Kainate-receptor-mediated sensory synaptic transmission in mammalian spinal cord

Glutamate, the major excitatory neurotransmitter in the central nervous system, activates three different receptors that directly gate ion channels, namely receptors for AMPA (alpha-amino-3-hydroxy-5-methyl isoxozole propionic acid), NMDA (N-methyl-D-aspartate), and kainate, a structural analogue of glutamate. The contribution of AMPA and NMDA receptors to synaptic transmission and plasticity is well established. Recent work on the physiological function of kainate receptors has focused on the hippocampus, where repetitive activation of the mossy-fibre pathway generates a slow, kainate-receptor-mediated excitatory postsynaptic current (EPSC). Here we show that high-intensity single-shock stimulation (of duration 200 microseconds) of primary afferent sensory fibres produces a fast, kainate-receptor-mediated EPSC in the superficial dorsal horn of the spinal cord. Activation of low-threshold afferent fibres generates typical AMPA-receptor-mediated EPSCs only, indicating that kainate receptors may be restricted to synapses formed by high-threshold nociceptive (pain-sensing) and thermoreceptive primary afferent fibres. Consistent with this possibility, kainate-receptor-mediated EPSCs are blocked by the analgesic mu-opiate-receptor agonist Damgo and spinal blockade of both kainate and AMPA receptors produces antinociception. Thus, spinal kainate receptors contribute to transmission of somatosensory inputs from the periphery to the brain.     

Down-regulation of mu-opioid receptors in rat and monkey dorsal root ganglion neurons and spinal cord after peripheral axotomy

To understand the role of opioids and their receptors in chronic pain following peripheral nerve injury, we have studied the mu-opioid receptor in rat and monkey lumbar 4 and 5 dorsal root ganglion neurons and the superficial dorsal horn of the spinal cord under normal circumstances and after peripheral axotomy. Our results show that many small neurons in rat and monkey dorsal root ganglia, and some medium-sized and large neurons in rat dorsal root ganglia, express mu-opioid receptor-like immunoreactivity. Most of these neurons contain calcitonin gene-related peptide. The mu-opioid receptor was closely associated with the somatic plasmalemma of the dorsal root ganglion neurons. Both mu-opioid receptor-immunoreactive nerve fibers and cell bodies were observed in lamina II of the dorsal horn. The highest intensity of mu-opioid receptor-like immunoreactivity was observed in the deep part of lamina II. Most mu-opioid receptor-like immunoreactivity in the dorsal horn originated from spinal neurons. A few mu-opioid receptor-positive peripheral afferent terminals in the rat and monkey dorsal horn were calcitonin gene-related peptide-immunoreactive. In addition to pre- and post-junctional receptors in rat and monkey dorsal horn neurons, mu-opioid receptors were localized on the presynaptic membrane of some synapses of primary afferent terminals in the monkey dorsal horn. Peripheral axotomy caused a reduction in the number and intensity of mu-opioid receptor-positive neurons in the rat and monkey dorsal root ganglia, and of mu-opioid receptor-like immunoreactivity in the dorsal horn of the spinal cord. The decrease in mu-opioid receptor-like immunoreactivity was more pronounced in the monkey than in the rat dorsal root ganglia and spinal cord. It is probable that there was a parallel trans-synaptic down-regulation of mu-opioid-like immunoreactivity in local dorsal horn neurons of the monkey. These data suggest that one factor underlying the well known insensitivity of neuropathic pain to opioid analgesics could be due to a marked reduction in the number of mu-opioid receptors in the axotomized sensory neurons and in interneurons in the dorsal horn of the spinal cord.     

Substance P and enkephalin immunoreactivities in axonal boutons presynaptic to physiologically identified dorsal horn neurons. An ultrastructural multiple-labelling study in the cat

A combination of intracellular electrophysiological recording and injection of horseradish peroxidase with ultrastructural immunocytochemistry was used to investigate the synaptic interplay between substance P- and enkephalin-immunoreactive axonal boutons and three types of functionally characterized dorsal horn neurons in the cat spinal cord. The dorsal horn neurons were classified as nociceptive specific, wide dynamic range and non-nociceptive based on their responses to innocuous and noxious stimuli. Most of the nociceptive neurons (either nociceptive specific or wide dynamic range) contained enkephalin immunoreactivity, but none of the non-nociceptive neurons were positive for enkephalin. Three types of immunoreactive boutons were found in contact with the functionally characterized dorsal horn neurons. These boutons were positive for either substance P, enkephalin, or substance P+enkephalin. Quantitative analysis revealed that the percentages of substance P-immunoreactive boutons apposed to the cell bodies, proximal dendrites and distal dendrites of nociceptive neurons were significantly higher than those of non-nociceptive neurons. Furthermore, the percentages of substance P+enkephalin-immunoreactive axonal boutons apposed to the distal dendrites of nociceptive neurons were significantly higher than those of non-nociceptive neurons and the percentages of enkephalin-immunoreactive boutons apposed to the cell bodies and proximal dendrites of nociceptive neurons were significantly higher than in non-nociceptive neurons. Finally, neither enkephalin-immunoreactive nor substance P+enkephalin-immunoreactive boutons were ever seen presynaptic to substance P-immunoreactive boutons. These results provide evidence of an anatomical substrate within the dorsal horn for the interaction of substance P-mediated with enkephalin-mediated mechanisms. The data support the idea that the modulation of nociceptive input in the dorsal horn by enkephalinergic neurons occurs mainly via a postsynaptic mechanism, and thus suggest that dorsal horn enkephalinergic neurons participate in a local inhibitory feedback loop in a distinct pathway from the previously postulated opioid-mediated depression of substance P release from primary afferent terminals.     

Activation of spinal N-methyl-D-aspartate or neurokinin receptors induces long-term potentiation of spinal C-fibre-evoked potentials

The use-dependent increase in synaptic strength between primary afferent C-fibres and second-order neurons in superficial spinal dorsal horn may be an important cellular mechanism underlying central hyperalgesia. This long-term potentiation can be blocked by antagonists of the N-methyl-D-aspartate subtype of glutamate receptor, the neurokinin 1 or the neurokinin 2 receptor. We have tested here whether activation of these receptors by superfusion of the spinal cord with corresponding agonists in the absence of presynaptic activity is sufficient to induce long-term potentiation. In urethane anaesthetized rats C-fibre-evoked field potentials were elicited in superficial laminae of lumbar spinal cord by electrical stimulation of the sciatic nerve. In rats with intact spinal cord, controlled superfusion of the spinal cord at recording segments for 60 min with N-methyl-D-aspartate, substance P or neurokinin A never induced long-term potentiation. Spinal superfusion with a mixture of N-methyl-D-aspartate, substance P and neurokinin A also failed to induce long-term potentiation in four rats tested. In spinalized rats, however, long-term potentiation was induced by either N-methyl-D-aspartate (at 10 microM, to 173 +/- 16% of control) substance P (at 10 microM, to 176 +/- 13% of control) or by neurokinin A (at 1 microM, to 198 +/- .20% of control). The induction of long-term potentiation by N-methyl-D-aspartate, substance P or neurokinin A was blocked by intravenous application of the receptor antagonists dizocilpine maleate (0.5 mg/kg), RP67580 (2 mg/kg) or SR48968 (0.2 mg/kg), respectively. Thus, activation of N-methyl-D-aspartate or neurokinin receptors may induce long-lasting plastic changes in synaptic transmission in afferent C-fibres and this effect may be prevented by tonic descending inhibition.     

Localization of NADPH diaphorase in the thoracolumbar and sacrococcygeal spinal cord of the dog

The distribution of NADPH-d activity and NOS-immunoreactivity in the spinal cord of the dog was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord examined, NADPH-d staining and NOS-immunoreactivity were present in neurons and fibers in the superficial dorsal horn, dorsal commissure and in neurons around the central canal. Sympathetic preganglionic neurons in the rostral lumbar segments identified by choline acetyl transferase (ChAT) immunoreactivity exhibited prominent NADPH-d and and NOS-immunoreactive staining; whereas the ChAT-immunoreactive parasympathetic preganglionic neurons in the sacral segments were not stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending form Lissauer's tract through lamina I along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These fibers were prominent in the S1-S3 segments but not in adjacent segments (L5-L7 and Cx1 or in thoracolumbar segments. The NADPH-d fibers were not NOS-immunoreactive, but did overlap with a prominent fiber bundle containing vasoactive intestinal polypeptide immunoreactivity in the sacral spinal cord. These results indicate that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic neurons, but not in sacral parasympathetic preganglionic neurons. The functional significance of the NADPH-d positive, NOS-negative fiber bundle on the lateral edge of the sacral dorsal horn remains to be determined. However, based on anatomical studies in other species it seems reasonable to speculate that the fiber tract represents, in part, visceral afferent projections to the sacral parasympathetic nucleus.     

Sprouting of primary afferent fibers after spinal cord transection in the rat

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I-V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III-V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.     

Endogenous opioid peptides acting at mu-opioid receptors in the dorsal horn contribute to midbrain modulation of spinal nociceptive neurons

Activation of neurons in the midbrain periaqueductal gray (PAG) inhibits spinal dorsal horn neurons and produces behavioral antinociception in animals and analgesia in humans. Although dorsal horn regions modulated by PAG activation contain all three opioid receptor classes (mu, delta, and kappa), as well as enkephalinergic interneurons and terminal fields, descending opioid-mediated inhibition of dorsal horn neurons has not been demonstrated. We examined the contribution of dorsal horn mu-opioid receptors to the PAG-elicited descending modulation of nociceptive transmission. Single-unit extracellular recordings were made from rat sacral dorsal horn neurons activated by noxious heating of the tail. Microinjections of bicuculline (BIC) in the ventrolateral PAG led to a 60-80% decrease in the neuronal responses to heat. At the same time, the responses of the same neurons to iontophoretically applied NMDA or kainic acid were not consistently inhibited. The inhibition of heat-evoked responses by PAG BIC was reversed by iontophoretic application of the selective mu-opioid receptor antagonists, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP). A similar effect was produced by naloxone; however, naloxone had an excitatory influence on dorsal horn neurons in the absence of PAG-evoked descending inhibition. This is the first demonstration that endogenous opioids acting via spinal mu-opioid receptors contribute to brain stem control of nociceptive spinal dorsal horn neurons. The inhibition appears to result in part from presynaptic inhibition of afferents to dorsal horn neurons.     

Corticotropin releasing factor-like immunoreactivity in afferent projections to the sacral spinal cord of the cat

Distribution and origin of corticotropin releasing factor (CRF) in the thoraco-lumbar and sacral spinal cord of the cat has been studied using immunohistochemical method. CRF immunoreactive (CRF-IR) nerve fibers and terminals were most prominent in dorsal part of sacral spinal cord. In the sacral segments of the spinal cord, immunoreactivity for CRF was detected in a prominent bundle of axons and varicosities extending from Lissauer's tract (LT) along the lateral edge of the superficial dorsal horn (laminae I and II) to laminae V at the base of the dorsal horn. Individual CRF-IR fibers passed from the bundle in ventral medial and ventrolateral directions to the dorsal commissure and the sacral preganglionic nucleus (SPN), respectively. The bundle of CRF-IR axons closely resembled vasoactive intestinal polypeptide (VIP) containing fibers in LT and on the lateral edge of the dorsal horn. Sacral dorsal root transection eliminated both the CRF and VIP fiber staining in the dorsal horn. Spinal transection at the T12-T13 segmental level did not influence the CRF- or VIP-IR. Less intense CRF-IR was also present in fibers in: (1) the dorsal lateral funiculus adjacent to LT, (2) the superficial layers of the dorsal horn and intermediolateral nucleus at thoracolumbar spinal levels, (3) the ventral horn, including Onuf's nucleus, (4) the intermediate gray matter including the dorsal gray commissure, and (5) the SPN. The similarity in the distribution of CRF-IR and pelvic nerve afferent projections in the sacral spinal cord raises the possibility that CRF may be a transmitter in afferent neurons innervating the pelvic viscera.     

Distribution of trigeminohypothalamic and spinohypothalamic tract neurons displaying substance P receptor-like immunoreactivity in the rat

Primary afferent neurons containing substance P (SP) are apparently implicated in the transmission of noxious information from the periphery to the central nervous system, and SP released from primary afferent neurons acts on second-order neurons with the SP receptor (SPR). In the rat, nociceptive information reached the hypothalamus not only through indirect pathways but also directly through trigeminohypothalamic and spinohypothalamic pathways. Thus, in the present study, the distribution pattern of trigeminohypothalamic and spinohypothalamic tract neurons showing SPR-like immunoreactivity (SPR-LI) was examined in the rat by a retrograde tract-tracing method combined with immunofluorescence histochemistry for SPR. A substantial number of trigeminal and spinal neurons with SPR-LI were retrogradely labeled with Fluoro-Gold (FG) injected into the hypothalamic regions. These neurons were distributed mainly in lamina I of the medullary and spinal dorsal horns, lateral spinal nucleus, regions around the central canal of the spinal cord, and the lateral aspect of the deep part of the spinal dorsal horn. A number of SPR-LI neurons in the spinal parasympathetic nucleus were labeled with FG injected into the area around the paraventricular hypothalamic nucleus. Some SPR-LI neurons in the lateral spinal nucleus and the lateral aspect of the deep part of the spinal dorsal horn were also labeled with FG injected into the septal region. On the basis of the distribution areas of SPR-LI trigeminal and spinal neurons projecting to the hypothalamic and septal regions, it is likely that these neurons are involved in the transmission of somatic and/or visceral noxious information.     

Silent glutamatergic synapses and nociception in mammalian spinal cord

Neurons in the superficial dorsal horn of the spinal cord are important for conveying sensory information from the periphery to the central nervous system. Some synapses between primary afferent fibres and spinal dorsal horn neurons may be inefficient or silent. Ineffective sensory transmission could result from a small postsynaptic current that fails to depolarize the cell to threshold for an action potential or from a cell with a normal postsynaptic current but an increased threshold for action potentials. Here we show that some cells in the superficial dorsal horn of the lumbar spinal cord have silent synapses: they do not respond unless the holding potential is moved from -70 mV to +40 mV. Serotonin (5-hydroxytryptamine, 5-HT), an important neurotransmitter of the raphe-spinal projecting pathway, transforms silent glutamatergic synapses into functional ones. Therefore, transformation of silent glutamatergic synapses may serve as a cellular mechanism for central plasticity in the spinal cord.     

Evidence for the interaction of glutamate and NK1 receptors in the periphery

It is known that Substance P (SP) enhances glutamate- and N-methyl-D-aspartate (NMDA)-induced activity in spinal cord dorsal horn neurons and that this enhancement is important in the generation of wind-up and central sensitization. It is now known that SP and glutamate receptors are present on sensory axons in rat glabrous skin. This raises the issue as to whether SP and glutamate interact in the periphery. Using the tail skin in rats, the present study demonstrates 1) that unmyelinated axons at the dermal-epidermal junction immunostain for antibodies directed against NMDA, non-NMDA or SP (NK1) receptors; 2) that glutamate injected into the tail skin results in dose-dependent nociceptive behaviors interpreted as mechanical hyperalgesia, mechanical allodynia and thermal hyperalgesia, which are blocked following co-injection with glutamate antagonists; 3) that peripheral injection of SP potentiates glutamate-induced nociceptive behaviors in that the co-injection of SP+glutamate results in a significantly longer duration of behavioral responses compared to the responses seen following injection of either substance alone. These data provide support for the hypothesis that primary afferent neurons might well be subject to similar mechanisms that result in wind-up or central sensitization of spinal cord neurons.     

AMPA receptors at primary afferent synapses in substantia gelatinosa after sciatic nerve section

Increased excitability of superficial laminae of the spinal cord may contribute to the pathological pain consequent to peripheral nerve injury. Among several mechanisms that may be responsible for this occurrence is upregulation of receptors for glutamate in the spinal cord. To explore this possibility, we investigated changes in AMPA receptors in substantia gelatinosa of rats after section of the sciatic nerve. Immunofluorescence was performed on sections from the fourth lumbar segment. Quantitative analysis of digitally captured images suggested that staining for an antibody to a sequence shared by GluR2 and GluR3 (GluR2/3) was increased on the side ipsilateral to the lesion. To determine whether antigen accumulation was at synaptic sites and to probe whether it was selective for primary afferent terminals, we performed electron microscopy on immunogold-labelled material. Gold particles coding for GluR2/3 subunits were counted from synaptic active zones of glomerular terminals in substantia gelatinosa that originate from small calibre afferent fibres, and from active zones of terminals of probable intrinsic origin. Counts were significantly increased on the side ipsilateral to the lesion only at synapses of primary afferent terminals. These results document selective upregulation of receptor protein at the synapse. This upregulation may contribute to the increased sensitivity of dorsal horn neurons following peripheral nerve injury.     

ATP stimulates sympathetic transmitter release via presynaptic P2X purinoceptors

ATP is a fast transmitter in sympathetic ganglia and at the sympathoeffector junction. In primary cultures of dissociated rat superior cervical ganglion neurons, ATP elicits noradrenaline release in an entirely Ca2+-dependent manner. Nevertheless, ATP-evoked noradrenaline release was only partially reduced (by approximately 50%) when either Na+ or Ca2+ channels were blocked, which indicates that ATP receptors themselves mediated transmembrane Ca2+ entry. An "axonal" preparation was obtained by removing ganglia from explant cultures, which left a network of neurites behind; immunostaining for axonal and dendritic markers revealed that all of these neurites were axons. In this preparation, ATP raised intraaxonal Ca2+ and triggered noradrenaline release, and these actions were not altered when Ca2+ channels were blocked by Cd2+. Hence, Ca2+-permeable ATP-gated ion channels, i.e., P2X purinoceptors, are located at presynaptic sites and directly mediate Ca2+-dependent transmitter release. These presynaptic P2X receptors displayed a rank order of agonist potency of ATP >/= 2-methylthio-ATP > ATPgammaS > alpha,beta-methylene-ATP approximately beta,gamma-methylene-L-ATP and were blocked by suramin or PPADS. ATP, 2-methylthio-ATP, and ATPgammaS also evoked inward currents measured at neuronal somata, but there these agonists were equipotent. Hence, presynaptic P2X receptors resemble the cloned P2X2 subtype, but they appear to differ from somatodendritic P2X receptors in terms of agonist sensitivity. Suramin reduced depolarization-evoked noradrenaline release by up to 20%, when autoinhibitory mechanisms were inactivated by pertussis toxin. These results indicate that presynaptic P2X purinoceptors mediate a positive, whereas G-protein-coupled P2Y purinoceptors mediate a negative, feedback modulation of sympathetic transmitter release.     

Synaptic strengthening through activation of Ca2+-permeable AMPA receptors

Postsynaptic Ca2+ elevation during synaptic transmission is an important trigger for short- and long-term changes in synaptic strength in the vertebrate central nervous system. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) receptors, a subfamily of glutamate receptors, mediate much of the excitatory synaptic transmission in the brain and spinal cord. It has been shown that a subtype of the AMPA receptor is Ca2+-permeable and is present in the subpopulations of neurons. When synaptically localized, these receptors should mediate postsynaptic Ca2+ influx, providing a trigger for changes in synaptic strength. Here we show that Ca2+-permeable AMPA receptors are synaptically localized on a subpopulation of dorsal horn neurons, and that they provide a synaptically gated route of Ca2+ entry, and that activation of these receptors strengthens synaptic transmission mediated by AMPA receptors. This pathway for postsynaptic Ca2+ influx may provide a new form of activity-dependent modulation of synaptic strength.     

Human and rat primary C-fibre afferents store and release secretoneurin, a novel neuropeptide

Secretoneurin is a recently discovered neuropeptide derived from secretogranin II (SgII). Since this peptide could be detected in the dorsal horn of the spinal cord we studied whether it is localized in and released from primary afferent neurons. Secretoneurin was investigated with immunocytochemistry and radioimmunoassay in spinal cord, dorsal root ganglia and peripheral organs. SgII mRNA was determined in dorsal root ganglia. Normal rats and rats pre-treated neonatally with capsaicin to destroy selectively polymodal nociceptive (C-) fibres were used. Slices of dorsal spinal cord were perfused in vitro for release experiments. Immunocytochemistry showed a distinct distribution of secretoneurin-immunoreactivity (IR) in the spinal cord and, lower brainstem. A particularly high density of fibres was found in lamina I and outer lamina II of the caudal trigeminal nucleus and of the spinal cord. This distribution was qualitatively identical in rat and human post-mortem tissue. Numerous small diameter and some large dorsal root ganglia neurons were found to contain SgII mRNA. Capsaicin treatment led to a marked depletion of secretoneurin-IR in the substantia gelatinosa, but not in other immunopositive areas of the spinal cord and to a substantial loss of small (< 25 microns) SgII-mRNA-containing dorsal root ganglia neurons. Radioimmunoassay revealed a significant decrease of secretoneurin-IR in the dorsal spinal cord, the trachea, heart and urinary bladder of capsaicin-treated rats. Perfusion of spinal cord slices with capsaicin as well as with 60 mM potassium led to a release of secretoneurin-IR. In conclusion, secretoneurin is a neuropeptide which is stored in and released from capsaicin-sensitive, primary afferent (C-fibre) neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of dopamine D2 receptor in rat spinal cord identified with immunocytochemistry and in situ hybridization

In the present study the distribution of dopamine D2 receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D2 receptor and in situ hybridization (ISH) using a [35S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D2 receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D2 receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D2 receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D2 receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.     

Peripheral nerve insult induces NMDA receptor-mediated, delayed degeneration in spinal neurons

Injury of a peripheral nerve gives rise to adaptive functional and structural alterations in spinal neurons. We report that the rearrangement of the spinal circuitry in response to sciatic nerve transection in adult rats involves a delayed mode of degeneration of lumbar spinal cord neurons. Nuclear fragmentation was detected by the TUNEL technique 7 days after sciatic neurectomy but not after 3 or 14 days. Dying cells were preferentially located in the ipsilateral superficial dorsal horn and expressed the neuronal cytoskeletal marker SMI-31. Degeneration was prevented by continuous systemic treatment with the NMDA receptor-antagonist MK-801. These data are supportive that apoptosis is induced in spinal neurons in a transsynaptic manner by an early signal from injured afferent fibres via activation of spinal NMDA receptors.     

Synthesis of thiazole-4-carboxamide-adenine difluoromethylenediphosphonates substituted with fluorine at C-2'' of the adenosine

Stimulation in the nucleus raphe magnus (NRM) inhibits transmission of nociceptive information within the spinal cord through activation of bulbospinal pathways. This study used microdialysis in combination with high pressure liquid chromatography to measure the release of serotonin (5HT) and several amino acids, including glutamate, aspartate and glycine, from the lumbar dorsal horn during electrical stimulation within the NRM in the alpha-chloralose anesthetized cat. Observed release of putative neurotransmitters was correlated with inhibition of nociceptive projection neurons recorded from sites within 800 microns rostral or caudal to the dialysis fiber. NRM stimulus parameters considered to preferentially activate myelinated fibers caused inhibition of nociceptive evoked activity, and increased the release of excitatory amino acids and glycine within the spinal cord, with no detectable release of 5HT. When pulse widths were lengthened and unmyelinated fibers were also activated, increases in 5HT in the spinal dialysate were observed as well. Strychnine administered through the dialysis fiber (0.02-1 mM) antagonized NRM-induced inhibition when 5HT release was not detected. Inhibition produced by stimulation that increased 5HT concentrations was relatively strychnine resistant. These results point to a raphe-spinal inhibitory pathway that is not dependent on 5HT, the activation of which results in the spinal release of glycine.     

NMDA EPSCs at glutamatergic synapses in the spinal cord dorsal horn of the postnatal rat

In rat dorsal horn, little is known about the properties of synaptic NMDA receptors during the first two postnatal weeks, a period of intense synaptogenesis. Using transverse spinal cord slices from postnatal day 0-15 rats, we show that 20% of glutamatergic synapses tested at low-stimulation intensity in spinal cord laminae I and II were mediated exclusively by NMDA receptors. Essentially all of the remaining glutamatergic EPSCs studied were attributable to the activation of both NMDA and AMPA receptors. Synaptic NMDA receptors at pure and mixed synapses showed similar sensitivity to membrane potential, independent of age, indicating similar Mg2+ sensitivity. Kinetic properties of NMDA EPSCs from pure and mixed synapses were measured at +50 mV. The 10-90% rise times of the pure NMDA EPSCs were slower (16 vs 10 msec), and the decay tau values were faster (tau1, 24 vs 42 msec; tau2, 267 vs 357 msec) than NMDA EPSCs at mixed synapses. Our results indicate that NMDA receptors are expressed at glutamatergic synapses at a high frequency, either alone or together with AMPA receptors, consistent with the prominent role of NMDA receptors in central sensitization (McMahon et al., 1993).