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Vesicular Zn2+, released in the brain and from small dorsal root ganglion neurons, interacts with opioid as well as N-methyl-D-aspartate (NMDA) receptors. We investigated the effect of Zn2+ on morphine antinociception in mice (tail flick assay), as well as acute tolerance and dependence, phenomena associated with NMDA activity. Administered intrathecally (i.t.), Zn2+ inhibited morphine antinociception in a dose-related fashion. Zn2+ also inhibited acute tolerance to morphine antinociception (5 h after 100 mg/kg of morphine). Injection i.t. of di-sodium calcium ethylenediamine tetra acetic acid (Na+Ca2+ EDTA), a chelator of divalent cations, had no effect on analgesia, acute tolerance or acute dependence. However, withdrawal jumps produced by naloxone (1 mg/kg s.c.) in morphine-pellet implanted mice (3 days) were potentiated by injections twice daily of 10 nmol of Na+Ca2+ EDTA, suggesting that endogenous Zn2+ tends to inhibit long-term development of withdrawal. These data suggest that the availability of Zn2+ is an important factor in opioid activity.

To monitor the possible effect of morphine on sigma sites, binding characteristics of [3H](+)-pentazocine and [3H]1,3-di-(2-tolyl)guanidine (DTG) to brain and spinal cord membranes of morphine-treated and control mice were compared. For morphine treatment, a single injection (100 mg/kg, s.c.) of morphine was followed 4 h later by pellet implantation (75 mg morphine free base). Animals were sacrificed 24, 72 h or 7 days later. The equilibrium dissociation value (Kd) and the density (Bmax) of [3H](+)-pentazocine binding remained unaffected by morphine treatment. Also, no change was found in Kd and Bmax values of [3H]DTG labeled sigma2 subtypes after any morphine treatment schedule when measured in the presence of 100 nM (+)-pentazocine. However, the Bmax of [3H]DTG binding in the spinal cord in the absence of 100 nM (+)-pentazocine, was significantly elevated 72 h after implantation of the morphine pellet and recovered by 7 days, a time when the antinociceptive effect produced by the morphine pellet had dissipated. These data suggest that one population of sigma sites, that has a high affinity for [3H]DTG, but is not equivalent with the [3H](+)-pentozocine labeled sigma1 subtype or the [3H]DTG labeled sigma2 subtype, is upregulated by morphine and, therefore, may play a role in the development of tolerance to or dependence on the effects of morphine.

Zn2+ inhibits NMDA-type excitatory amino acid activity by a non-competitive action. Based on regional differences in the central nervous system (CNS) in binding characteristics of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) and other non-competitive antagonists of NMDA used to label open channels in the receptor complex, we compared the inhibitory influence of Zn2+ on [3H]MK-801 binding in whole mouse brain and spinal cord membranes. Radioligand binding techniques were used in the presence and absence of maximally effective concentrations of glycine and glutamate. Using extensively washed membranes without exogenous glycine and glutamate, Zn2+ was found to be a weaker inhibitor of the [3H]MK-801-labeled site in the spinal cord than in the whole brain. In contrast, exogenous glycine and glutamate decreased the inhibitory effect of Zn2+ in the brain but dramatically increased the inhibitory effect of Zn2+ in the spinal cord. Thus the inhibitory effect of Zn2+ in the spinal cord appears to be magnified by glutamatergic and glycinergic activity while that in the brain is not. The different actions of Zn2+ may be attributable to the differential distribution of NMDA receptor subunits in the mouse brain and spinal cord.

Behaviors induced in mice by intrathecal injections of either N-methyl-D-aspartate (NMDA) or kainic acid are modulated by NH2-terminal fragments of substance P, such as substance P-(1-7). The action of substance P-(1-7) on kainic acid depends on sigma receptor activity. The present study was designed to test the hypothesis that sigma receptor activity is also necessary for modulation of NMDA by substance P-(1-7). Intrathecal injection of mice with NMDA results in a brief burst of biting and scratching behaviors which decrease in intensity when NMDA is injected repeatedly at 2 min intervals. Pretreatment with 1,3-di-O-tolylguanidine (DTG), a ligand at both sigma 1 and sigma 2 sites, converted NMDA-induced desensitization to sensitization, thereby enhancing tonic NMDA receptor activity. Although haloperidol (30 min) alone was without effect, the potentiation of NMDA-induced activity by DTG was abolished by haloperidol but unaffected by an equimolar dose of either spiperone or thiothixine, two dopamine receptor antagonists. When mice received substance P-(1-7), NMDA-induced behaviors were initially inhibited but then potentiated. Pretreatment with haloperidol prevented both inhibitory and potentiative effects of substance P-(1-7) whereas thiothixine did not, suggesting inhibitory as well as potentiative modulation of NMDA by sigma receptor activity. Endogenous sigma 1 receptor activity may enhance NMDA receptor activity as a treatment regimen that down-regulates sigma 1 binding also inhibited responses to NMDA. In contrast, pretreatment with haloperidol just 5 min prior to challenge, which blocks both sigma 1 and sigma 2 receptor activity, increased responses to NMDA suggesting an inhibitory effect of sigma 2 receptor activity. In summary, modulation of NMDA by substance P-(1-7) appears to depend on activity at sigma sites as substance P-(1-7) mimicked the potentiative effects of DTG, while haloperidol inhibited the effects of both DTG and substance P-(1-7).

The characteristics of [3H](+)-pentazocine and [3H]1,3-di(2-tolyl) guanidine (DTG) binding to mouse whole brain, cortex, cerebellum and spinal cord membranes were investigated in radioreceptor assays. [3H](+)-Pentazocine bound to a single, high affinity site (Kd = 1.2-1.6 nM) with increasing density along the neuraxis from the cortex (Bmax = 543 fmol/mg protein) to the spinal cord (Bmax = 886 fmol/mg protein). Hot saturation studies resolved the presence of one binding site for [3H]DTG showing no tissue variations in terms of density (Bmax = 1075-1264 fmol/mg protein) or affinity (Kd = 16.6-22.3 nM). Incubation with 100 nM (+)-pentazocine revealed two classes of high affinity [3H]DTG labeled binding sites corresponding to sigma 1 and sigma 2 subtypes. A preponderance of sigma 2 sites was revealed in all investigated tissues. Different pharmacological profiles were demonstrated for the sigma 2 sites in mouse whole bain compared to mouse spinal cord. However, competition studies indicated that the whole brain and spinal [3H](+)-pentazocine labeled sigma 1 binding sites exhibited similar pharmacological properties. The density of [3H](+)-pentazocine labeled sigma 1 population was found not to match that of [3H]DTG labeled sigma 1 site throughout the mouse central nervous system. The presence of low affinity [3H]DTG labeled sites was demonstrated in cold saturation experiments. Equilibrium binding data for the low affinity [3H]DTG binding site resulted in an increasing density (Bmax = 1973-11,369 fmol/mg protein) with a decreasing affinity (Kd = 242-943 nM) in mouse cortex through the spinal cord.

The role of nitric oxide (NO) in responses of spinal dorsal horn neurons to excitatory amino acids and to cutaneous mechanical stimuli was examined. Extracellular recordings were made from wide dynamic range neurons excited with iontophoretically applied excitatory amino acid agonists, N-methyl-D-aspartate (NMDA) and (R,S)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) or kainic acid. Nitric oxide availability was decreased by iontrophoretic application of NO synthase inhibitors, N omega-nitro-L-arginine methyl ester (L-NAME) or L-N5-(1-iminoethyl)ornithine (L-NIO), or elevated by the NO donating compound, S-nitroso-N-penicillamine (SNAP). When cells were excited with successive application of NMDA and non-NMDA excitatory amino acid receptor agonists, application of NO synthase inhibitors led to a decrease in responses to NMDA in 60% of neurons. In more than a third of the cells tested, inhibition of NO synthase caused reciprocal changes in responses to glutamate receptor agonists: NMDA-evoked responses were significantly decreased whereas responses to the non-NMDA receptor agonists (AMPA or kainic acid) were increased. Application of the NO donating compound, S-nitroso-N-penicillamine, revealed an opposite tendency, increasing responses to NMDA in more than half of the neurons tested. In approximately 40% of the cells, reciprocal changes in responses to excitatory amino acid receptor agonists of NMDA versus non-NMDA types were observed after application of S-nitroso-N-penicillamine, such that the increase in NMDA responses was accompanied by decreases in the responses to kainic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P injected intrathecally or a conditioning stimulus (1 Hz, 20 s) at C-fiber strength, which releases substance P from intraspinal primary afferent terminals, each enhance the hamstring muscle flexor reflex elicited by electrical stimulation of the sural nerve in decerebrate, spinalized rats. This suggests a role for substance P in pain transmission. Since substance P N-terminal metabolites are biologically active, we examined the effect of the metabolite substance P-(1-7) on the flexor reflex and the enhancement of the flexor reflex following a conditioning stimulus of the sural nerve. In contrast to the excitatory effect of substance P, intrathecal injection of substance P-(1-7) had no effect on the flexor reflex. However, 10 and 30 min after injection, 0.6 or 6 micrograms of substance P-(1-7) attenuated the facilitation of the flexor reflex by the conditioning stimulus. These data indicate that substance P-(1-7) may modulate the expression of nociception involving repetitive firing of C-fibers while having no significant effect on acute or phasic responses.

Substance P has been previously shown to inhibit the intensity of the morphine abstinence syndrome in mice. In view of the rapid degradation of substance P after its release from nerve terminals, we hypothesized that this inhibition is mediated by the N-terminus of substance P and its metabolites rather than via the C-terminus interacting with neurokinin receptors. Intrathecal injection of substance P-(1-7) (1 nmol) 30 min prior to naloxone challenge, in mice that had received 25 micrograms of morphine sulfate intrathecally once daily for three days, caused a dose-related attenuation of withdrawal jumping. In contrast, administration of the substance P-(1-7) antagonist, [D-Pro2,D-Phe7]substance P-(1-7), 15 min prior to naloxone increased withdrawal jumping behaviors. Equimolar doses of morphine and naloxone at 30 min had no effect. From these data, it appears that substance P N-terminal metabolites modulate withdrawal behaviors in morphine-dependent mice.

To determine whether opioid receptors or the more recently characterized naloxone-sensitive substance P (SP) N-terminal binding sites play a role in desensitization to the behavioral effects of SP, we assessed the effects of selective antagonists at mu-(naloxonazine and beta-funaltrexamine), delta- (naltrindole) and kappa- (nor-binaltorphimine) opioid receptors, as well as the effect of [D-Pro2,D-Leu7]SP-(1-7) D-SP-(1-7) (D-SP (1-7)), an inhibitor of [3H]SP-(1-7) binding, on behaviors induced by intrathecally administered SP in mice. Whereas naloxone, a non-selective opioid antagonist, inhibited the development of behavioral desensitization to SP, the response to repeated SP administration remained unaffected by pretreatment with selective opioid antagonists. Like naloxone, however, the SP-(1-7) antagonist inhibited SP-induced desensitization. The protection against desensitization to SP by D-SP-(1-7), but not by selective antagonists of mu, delta or kappa receptors, suggests that desensitization to the behavioral effects of SP does not appear to be mediated by an action at an opioid receptor but by an action at the SP-(1-7) binding site.

The effects of an N-terminal fragment of substance P, substance P-(1-7) [SP-(1-7)], on the responses of dorsal horn nociceptive neurons to N-methyl-D-aspartate (NMDA) and (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) were tested by combined single-unit extracellular recordings/microiontophoresis. While SP-(1-7) had no effects when applied by itself, it was a potent and long-lasting modulator of both NMDA- and AMPA-mediated excitation of spinal dorsal horn nociceptive neurons. NMDA responses were transiently decreased (by an average of 36% of control at minimum) by SP-(1-7) followed by a more sustained increase (by 76% at maximum). In contrast, AMP responses were only increased by SP-(1-7) (by 81% at maximum). It is hypothesized that the actions of SP-(1-7) on excitatory amino acid (EAA) responses of dorsal horn nociceptive neurons reflect a novel mechanism by which SP and EAAs interact to modulate pain transmission.

Urethane and ketamine were tested for their ability to alter the caudally directed binding and scratching response elicited by the intrathecal (i.t.) injection of excitatory amino acids (EAAs) or substance P (SP). EAAs, such as N-methyl-D-aspartate (NMDA), kainate acid and quisqualic acid, but not SP, were inhibited by subanesthetic doses of urethane. In contrast, SP was more sensitive than NMDA to the inhibitory effect of (+)-ketamine. (-)-Ketamine produced much less inhibition of the SP-induced behaviors than the (+)isomer. These results have important implications regarding the use of urethane and ketamine as anesthetics for studies in which these excitatory compounds are potential mediators.

Fluoroacetic and fluorocitric acid toxicity is often characterized by seizures, however the mechanism of this activity is unknown. Intrathecal (i.t.) injection of fluorocitrate in mice resulted in seizures after an average latency of 15 s, while intracerebroventricular (i.c.v.) injection produced seizures after 36.5 min, and required higher doses to achieve this effect. This indicates the probable site of fluoroacetate and fluorocitrate neurotoxicity is the spinal cord. To mimic citrate accumulation, characteristic of fluoroacetate and fluorocitrate poisoning, citric acid was injected i.t. and also found to produce seizures. The structurally unrelated compounds EDTA, EGTA, glutamic acid and lactic acid also produced seizures identical to fluorocitrate. The ability of these compounds to chelate Ca2+ correlates well with their ability to cause seizures when administered i.t. and coadministration of calcium greatly attenuated the neurotoxicity of these compounds as well as fluoroacetate and fluorocitrate. In contrast, Ca2+ was unable to inhibit seizures elicited by strychnine, suggesting calcium's ability to inhibit chelators of divalent cations is not due to a general anticonvulsant effect. These results suggest that changes in Ca2+ concentration in the spinal cord may be responsible for some forms of seizure activity.

In two strains of rats, intrathecal administration of morphine and met-enkephalin was found to produce a dose-dependent analgesic effect as measured by the tail-flick assay. Prior administration of leu-enkelphalin at a dose which had no analgesic effect was found to reduce significantly the analgesic ED50 of both morphine and met-enkephalin. Met-enkephalin pretreatment, at a similar sub-analgesic dose, was without effect. Thus, a differential interaction of the enkephalins with morphine is evident at spinal structures.

Pipecolic acid (PA) is an alicyclic amino acid and putative neurotransmitter which may modulate GABAergic transmission in the central nervous system. The present study was designed to investigate the anticonvulsant effect of intrathecally (i.t.) injected PA on picrotoxin- and bicuculline-induced convulsions which are thought to be produced by interactions with GABAergic systems. Intrathecal injections of picrotoxin and bicuculline in mice produced convulsions which were characterized by a rapid onset and short duration. Coadministration of GABA with either bicuculline or picrotoxin, but not strychnine, attenuated the severity of the convulsions. Coadministration of PA also protected against bicuculline- and picrotoxin-induced convulsions. Intrathecal injections of PA produced a dose-related increase in the latency to the onset of these convulsions as well as a decrease in their duration, however PA failed to inhibit the duration of strychnine-induced seizures. The D isomer of PA was found to be more effective than the L isomer as an anticonvulsant in this study. When administered in a high dose (500 micrograms i.t.), the D isomer produced flaccid paralysis while injection of high doses (100-500 micrograms i.t.) of the L isomer actually elicited convulsions. These results further support an interaction between PA and GABAergic activity.