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.     

N-methyl-D-aspartate enhancement of the glycine response in the rat sacral dorsal commissural neurons

The effect of N-methyl-D-aspartate (NMDA) on the glycine (Gly) response was examined in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The application of 100 microM NMDA to SDCN neurons reversibly potentiated Gly-activated Cl- currents (IGly) without affecting the Gly binding affinity and the reversal potential of IGly. A selective NMDA receptor antagonist, APV (100 microM), blocked the NMDA-induced potentiation of IGly, whereas 50 microM CNQX, a non-NMDA receptor antagonist, did not. The potentiation effect was reduced when NMDA was applied in a Ca2+-free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels. Pretreatment with KN-62, a selective Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the NMDA action. Inhibition of calcineurin (CaN) further enhanced the NMDA-induced potentiation of IGly. In addition, the GABAA receptor-mediated currents were suppressed by NMDA receptor activation in the SDCN neurons. The present results show that Ca2+ entry through NMDA receptors modulates the Gly receptor function via coactivation of CaMKII and CaN in the rat SDCN neurons. This interaction may represent one of the important regulatory mechanisms of spinal nociception. The results also suggest that GABAA and Gly receptors may be subject to different intracellular modulatory pathways.     

Neurotensin inhibits background K+ channels and facilitates glutamatergic transmission in rat spinal cord dorsal horn

Neurotensin (NT) is a neuropeptide involved in the modulation of nociception. We have investigated the actions of NT on cultured postnatal rat spinal cord dorsal horn (DH) neurons. NT induced an inward current associated with a decrease in membrane conductance in 46% of the neurons and increased the frequency of glutamatergic miniature excitatory synaptic currents in 37% of the neurons. Similar effects were observed in acute slices. Both effects of NT were reproduced by the selective NTS1 agonist JMV449 and blocked by the NTS1 antagonist SR48692 and the NTS1/NTS2 antagonist SR142948A. The NTS2 agonist levocabastine had no effect. The actions of NT persisted after inactivation of G(i/o) proteins by pertussis toxin but were absent after inactivation of protein kinase C (PKC) by chelerythrine or inhibition of the MAPK (ERK1/2) pathway by PD98059. Pre- and postsynaptic effects of NT were insensitive to classical voltage- and Ca(2+) -dependent K(+) channel blockers. The K(+) conductance inhibited by NT was blocked by Ba(2+) and displayed no or little inward rectification, despite the presence of strongly rectifying Ba(2+) -sensitive K(+) conductance in these neurons. This suggested that NT blocked two-pore domain (K2P) background K(+) -channels rather than inwardly rectifying K(+) channels. Zn(2+) ions, which inhibit TRESK and TASK-3 K2P channels, decreased NT-induced current. Our results indicate that in DH neurons NT activates NTS1 receptors which, via the PKC-dependent activation of the MAPK (ERK1/2) pathway, depolarize the postsynaptic neuron and increase the synaptic release of glutamate. These actions of NT might modulate the transfer and the integration of somatosensory information in the DH.     

BDNF induces late-phase LTP of C-fiber evoked field potentials in rat spinal dorsal horn

Several lines of evidence have shown that in some brain regions brain-derived neurotrophic factor (BDNF) is important for long-term potentiation (LTP), a synaptic model of memory storage. In the present work we evaluate the role of BDNF in LTP of C-fiber evoked field potentials in spinal dorsal horn, a synaptic model of pain memory. We found that spinal application of BDNF-induced LTP of C-fiber evoked field potentials with a long latency, lasting for > 8 h, and the effect was blocked by either tyrosine kinase inhibitor (K252a) or BNDF scavenger (TrkB-Fc). The potentiation produced by BDNF was occluded by late-phase LTP (L-LTP) but not by early-phase LTP (E-LTP) induced by electrical stimulation. Pretreatment of K252a or TrkB-Fc selectively blocked spinal L-LTP induced by low-frequency stimulation (LFS) but not E-LTP. BDNF-induced LTP was completely abolished by the protein synthesis inhibitor (anisomycin), by N-methyl-D-aspartate (NMDA) receptor blocker (MK-801), by extracellular signal-regulated protein kinase (ERK) inhibitor (PD98059) or by p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) but not by c-Jun N-terminal kinase (JNK) inhibitor (SP600125). Nuclear factor-kappaB (NF-κB) inhibitor (PDTC) also suppressed spinal BDNF-LTP. The results suggest that BDNF play a crucial role in protein synthesis-dependent L-LTP in spinal dorsal horn via activation of ERK, p38 MAPK and NF-κB signal pathways.     

Long-term synaptic plasticity in the spinal dorsal horn and its modulation by electroacupuncture in rats with neuropathic pain

Our previous study has reported that electroacupuncture (EA) at low frequency of 2 Hz had greater and more prolonged analgesic effects on mechanical allodynia and thermal hyperalgesia than that EA at high frequency of 100 Hz in rats with neuropathic pain. However, how EA at different frequencies produces distinct analgesic effects on neuropathic pain is unclear. Neuronal plastic changes in spinal cord might contribute to the development and maintenance of neuropathic pain. In the present study, we investigated changes of spinal synaptic plasticity in the development of neuropathic pain and its modulation by EA in rats with neuropathic pain. Field potentials of spinal dorsal horn neurons were recorded extracellularly in sham-operated rats and in rats with spinal nerve ligation (SNL). We found for the first time that the threshold for inducing long-term potentiation (LTP) of C-fiber-evoked potentials in dorsal horn was significantly lower in SNL rats than that in sham-operated rats. The threshold for evoking the C-fiber-evoked field potentials was also significantly lower, and the amplitude of the field potentials was higher in SNL rats as compared with those in the control rats. EA at low frequency of 2 Hz applied on acupoints ST 36 and SP 6, which was effective in treatment of neuropathic pain, induced long-term depression (LTD) of the C-fiber-evoked potentials in SNL rats. This effect could be blocked by N-methyl-d-aspartic acid (NMDA) receptor antagonist MK-801 and by opioid receptor antagonist naloxone. In contrast, EA at high frequency of 100 Hz, which was not effective in treatment of neuropathic pain, induced LTP in SNL rats but LTD in sham-operated rats. Unlike the 2 Hz EA-induced LTD in SNL rats, the 100 Hz EA-induced LTD in sham-operated rats was dependent on the endogenous GABAergic and serotonergic inhibitory system. Results from our present study suggest that (1) hyperexcitability in the spinal nociceptive synaptic transmission may occur after nerve injury, which may contribute to the development of neuropathic pain; (2) EA at low or high frequency has a different effect on modulating spinal synaptic plasticities in rats with neuropathic pain. The different modulation on spinal LTD or LTP by low- or high-frequency EA may be a potential mechanism of different analgesic effects of EA on neuropathic pain. LTD of synaptic strength in the spinal dorsal horn in SNL rats may contribute to the long-lasting analgesic effects of EA at 2 Hz.     

Hypoxic regulation of Ca2+ signaling in cultured rat astrocytes

Acute hypoxia modulates various cell processes, such as cell excitability, through the regulation of ion channel activity. Given the central role of Ca2+ signaling in the physiological functioning of astrocytes, we have investigated how acute hypoxia regulates such signaling, and compared results with those evoked by bradykinin (BK), an agonist whose ability to liberate Ca2+ from intracellular stores is well documented. In Ca2+-free perfusate, BK evoked rises of [Ca2+]i in all cells examined. Hypoxia produced smaller rises of [Ca2+]i in most cells, but always suppressed subsequent rises of [Ca2+]i induced by BK. Thapsigargin pre-treatment of cells prevented any rise of [Ca2+]i evoked by either BK or hypoxia. Restoration of Ca2+ to the perfusate following a period of acute hypoxia always evoked capacitative Ca2+ entry. During mitochondrial inhibition (due to exposure to carbonyl cyanide p-trifluromethoxyphenyl hydrazone (FCCP) and oligomycin), rises in [Ca2+]i (observed in Ca2+-free perfusate) evoked by hypoxia or by BK, were significantly enhanced, and hypoxia always evoked responses. Our data indicate that hypoxia triggers Ca2+ release from endoplasmic reticulum stores, efficiently buffered by mitochondria. Such liberation of Ca2+ is sufficient to trigger capacitative Ca2+ entry. These findings indicate that the local O2 level is a key determinant of astrocyte Ca2+ signaling, likely modulating Ca2+-dependent astrocyte functions in the central nervous system.     

Effects of electrical stimulation of thalamic nucleus submedius and periaqueductal gray on the visceral nociceptive responses of spinal dorsal horn neurons in the rat

Electrical stimulation of the nucleus submedius (Sm) has been shown to suppress the viscerosomatic reflex (VSR), which is evoked by colorectal distension (CRD). We have examined the effects of focal electrical stimulation (0.3 ms, 50 Hz, 100 microA, 10 s) of the Sm and the periaqueductal gray (PAG) on the excitatory responses evoked by CRD in spinal dorsal horn neurons within the L6-S1 region in the urethane-anesthetized Wistar rats. Extracellular recordings were made from 32 spinal excitatory CRD responses. All of these neurons were convergent neurons with cutaneous receptive fields. The majority of the neurons (27/32) were wide dynamic range (WDR) neurons (responding to noxious and non-noxious cutaneous stimuli) while the remaining five neurons were nociceptive specific (NS) neurons (responding only to noxious cutaneous stimuli). The effects of electrical stimulation applied to 28 sites within the Sm were assessed for spinal neurons. Electrical stimulation in seven sites within the Sm (25%) inhibited the CRD excitatory response of dorsal horn neurons, while in two sites (7%) the same stimulation yielded facilitation. Electrical stimulation in the majority of the sites in the Sm (19/28, 68%) did not affect spinal excitatory CRD responses. On the other hand, electrical stimulation of the PAG clearly inhibited 20 of 22 (90%) CRD excitatory responses. These results suggest that the majority of Sm neurons may suppress VSR activity at a supraspinal reflex center rather than via a descending inhibition of spinal visceral nociceptive transmission, as is the case for the PAG.     

Neuronal input triggers Ca2+ influx through AMPA receptors and voltage-gated Ca2+ channels in oligodendrocytes

Communication between neurons and developing oligodendrocytes (OLs) leading to OL Ca²⁺ rise is critical for axon myelination and OL development. Here, we investigate signaling factors and sources of Ca²⁺ rise in OLs in the mouse brainstem. Glutamate puff or axon fiber stimulation induces a Ca²⁺ rise in pre-myelinating OLs, which is primarily mediated by Ca²⁺-permeable AMPA receptors. During glutamate application, inward currents via AMPA receptors and elevated extracellular K⁺ caused by increased neuronal activity collectively lead to OL depolarization, triggering Ca²⁺ influx via P/Q- and L-type voltage-gated Ca²⁺ (Ca_v) channels. Thus, glutamate is a key signaling factor in dynamic communication between neurons and OLs that triggers Ca²⁺ transients via AMPARs and Ca_v channels in developing OLs. The results provide a mechanism for OL Ca²⁺ dynamics in response to neuronal input, which has implications for OL development and myelination.     

The contralateral input to the dorsal horn of the spinal cord in the decerebrate spinal rat

Input from the contralateral limb and tail was examined in the lumbar dorsal horn of decerebrate spinal rats. Fifty-three cells were recorded from laminae 4, 5 and 6 and classified according to their ipsilateral response to natural and electrical stimulation. Twenty-nine (54%) of these cells were found to have inhibitory contralateral fields. This inhibition was evoked by noxious pinching or heating of the skin. In most cases the inhibitory field was a mirror image of the excitatory ipsilateral field although it also often included the tail. Activity evoked by natural and electrical stimulation as well as spontaneous activity was inhibited by contralateral skin stimulation. Noxious specific and wide dynamic range cells displayed these fields but low threshold mechanoreceptive cells did not. Twenty-six cells (49%) received direct short-latency excitatory input from the contralateral sciatic nerve; this correlated well with the presence of contralateral fields. Trains of stimuli applied to the contralateral sciatic nerve at Aδ- and C-fibre strength resulted in inhibition of the cell whereas trains of Aβ strength had no effect. The results demonstrate the existence of segmental contralateral control over dorsal horn cell activity, not involving supraspinal pathways.     

Immunoreactive TRPV-2 (VRL-1), a capsaicin receptor homolog, in the spinal cord of the rat

The vanilloid receptor-like 1 protein (VRL-1, also called TRPV2) is a member of the TRPV family of proteins and is a homolog of the capsaicin/vanilloid receptor (VR1, or TRPV1). Although VRL-1 does not bind capsaicin, like VR1 it is activated by noxious heat (>52 degrees C). Unlike VR1, however, VRL-1 is primarily expressed by medium- and large-diameter primary afferents, which suggests that nociceptive processing is but one of the functions to which VRL-1 contributes. To provide information on the diverse spinal circuits that are engaged by these VRL-1-expressing primary afferents, we completed a detailed immunocytochemical map of VRL-1 in rat spinal cord, including light and electron microscopic analysis, and generated a more comprehensive neurochemical characterization of VRL-1-expressing primary afferents. Consistent with previous reports, we found that VRL-1 and VR1 are expressed in different dorsal root ganglion (DRG) cell bodies. Almost all VRL-1-expressing cells labeled for N52 (a marker of myelinated afferents), consistent with VRL-1 expression in Adelta and Abeta fibers. EM analysis of the DRG and dorsal roots confirmed this and revealed two categories of neurons based on the intensity of immunolabeling. The densest VRL-1 immunoreactivity in the spinal cord was found in lamina I, inner lamina II, and laminae III/IV. This is consistent with the expression of VRL-1 by myelinated nociceptors that target laminae I and IIi and in nonnociceptive Abeta fibers that target laminae III/IV. Dorsal rhizotomy reduced, but did not eliminate, the immunostaining in all dorsal horn laminae, which indicates that VRL-1 expression derives from both DRG cells and from neurons intrinsic to the brain or spinal cord. Spinal hemisection reduced immunostaining of the ipsilateral dorsal columns in segments rostral to the lesion and in the dorsal column nuclei, presumably from the loss of ascending Abeta afferents, but there was no change caudal to the lesion. Thus, supraspinal sources of dorsal horn VRL-1 immunoreactivity are likely not significant. Although we never observed VRL-1 immunostaining in cell bodies in the superficial dorsal horn, there was extensive labeling of motoneurons and ventral root efferents-in particular, in an extremely densely labeled population at the lumbosacral junction. Finally, many ependymal cells surrounding the central canal were intensely labeled. These results emphasize that VRL-1, in contrast to VR1, is present in a diverse population of neurons and undoubtedly contributes to numerous functions in addition to nociceptive processing.     

A comparative study of selective stimulation of raphe nuclei in the cat in inhibiting dorsal horn neuron responses to noxious stimulation

Consistent inhibition of cord nociceptive neurons was obtained at low levels of stimulation (5 V or 50–100 μA) within raphe magnus. Less consistently and with higher stimulus intensities, inhibition was observed on stimulating raphe pallidus. Still less frequently, and generally only with stimulation in the 20 V or 500 μA range, inhibition was observed in raphe dorsalis, raphe obscurus and centralis superior. No inhibition could be obtained by stimulation in linearis intermedias or linearis rostralis. Nearly all midline sites where inhibition of cord nociceptive neurons was observed were those within or in immediate proximity to raphe nuclei.     

AMPA receptor trafficking in inflammation-induced dorsal horn central sensitization

Activity-dependent postsynaptic receptor trafficking is critical for long-term synaptic plasticity in the brain, but it is unclear whether this mechanism actually mediates the spinal cord dorsal horn central sensitization (a specific form of synaptic plasticity) that is associated with persistent pain. Recent studies have shown that peripheral inflammation drives changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit trafficking in the dorsal horn and that such changes contribute to the hypersensitivity that underlies persistent pain. Here, we review current evidence to illustrate how spinal cord AMPARs participate in the dorsal horn central sensitization associated with persistent pain. Understanding these mechanisms may allow the development of novel therapeutic strategies for treating persistent pain.     

Removal of Ca(2+) following depolarization-evoked cytoplasmic Ca(2+) transients in freshly dissociated pyramidal neurones of the rat dorsal cochlear nucleus

Cytoplasmic [Ca²⁺] ([Ca²⁺]_i) was measured using Fura-2 in pyramidal neurones isolated from the rat dorsal cochlear nucleus (DCN). The kinetic properties of Ca²⁺ removal following K⁺ depolarization-induced Ca²⁺ transients were characterized by fitting exponential functions to the decay phase. The removal after small transients (<82 nM peak [Ca²⁺]_i) had monophasic time course (time constant of 6.43±0.48 s). In the cases of higher Ca²⁺ transients biphasic decay was found. The early time constant decreased (from 3.09±0.26 to 1.46±0.11 s) as the peak intracellular [Ca²⁺] increased. The value of the late time constant was 18.15±1.60 s at the smallest transients, and showed less dependence on [Ca²⁺]_i. Blockers of Ca²⁺ uptake into intracellular stores (thapsigargin and cyclopiazonic acid) decreased the amplitude of the Ca²⁺ transients and slowed their decay. La³⁺ (3 mM) applied extracellularly during the declining phase dramatically changed the time course of the Ca²⁺ transients as a plateau developed and persisted until the La³⁺ was present. When the other Ca²⁺ removal mechanisms were available, reduction of the external [Na⁺] to inhibit the Na⁺/Ca²⁺ exchange resulted in a moderate increase of the time constants. It is concluded that in the isolated pyramidal neurones of the DCN the removal of Ca²⁺ depends mainly on the activity of Ca²⁺ pump mechanisms.     

Expression of 5-HT2A receptor mRNA in rat spinal dorsal horn and some nuclei of brainstem after peripheral inflammation

The expression of 5-hydroxytryptamine 5-HT2A receptor mRNA was studied in the lumbar spinal dorsal horn, nucleus of raphe magnus (NRM), ventrolateral periaqueductal gray (vlPAG) and dorsal raphe nucleus (DRN) following carrageenan inflammation using in situ hybridization technique. The findings of this study demonstrated that 5-HT2A receptor mRNA was expressed with low to moderate levels in lumbar spinal dorsal horn, NRM, vlPAG and DRN. Following carrageenan inflammation, the expression of 5-HT2A receptor mRNA in ipsilateral dorsal horn, bilateral NRM, vlPAG and DRN was significantly increased. The peak occurred at 3 h and then there was a clear decrease but still a substantial number of labeled cells at 24 h after injection of carrageenan. This result suggested that the synthesis of 5-HT2A receptor is enhanced in spinal dorsal horn, NRM, vlPAG and DRN during inflammatory pain.     

The properties of neurones recorded in the superficial dorsal horn of the rat spinal cord

The physiological properties of neurones in the superficial laminae of the dorsal horn of the fourth and fifth lumbar segments of the rat spinal cord have been investigated in decerebrate spinal animals. Both extracellular recordings with platinum-plated tungsten microelectrodes (n = 72) and intracellular recordings with glass microelectrodes (N = 79) were made. Attempts were made to fill cells intracellularly with horseradish peroxidase or Lucifer Yellow. Thirty-seven percent of the intracellularly injected neurones were recovered after histological processing and their cell bodies found to be in lamina 1 or 2 and in the dorsal white matter overlying lamina 1. The dendritic spread of the stained neurones was maximal in the rostrocaudal plane with a restricted mediolateral spread. The physiological properties of the extracellularly recorded units, the intracellularly unidentified units, and the intracellularly stained units were the same. The neurones were characterized by low background activity and all had excitatory receptive fields on the lower limb. Some neurones responded only to low-threshold mechanical stimulation of the skin or only to noxious skin stimulation but the majority of units (58%) were wide-dynamic-range cells responding to both types of stimuli. Receptive field classification was made questionable, however, by the existence of cells (9%) that exhibited a spontaneous shift in the size of their receptive fields and in the type of stimulus that elicited a response. The neurones in the superficial dorsal horn commonly showed a marked inhibition to repeated cutaneous stimuli (27%) or a prolonged afterdischarge followed a single stimulus (20%). Afferent input from the sural nerve was found to be from A and C fibres in both extra- and intracellular recordings. Aδ- and C-mediated excitations were most common although convergent inputs from Ab?-fibres occurred in 40% of units. No correlation was found between cell structure or distribution of dendritic fields and physiological properties in our small sample of intracellularly stained cells. The morphology of the cells was highly diverse, as were the different receptive fields. There was, however, some correlation between the location of cell bodies and their responses. Neurones responding only to low-threshold stimuli were distributed either in the dorsal white matter or in inner lamina 2. Wide-dynamic-range cells were distributed throughout the superficial dorsal horn. These results suggest that neurones of different shapes and positions may subserve the same function and, conversely, that neurones of the same shape and position may subserve different functions.     

Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters

Measurements of local Ca2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto-fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution. Aequorin is a genetically encoded Ca(2+)-sensitive bioluminescent protein, however, its low quantum yield prevents dynamic measurements of Ca2+ responses in single cells. To overcome this limitation, we recently reported the bi-functional Ca2+ reporter gene, GFP-aequorin (GA), which was developed specifically to improve the light output and stability of aequorin chimeras [V. Baubet, et al., (2000) PNAS, 97, 7260-7265]. In the current study, we have genetically targeted GA to different microdomains important in synaptic transmission, including to the mitochondrial matrix, endoplasmic reticulum, synaptic vesicles and to the postsynaptic density. We demonstrate that these reporters enable 'real-time' measurements of subcellular Ca2+ changes in single mammalian neurons using bioluminescence. The high signal-to-noise ratio of these reporters is also important in that it affords the visualization of Ca2+ dynamics in cell-cell communication in neuronal cultures and tissue slices. Further, we demonstrate the utility of this approach in ex-vivo preparations of mammalian retina, a paradigm in which external light input should be controlled. This represents a novel molecular imaging approach for non-invasive monitoring of local Ca2+ dynamics and cellular communication in tissue or whole animal studies.     

Numbers of synapses in laminae I-IV of the rat dorsal horn

The present study determines numerical densities (NVsyn) and total numbers of synaptic discs in laminae I-IV of the rat S2 dorsal horn. Previous methods for NVsyn have the advantage of being relatively simple, but these assume that the discs are round, flat, and of uniform size. In our material, serial reconstructions indicate that these assumptions are not met. Accordingly we use a stereological method that is not as dependent on these assumptions. This method is to divide the surface density of the discs by the mean surface area of a disc (NVsyn = SVsyn/Ssyn). We refer to this as a reconstruction method because synaptic discs are reconstructed from serial sections. We also calculate numerical densities by several previously used standard methods, and the findings are similar but not identical. We find that numerical density and total synaptic numbers are smallest in lamina I, and densities and total numbers are not significantly different when lamina II is compared to laminae III and IV. Thus the intense labeling of terminals with certain compounds that characterize lamina I and II does not imply an increase in total synaptic numbers or in synaptic density. In addition there is a general increase in synaptic densities and numbers as one proceeds from lamina I to lamina IV. Another point is that the numerical density of synapses in the dorsal horn is approximately that of the cerebral cortex. These data will serve as a basis from which to judge the effects of denervations and other manipulations that purportedly change synaptic numbers.     

ATP-sensitive K+ channels mediate an IPSP in dorsal horn neurones elicited by sensory stimulation.

Nociceptive dorsal horn neurones, which are involved in the processing of pain-related information, are inhibited by input from vibration-sensitive, large diameter primary sensory fibres (Wall and Cronly-Dillon, 1960; Salter and Henry, 1990a,b). We have reported previously that the inhibition of spinal nociceptive neurones by vibration is mediated by adenosine acting through P1-purinergic receptors (Salter and Henry, 1987). In a number of different types of cell, adenosine is known to activate K+ currents (Gerber et al., 1989; Greene and Haas, 1985; Proctor and Dunwiddie, 1987; Segal, 1982; Trussell and Jackson, 1987) and we have recently found that the adenosine-mediated inhibition of nociceptive neurones by vibration is the result of an inhibitory postsynaptic potential (IPSP), which is, indeed, caused by a K+ conductance (De Koninck and Henry, 1988, 1992). It has been reported that adenosine-activated K+ channels in cardiac muscle cells are the ATP-sensitive K+ channels (Kirsch et al., 1990). Therefore, we questioned whether these channels might mediate the purinergic IPSP we have observed in nociceptive dorsal horn neurones. We report here that glibenclamide, a blocker of ATP-sensitive K+ channels (Ashcroft, 1988; Schmid Antomarchi et al., 1987a,b), blocks the inhibition of nociceptive neurones by vibratory stimulation when this compound is administered locally by iontophoresis or systemically by intravenous injection. In addition, direct intracellular injection of ATP was found to block the IPSP evoked by vibratory stimulation. These data indicate that the purinergic IPSP in nociceptive spinal neurones is mediated via ATP-sensitive K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)