Segmental analysis of neuropeptide concentrations in normal human spinal cord.

We concurrently measured, by radioimmunoassay, levels of substance P (SP), somatostatin (SST), methionine-enkephalin (Met-Enk), cholecystokinin (CCK), peptide hystidyl-isoleucine (PHI), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) in the ventral and dorsal gray matter at each segment of the cervical, thoracic, lumbar, and sacral spinal cord, obtained within 6 hours of death from 4 subjects (ages 17 to 55) with no neurologic disease. Levels (pmol/g gray matter) of SP, SST, and Met-Enk throughout and PHI, VIP, and NPY in lumbar and sacral cord were significantly higher in dorsal than in ventral gray matter. PHI, VIP, and NPY were significantly higher in lumbar and especially sacral cord than in cervical and thoracic segments. In rats, a postmortem delay of up to 8 hours did not affect SP, Met-Enk, PHI, or NPY and decreased SST, CCK, and VIP levels. Thus, there is a characteristic profile of neuropeptide distribution in gray matter, which emphasizes the neurochemical heterogeneity along the rostrocaudal and dorsoventral extent of normal human spinal cord.

Stimulation-produced descending inhibition from the periaqueductal gray and nucleus raphe magnus in the rat: mediation by spinal monoamines but not opioids.

Focal electrical stimulation in the midbrain periaqueductal gray (PAG) or medullary nucleus raphe magnus (NRM) inhibits spinal nociceptive transmission and nociceptive reflexes. The purpose of this study was to evaluate, in lightly pentobarbital-anesthetized rats, the spinal neurotransmitter(s) mediating descending inhibition of the nociceptive tail-flick (TF) reflex produced by focal electrical stimulation in the PAG or NRM. To characterize the neurotransmitter(s) mediating inhibition of the TF reflex, selective pharmacologic antagonists were administered into the lumbar intrathecal space. Stimulation thresholds in the PAG or NRM for inhibition of the TF reflex were established and the effects of intrathecally administered phentolamine, yohimbine, prazosin, methysergide (15 micrograms initially, 30 micrograms cumulative) or naloxone (10 micrograms initially, 20 micrograms cumulative) on TF inhibitory thresholds determined. Phentolamine, yohimbine and methysergide increased the intensity of stimulation in the PAG and the NRM for inhibition of the TF reflex; prazosin and naloxone had no effect. Descending inhibition produced by focal electrical stimulation in the PAG or NRM is mediated in part by spinal serotonergic and/or alpha 2-adrenergic receptors. Naloxone was administered both intrathecally and intravenously; however, a role for opioid receptors in descending inhibition from the midbrain or medulla was not found.

Discovery of 6,11-ethano-12,12-diaryl-6,11-dihydrobenzo[b]quinolizinium cations, a novel class of N-methyl-D-aspartate antagonists.

6,11-Ethano-12,12-diaryl-6,11-dihydrobenzo[b]quinolizinium cations 8, a novel class of N-methyl-D-aspartate (NMDA) antagonists acting at the phencyclidine site, have been identified. Structure-activity relationship studies around the lead compound 8a led to the identification of 12g (WIN 67870-2), one of the most potent compounds in this series. Compound 12g has a Ki = 1.8 +/- 0.2 nM vs [3H]TCP binding, has 700-fold selectivity for binding to the open state of the NMDA receptor-ionophore, and was devoid of MK-801- and PCP-like behavioral effects in rats. Compound 12g was neuroprotective in cultured mouse cortical neurons and exhibited antiischemic activity in a rat middle cerebral artery occlusion/reperfusion model of focal ischemia.

Novel NMDA antagonists: replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with heteroisoquinolinium cations.

Replacement of the pyridinium ring of 6,11-ethanobenzo[b]quinolizinium cations with thiazolium (4a and 4b) and N-methylimidazolium (4c and 4d) resulted in equipotent compounds in the [3H]TCP binding assay. The corresponding N-methyl-1,2,4-triazolium analogs were less potent in this assay. The thiazolium derivative 4b, with a Ki = 2.9 nM, is being evaluated as a possible neuroprotective N-methyl-D-aspartic acid (NMDA) antagonist.

Synthesis and substance P receptor binding activity of androstano[3,2-b]pyrimido[1,2-a]benzimidazoles.

Several heterosteroids containing a dihydroethisterone skeleton were prepared and shown to displace substance P in a receptor binding assay. Further biochemical (kinetic and Scatchard analyses) and pharmacological evaluation (substance P-induced plasma extravasation and salivation in the rat) of a representative example in this series (5a) established that these compounds are competitive antagonists at the substance P receptor.

Novel benzo[b]quinolizinium cations as uncompetitive N-methyl-D-aspartic acid (NMDA) antagonists: the relationship between log D and agonist independent (closed) NMDA channel block.

A series of permanently charged benzo[b]quinolizinium cations having lower lipophilicity than MK-801 or phencyclidine (PCP) were synthesized. Data relating agonist independent block of N-methyl-D-aspartic acid (NMDA) ion channels to log D are described. Closed channel access is predicted to result in a more noncompetitive profile of antagonism compared to selective open channel blockers, which are uncompetitive inhibitors. Reduced closed channel block may underlie the absence of PCP or MK-801-like behavioral side effects observed for benzo[b]-quinolizinium cations.

Opioid modulation of capsaicin-evoked release of substance P from rat spinal cord in vivo.

Capsaicin has been shown to evoke the release of substance P (SP) from small diameter primary afferent fibers. Using an in vivo perfusion of the rat spinal cord, this study examined the pharmacology of opioid receptor systems which modulate the capsaicin-evoked release of SP. The addition of capsaicin (200 microM) to the perfusate raised SP-like immunoreactivity (SP-LI) from resting levels of 31 +/- 5 to 74 +/- 14 pg/ml or an increase of 139% above the baseline. Using high pressure liquid chromatography (HPLC) the identity of the released SP-LI was determined to coelute primarily with authentic SP or the oxidized form of SP. Opioid receptor agonists were added to the perfusate and their ability to inhibit capsaicin-evoked release of SP-LI was assessed. Morphine (10-100 microM), DAGO (1-100 microM), DPLPE (10-100 microM), but not U50488H (100 microM) produced a dose-dependent reduction in the capsaicin-evoked release of SP-LI. Pretreatment with the opioid receptor antagonist naloxone (1 mg/kg, IP) had no effect on the basal or capsaicin-evoked release of SP-LI. Naloxone pretreatment was able to antagonize completely the opioid-produced inhibition of capsaicin-evoked SP-LI release. These data indicate that the release of SP from primary afferent fibers can be modulated by the activation of mu or delta but not kappa opioid receptors. Further, these data support the hypothesis that spinally administered mu and delta opioid agonists may produce their antinociceptive effect through the presynaptic inhibition of neuropeptide release from small diameter primary afferent fibers.

Distribution of galanin-like immunoreactivity in the pig, rat and human central nervous system.

The distribution of galanin-like immunoreactivity in various regions of the central nervous system was assessed in three mammalian species, pig, rat, and human, by radioimmunoassay. Galanin concentrations were highest in the hypothalamus and pituitary region. In spinal cord, there was a rostrocaudal/dorsoventral gradient with highest levels observed in the sacral dorsal horn. Serial dilutions of porcine tissue extracts diluted parallel to the porcine standard curve, while the rat and human tissue extracts did not. In all tissues examined by high pressure liquid chromatography, the principal peak of immunoreactivity coeluted with the authentic porcine galanin standard and was decreased by trypsin cleavage. These results suggest a role for galanin in the central nervous system and support species differences in the structure of galanin.

Distribution of neuropeptide Y in the spinal cords of cat, dog, rat, man and pig.

Regional distribution of neuropeptide Y (NPY) in spinal cord, dorsal root ganglia (DRG) and peripheral nerves was quantitated in rat, cat, dog, pig, and man. Spinal cords were harvested post mortem and dissected into regions or individual segments. A further dissection into dorsal and ventral horns was carried out, and DRG were harvested in all species except rat. Tissues were extracted into boiling 0.1 M HCl, and NPY was measured by radioimmunoassay using a specific antibody and I125-labeled NPY. Highest concentrations of NPY were consistently found in the dorsal horn of the lumbo-sacral cord (200-800 ng/g). DRG concentrations, in contrast, were routinely low or undetectable. Sciatic nerve concentrations in rat and pig were considerable. High performance liquid chromatography (HPLC) confirmed that the NPY immunoreactivity measured in dorsal horns of each species coeluted with authentic NPY (1-36) as a single peak.

Serotonin and/or an excitatory amino acid in the medial medulla mediates stimulation-produced antinociception from the lateral hypothalamus in the rat.

Several lines of evidence have demonstrated a role for the lateral hypothalamus (LH) in an endogenous system of descending inhibition. Descending inhibition from the LH relies, at least in part, on a relay(s) in the midbrain and/or medulla. The medullary nucleus raphe magnus (NRM) serves as one such relay. The present study, in rats lightly anesthetized with pentobarbital, was undertaken to systematically examine the transmitter(s) in the medial medulla mediating descending inhibition of the nociceptive tail flick (TF) reflex produced by focal electrical stimulation in the LH. The microinjection of pharmacologic receptor antagonists (5 micrograms) into the NRM revealed that the glutamate receptor antagonists, gamma-D-glutamylglycine and 2-amino-5-phosphonovalerate produced the largest increases in stimulation thresholds in the LH for inhibition of the TF reflex (107.6% and 102.6%, respectively). Methysergide, a serotonin receptor antagonist, also produced a significant increase (81.5%) in the stimulation threshold in the LH for inhibition of the TF reflex. The opioid receptor antagonist, naloxone, however, was without effect, producing only a 4.0% increase in the LH stimulation threshold. These results suggest that serotonin and/or an excitatory amino acid are transmitters at the bulbar relay in the medial medulla mediating descending inhibition of the TF reflex produced by focal electrical stimulation in the LH.

Spinal monoamine mediation of stimulation-produced antinociception from the lateral hypothalamus.

Stimulation-produced antinociception can be evoked from a wide variety of sites in the brain, including the lateral hypothalamus (LH). The present study, in rats lightly anesthetized with pentobarbital, examined descending inhibition of the nociceptive tail flick (TF) reflex produced by focal electrical stimulation in the LH and the neurotransmitter(s), at the level of the lumbar enlargement, mediating the inhibition. Systematic tracking studies demonstrated that stimulation in the diencephalon dorsal to the hypothalamus did not reliably inhibit the TF reflex. Inhibition of the TF reflex was produced, however, throughout the hypothalamus at intensities of stimulation typically between 50 and 200 microA. The area requiring low intensities of stimulation (50-100 microA) to inhibit the TF reflex was a diffuse region of the LH, inferior to the mammillothalamic tract and internal capsule, medial to the supraoptic decussation and including the medial forebrain bundle. Microinjections of S-glutamate (100 mM, 0.5 microliter) in the LH did not inhibit the TF reflex, suggesting that activation of fibers of passage by stimulation was responsible for inhibition of the TF reflex produced from the LH. The intrathecal administration of pharmacologic antagonists (15-30 micrograms; naloxone, methysergide, phentolamine, prazosin or yohimbine) revealed that the alpha-adrenoceptor antagonists phentolamine and yohimbine produced the greatest increases in stimulation thresholds in the LH for inhibition of the TF reflex (83.7% and 89.8%, respectively). The intrathecal administration of methysergide produced a lesser, but statistically significant 11% increase in the stimulation threshold for inhibition of the TF reflex. These results indicate that spinal alpha 2-adrenoceptors primarily mediate the descending inhibition of the TF reflex produced by electrical stimulation in the LH.

Medullary substrates of descending spinal inhibition activated by intravenous administration of [D-Ala2]methionine enkephalinamide in the rat.

Two experiments were performed in pentobarbital-anesthetized rats to provide information about the locus of the descending inhibitory system(s) in the brainstem engaged by [D-Ala2]methionine enkephalinamide (DALA) activation of vagal afferents. In Expt. 1, local anesthetic block of either the medial medullary nucleus raphe magnus (NRM) or the combined NRM-lateral medullary reticular formation (MRF) region failed to affect DALA-induced inhibition of the tail-flick reflex or reflex bradycardia. The hypotensive action of DALA was marginally enhanced in rats with a combined NRM-MRF local anesthetic block. However, inhibition of the tail-flick reflex produced by electrical stimulation in the NRM-MRF was eliminated by the local anesthetic. In Expt. 2, local anesthetic blocks of regions at the level of and caudal to obex were effective in blocking DALA-induced inhibition of the tail-flick reflex. These regions included lateral sites approximately 0.4 mm rostral to obex to -2.4 mm caudal to obex, and midline sites approximately -2.4 to -3.2 mm caudal to obex. These findings suggest that brainstem areas mediating DALA-induced antinociception are lateral to the midline at the level of the obex. The spinopetal efferents mediating antinociception then course medially and ventrally, and finally course bilaterally in the dorsolateral funiculi before terminating in the spinal dorsal horn.

Stimulation-produced spinal inhibition from the midbrain in the rat is mediated by an excitatory amino acid neurotransmitter in the medial medulla.

It has been previously established that a bulbar relay plays an important role in descending inhibition of spinal dorsal horn nociceptive neurons and nociceptive reflexes produced by stimulation in the midbrain periaqueductal gray (PAG). In the present study, selected receptor antagonists were microinjected into the medial medullary nucleus raphe magnus (NRM) to determine whether descending inhibition of the tail flick (TF) reflex in the rat produced by focal electrical stimulation in the midbrain PAG was mediated by serotonin, opioid, or glutamate receptors on bulbospinal neurons in the NRM. It was determined in initial experiments that the serotonin receptor antagonist methysergide, the opioid receptor antagonist naloxone, the local anesthetic lidocaine, and the glutamate receptor antagonists gamma-D-glutamylglycine (DGG) and DL-2-amino-5-phosphonovalerate (APV) microinjected into the medulla all significantly increased the threshold of focal electrical stimulation in the medulla required to inhibit the TF reflex. The antinociceptive efficacy of agonists at opioid, serotonin, and glutamate receptors was also tested in other experiments. The microinjection of morphine (2.5-10 micrograms) into the NRM increased significantly TF latencies in a dose-dependent manner in rats in the awake or lightly anesthetized state; morphine was more potent in awake rats. Inhibition of the TF reflex produced by the microinjection of morphine was reversed by a subsequent microinjection of naloxone into the same site in the medulla. The microinjection of serotonin (5 and 10 micrograms), however, did not affect the latency of the TF reflex in either awake or lightly anesthetized rats. Glutamate (100 microM, 0.5 microliter) microinjected into the rostral ventral medulla produced an inhibition of the TF reflex of short duration that could be blocked or attenuated significantly by the glutamate receptor antagonists DGG or APV microinjected into the same site. In subsequent experiments, a nonspecific functional block was introduced adjacent to the NRM bilaterally in the medullary reticular formations (MRFs) by the microinjection of the local anesthetic lidocaine; receptor antagonists were then microinjected into the NRM and their effect on the threshold of focal electrical stimulation in the PAG to inhibit the TF reflex determined. No increase was seen in stimulation thresholds in the PAG following the microinjection of either methysergide or naloxone into the NRM. Following the microinjection of lidocaine, DGG or APV into the NRM, the stimulation threshold in the PAG for inhibition of the TF reflex was increased significantly.(ABSTRACT TRUNCATED AT 400 WORDS)

Brain-stem relays mediating stimulation-produced antinociception from the lateral hypothalamus in the rat.

Several lines of evidence have demonstrated a role for the lateral hypothalamus (LH) in an endogenous system of descending inhibition. The present study, in rats lightly anesthetized with pentobarbital, was undertaken to examine systematically the organization in the brain stem of pathways mediating descending inhibition of the nociceptive tail flick (TF) reflex produced by focal electrical stimulation in the LH. The microinjection of lidocaine into the midbrain, dorsolateral pons, or medial medulla resulted in significant increases in stimulation thresholds in the LH for inhibition of the TF reflex (89.1, 67.4, and 73.6%, respectively). Selective lesions of cell bodies in the midbrain or medulla by the neurotoxin ibotenic acid also produced significant increases in stimulation thresholds in the LH for inhibition of the TF reflex (31.6 and 131.6%, respectively), thus revealing relays in the periaqueductal gray and the nucleus raphe magnus located between the LH and the lumbar spinal cord. The failure of ibotenic acid to affect LH-produced descending inhibition when microinjected into the dorsolateral pons, and the significant effect produced by lidocaine microinjected into the same area, implicates fibers of passage in the dorsolateral pons in descending inhibition of the TF reflex produced by focal electrical stimulation in the LH. The fluorescent dye Fast blue and HRP conjugated to wheat germ agglutinin were used to confirm that the area stimulated in the LH has reciprocal connections with the periaqueductal gray and nucleus raphe magnus.