James A. Bell

Regulation of the NMDA receptor by redox phenomena: inhibitory role of ascorbate

Redox phenomena seem to modulate activity of the N-methyl-D-aspartate receptor. Some reductants (ascorbate, hydroquinone) inhibit, while others (dithiothreitol, mercaptoethanol, penicillamine) potentiate NMDA receptor function. Ascorbate inhibits binding of [3H]glutamate and [3H]thienylcycohexylpiperidine to the NMDA receptor complex, and impedes NMDA-gated currents in isolated neurons; dithiothreitol-like reductants enhance NMDA-induced currents. The ability of reductants to alter function of the NMDA receptor is abolished by oxidation.

The effect of the narcotic antagonists naloxone, naltrexone and nalorphine on spinal cord c-fiber reflexes evoked by electrical stimulation or radiant heat☆

C-fiber reflexes were recorded from an S1 ventral root in the acute decerebrate low spinal cat following stimulation of the ipsilateral superficial peroneal nerve or application of radiant heat to the metacarpel footpad. Naloxone when administered i.v. increased the electrically evoked C-fiber reflex to 158% (+/- 23.8% S.E.M.) of control 10 min after administration; whereas, naltrexone, 0.0025 mg/kg, increased the C-fiber reflex to 206 +/- 26.1% (S.E.M.) of control. Naloxone in a dose of 0.050 mg/kg increased the radiant heat evoked ventral root reflext to 161 +/- 19.5% of control. Nalorphine, 1 mg/kg, facilitated the electrically evoked C-fiber reflex to 282 +/- 75% of control. These findings that naloxone and naltrexone facilitated these reflexes in doses too small to have non-specific excitatory effects and that nalorphine facilitated the C-fiber reflex at a dose level that is depressant to the flexor reflex in the chronic spinal dog are consistent with a hypothesis that these effects are due to antagonism of a naturally occurring opiate-like inhibitory substance.

Inhibitory effects of dorsal horn and excitant effects of ventral horn intraspinal microinjections of norepinephrine and serotonin in the cat.

Abstract Norepinephrine and serotonin microinjections (10 μg) into the dorsal spinal gray matter depressed C-fiber reflexes induced by electrical stimulation of the superficial peroneal nerve or radiant heat stimulation of the footpad. Microinjection of norepinephrine or serotonin into the ventral gray matter facilitated C-fiber reflexes. These studies lend support to the suggestion that dorsal horn projections of neuronal systems which utilized norepinephrine or serotonin as neurotransmitters inhibit nociceptive spinal reflexes, and ventral horn projections facilitate spinal reflexes.

Clonidine attenuates increased brain glucose metabolism during naloxone-precipitated morphine withdrawal

The effect of two doses of clonidine on regional cerebral metabolic rates for glucose were measured during morphine withdrawal in rats. In the first study, 0 or 200 micrograms/kg clonidine was administered to rats subjected to naloxone-precipitated morphine withdrawal (naloxone, 0.5 mg/kg, s.c.), and to non-dependent control rats. In a second study of similar design, 0 or 20 micrograms/kg clonidine were administered. Withdrawal signs in rats subjected to naloxone-precipitated morphine withdrawal and receiving 0, 20 or 200 micrograms/kg clonidine were also assessed. Naloxone-precipitated morphine withdrawal stimulated regional cerebral metabolic rates for glucose (59 of 83 regions in study no. 1; 73 of 83 regions in study no. 2). At 200 micrograms/kg, clonidine attenuated this effect (33 of 59 regions). Although 200 micrograms/kg clonidine directly suppressed regional cerebral metabolic rates for glucose in many regions (significant main effect of clonidine), it attenuated the naloxone-precipitated morphine withdrawal effect specifically in the lateral septal nucleus, medial habenula, subiculum and gracile nucleus (significant interactions between clonidine and morphine withdrawal). The 20 micrograms/kg dose of clonidine had no statistically significant effect. In behavioral experiments, both doses of clonidine diminished withdrawal in that there was no diarrhea, fewer wet-dog shakes and less abnormal posturing. However, locomotion, grooming and jumping were increased by clonidine. Most of these effects were statistically significant only with the 200 micrograms/kg dose. The results of these studies show that clonidine reduces morphine withdrawal-induced increases in regional cerebral metabolic rates for glucose in many brain regions, irrespective of the distribution of alpha 2-adrenoceptors. Although clonidine has been thought to ameliorate morphine withdrawal by actions primarily at the locus coeruleus and central amygdala, it may play a major role in other regions as well.

Responses of the flexor reflex to LSD, tryptamine, 5-hydroxytryptophan, methoxamine, and d-amphetamine in acute and chronic spinal rats

The flexor reflex of acute (40–48 h after mid-thoracic spinal transection) and chronic (at least 2 months after transection) spinal rats was evoked by tetanic electrical stimulation of both hindfeet and recorded on a polygraph using a transducer connected to the left hindfoot. The flexor reflex in the chronic spinal rat was more responsive to electrical stimulation and to the actions of drugs studied than was the flexor reflex in the acute spinal rat. In chronic spinal rats, d-amphetamine, methoxamine, LSD, tryptamine, and 5-hydroxytryptophan (5-HTP) facilitated the flexor reflex and induced spontaneous movements. These facilitative effects were seen in acute spinal rats only when much larger i.p. doses of amphetamine, methoxamine, and LSD were used. Small i.v. doses of tryptamine also produced the facilitation. The facilitation caused by LSD and tryptamine, but not 5-HTP, in chronic spinal rats was antagonized by cyproheptadine. These observations suggest that chronic spinal rats were more sensitive to the drugs than acute spinal rats and support the hypothesis that the mode of action of LSD is similar to that of tryptamine but different from that of 5-HTP since cyproheptadine antagonized the facilitative effects of LSD and tryptamine but not those of 5-HTP.

Functional corticotropin-releasing factor receptors in neonatal rat spinal cord

The present study localized corticotropin-releasing factor (CRF) receptors and studied the action of CRF in the neonatal rat spinal cord preparation. Lumbar CRF receptors were present in highest concentrations in laminae I and II with progressively lower concentrations in lamina IX and intermediate and central zones respectively. CRF directly and indirectly depolarized lumbar motoneurons in a concentration-related manner and the putative receptor antagonist, alpha helical oCRF(9-41), partially blocked the depolarizing response to CRF. The electrophysiological responses to CRF and the distribution of receptors within the spinal cord suggest that CRF may play a physiological role in regulating spinal cord reflex function.

Depressant and excitant effects of intraspinal microinjections of morphine and methionine-enkephalin in the cat

The effects of intraspinal microinjectins of morphine (10 microgram) and methionine-enkephalin (Met-enkephalin) (5 microgram) on the C-fiber and polysynaptic reflexes in the acute decerebrate low spinal cat were investigated. Microinjected into the dorsal horn, morphine and Met-enkephalin depressed the nociceptive C-fiber reflex (CFR) without altering the short latency polysynaptic reflex. Microinjected into the ventral horn, morphine and Met-enkephalin facilitated the C-fiber and polysynaptic reflexes. Pretreatment of the cats with intravenous naltrexone (2 mg/kg) antagonized the depressant effects produced by dorsal horn intraspinal microinjections of morphine and Met-enkephalin. The excitant effects of ventral horn microinjections of morphine were not antagonized by naltrexone (2 mg/kg). These results support a hypothesis that the analgesic effects of morphine at the spinal cord level are due to interactions with opiate receptors in the dorsal horn.

Co-treatment with MK-801 potentiates naloxone-predpitated morphine withdrawal in the isolated spinal cord of the neonatal rat

The effects of acute and chronic administration of (MK-801: [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate) were assessed on morphine dependence in the isolated spinal cord of the neonatal rat and on behavioral measures in intact adult rats. Neonatal rats were treated chronically (3 or 4 days) with injections of either morphine, morphine + MK-801, or saline. Naloxone (10 microM) which increased baseline ventral root spontaneous firing, induced more activity in spinal cords from morphine-treated neonates than in saline controls. In spinal cords from neonates receiving MK-801 with morphine, naloxone-induced spontaneous firing was significantly greater than in saline-treated and morphine alone-treated neonates. Acute MK-801 attenuated naloxone-induced firing in the morphine-treated group. Chronic co-treatment with MK-801 increased locomotor signs of withdrawal and decreased mastication in intact adult rats which had been treated chronically with morphine. MK-801-induced enhancement of morphine withdrawal is consistent with upregulation of NMDA receptors.

Electrophysiological evidence for a presynaptic mechanism of morphine withdrawal in the neonatal rat spinal cord

Dorsal and ventral root depolarizing responses to capsaicin (1 microM) and substance P (SP; 1 microM) were measured from the isolated, hemisected spinal cord of the neonatal rat. Capsaicin depolarized the dorsal and ventral roots. The mechanism of ventral root depolarization was presynaptic; since dorsal root depolarization preceded the ventral, and the ventral depolarization was eliminated when synaptic transmission was blocked in the absence of calcium. SP depolarized the ventral root without affecting the dorsal root. The SP-induced depolarization of the ventral root was reduced but not abolished by blocking synaptic transmission with low calcium, suggesting that SP acted postsynaptically on motoneurons and excitatory interneurons to depolarize the ventral root. Morphine (10 microM) abolished the capsaicin-induced ventral root depolarization, but only slightly suppressed the SP response (30%). The capsaicin-induced depolarization of the ventral root was enhanced greatly (238%) when morphine, which had been in the superfusion for 1 h, was removed and naloxone (1 microM) was added to the superfusion solution, whereas the SP response was not augmented during withdrawal from acute morphine. Furthermore, a putative SP antagonist ([D-Arg1, D-Pro2, D-Tryp7,9, Leu11]-SP) prevented the augmented capsaicin ventral root response during precipitated withdrawal. These data provide electrophysiological evidence for a presynaptic mechanism of acute morphine withdrawal in the neonatal rat spinal cord.

Locus coeruleus neurons from morphine-treated rats do not show opiate-withdrawal hyperactivity in vitro

In vitro studies have not consistently demonstrated naloxone-precipitated opiate-withdrawal hyperactivity of locus coeruleus neurons. The reason for this inconsistency may be because partial or complete withdrawal occurred during preparation of the locus coeruleus slice. The aim of the present study was to assay opiate withdrawal-related hyperactivity in neurons recorded from locus coeruleus slices while ensuring the maintenance of dependence until naloxone-precipitated withdrawal. Extracellular recordings were obtained from individual locus coeruleus neurons in slices from morphine-treated and drug-naive rats. Morphine 1 microM was present in all solutions during preparation and recording in slices from morphine-treated rats. The average firing rate of the drug-naive controls was 0.93 Hz (+/-0.04 Hz). Bath application of morphine (1 microM) almost completely suppressed firing in drug-naive controls (0.058 Hz, +/-0.04 Hz, n=12), whereas in solutions containing 1 microM morphine, the firing rate of cells from morphine-treated rats averaged 0.71 Hz (+/-0.05 Hz), indicating considerable, but incomplete tolerance. In the same slices, naloxone increased the average spontaneous firing of locus coeruleus cells to 0.96 Hz (+/-0. 04 Hz). Thus, naloxone did not produce withdrawal hyperactivity, but returned the cells from morphine-treated rats to control rates. We conclude that locus coeruleus cells in locus coeruleus slice preparations from morphine-treated rats did not demonstrate withdrawal-related hyperactivity even when dependence was maintained until naloxone-precipitated withdrawal. Thus, our results do not support a role for adaptations intrinsic to locus coeruleus neurons in withdrawal hyperexcitability, but instead imply the necessity of functional afferent activity.