micron). Statistical analysis of cell sizes revealed that substance P cells projecting to the pelvic nerve were smaller than substance P cells sending axons into the pudendal nerve. On the other hand, VIP cells in the two afferent pathways were not significantly different in size. Sacral visceral and somatic afferent neurons contain a wide spectrum of neuropeptides, some of which (e.g., VIP and cholecystokinin) seem to be preferentially distributed in the visceral afferent systems.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of fos-like immunoreactivity in the caudal brainstem of the rat following noxious chemical stimulation of the temporomandibular joint

Central expression of the protooncogene c-fos was used to examine areas receiving noxious sensory input from the rat temporomandibular joint (TMJ). Fos-like immunoreactivity (Fos-LI) in the caudal brainstem was visualized 2 hours after unilateral injection of the small-fiber-specific excitant /inflammatory irritant mustard oil into the TMJ region. Control animals received injection of either mustard oil into the subcutaneous fascia overlying the masseter muscle or mineral oil vehicle into the TMJ region. In all groups, Fos-LI was consistently observed ipsilaterally in the spinal trigeminal nucleus and cervical dorsal horn and, bilaterally, in the nucleus of the solitary tract and. the ventrolateral medulla. The expression of Fos-LI ipsilaterally in the paratrigeminal nucleus was variable. Within the trigeminal sensory complex, Fos-LI was restricted to subnucleus caudalis and the caudal portions of subnucleus interpolaris near the level of the obex. Approximately 12% of Fos-LI cells in subnucleus caudalis and in the cervical dorsal horn were found in laminae III-VI. Compared to TMJ mustard oil injection, mineral oil injection produced less Fos-LI at all rostrocaudal levels, whereas subcutaneous mustard oil injection produced less Fos-LI in caudal subnucleus caudalis but similar amounts in the cervical dorsal horn. Neither of these injections yielded significant ipsilateral responses in subnucleus caudalis, indicating that Fos-LI in this region following TMJ mustard oil injection could be ascribed solely to small-fiber stimulation in the deep TMJ region. The wide rostrocaudal distribution of Fos-LI within the caudal brainstem reflects the distribution of TMJ-responsive nociceptive neurons that may underlie the spread and referral of pain from the TMJ region. © 1995 Wiley-Liss, Inc.     

Segmental disinhibition suppresses C‐fiber inputs to the rat superficial medullary dorsal horn via the activation of GABAB receptors

Specialized primary afferents, although they terminate in different laminae within the dorsal horn (DH), are known to interact through local circuit excitatory and inhibitory neurons. That a loss of segmental inhibition probably contributes to persistent pain hypersensitivity during chronic pain raises the question as to how disinhibition‐induced changes in cross‐modal interactions account for chronic pain symptoms. We sought to characterize how pharmacological blockade of glycine and gamma‐aminobutyric acid (GABA) receptors modifies synaptic transmission between primary afferent fibers and second‐order neurons by recording field potentials in the superficial medullary dorsal horn (MDH) of anesthetized rats. Transcutaneous electrical stimulation evokes three negative field potentials elicited by, from earliest to latest, Aβ‐, Aδ‐ and C‐fiber primary afferents. Blocking segmental glycine and/or GABA_A receptors, with strychnine and bicuculline, respectively, strongly facilitates Aβ‐ and Aδ‐fiber‐evoked polysynaptic field potentials but, conversely, inhibits, or even abolishes, the whole C‐fiber field potential. Blocking segmental GABA_B receptors, with phaclofen, reverses such suppression of C‐fiber field potentials. Interestingly, it also potentiates C‐fiber field potentials under control conditions. Finally, activation of segmental GABA_B receptors, with baclofen, preferentially inhibits C‐fiber field potentials. Our results suggest that activation of A‐fiber primary afferents inhibits C‐fiber inputs to the MDH by the way of polysynaptic excitatory pathways, last‐order GABAergic interneurons and presynaptic GABA_B receptors on C‐fiber primary afferents. Under physiological conditions, activation of such local DH circuits is closely controlled by segmental inhibition but it might contribute to paradoxically reduced pain hypersensitivity under pathological disinhibition.     

Calcium-activated cationic channel in rat sensory neurons

Ion channels in sensory neurons are molecular sensors that detect external stimuli and transduce them to neuronal signals. Although Ca2+-activated nonselective cation (CAN) channels were found in many cell types, CAN channels in mammalian sensory neurons are not yet identified. In the present study, we describe an ion channel that is activated by intracellular Ca2+ in cultured rat sensory neurons. Half-maximal concentration of Ca2+ in activating the CAN channel was approximately 780 micro m. The current-voltage relationship of this channel was linear with a unit conductance of 28.8 +/- 0.4 pS at -60 mV in symmetrical 140 mm Na+ solution. The CAN channel was permeable to monovalent cations such as Na+, K+, Cs+, and Li+, but poorly permeable to Ca2+. The CAN channel in mammalian sensory neurons was reversibly blocked by intracellular adenine nucleotides, such as ATP, ADP, and AMP. Interestingly, single-channel currents activated by Ca2+ were blocked by fenamates, such as flufenamic acid, a class of nonsteroidal anti-inflammatory drugs. Thus, these results suggest that CAN channels in mammalian sensory neurons would participate in modulating nociceptive neural transmission in response to ever-changing intracellular Ca2+ in the local microenvironment.     

Neurokinin 1 receptor expression by neurons in laminae I, III and IV of the rat spinal dorsal horn that project to the brainstem

Large neurons in laminae III and IV of the spinal cord which express the neurokinin 1 receptor and have dendrites that enter the superficial laminae are a major target for substance P (SP)-containing (nociceptive) primary afferents. Although some of these neurons project to the thalamus, we know little about other possible projection targets. The main aim of this study was to determine whether all cells of this type are projection neurons and to provide information about brainstem sites to which they project. Injections of cholera toxin B subunit were made into four brainstem areas that receive input from the spinal cord, and the proportion of cells of this type in the L4 spinal segment that were retrogradely labelled was determined in each case. The results suggest that most of these cells (>90%) project to the contralateral lateral reticular nucleus (or to a nearby region), while many (>60%) send axons to the lateral parabrachial area and some to the dorsal part of the caudal medulla. However, few of these cells project to the periaqueductal grey matter. As lamina I neurons with the neurokinin 1 receptor appear to be important in the generation of hyperalgesia, we also examined projection neurons in this lamina and found that for each injection site the great majority possessed the receptor. These results demonstrate that dorsal horn neurons which express the neurokinin 1 receptor contribute to several ascending pathways that are thought to be important in pain mechanisms.