Bupivacaine suppresses [Ca(2+)](i) oscillations in neonatal rat cardiomyocytes with increased extracellular K+ and is reversed with increased extracellular Mg(2+).

UNLABELLED: Lidocaine is used to treat cardiac arrhythmias, whereas bupivacaine is noted for its cardiotoxicity. A precise mechanism for these differences is unclear, and there is no well defined antidote for local anesthetic cardiotoxicity. Our study compares the effect of lidocaine and bupivacaine on oscillations of intracellular Ca(2+) coupled with contractions in neonatal rat cardiomyocytes by using digital imaging. In medium containing 5.6 mM K(+), both 42 microM lidocaine and 5.5 microM bupivacaine significantly reduced the oscillation rate. The oscillatory patterns were highly irregular, and the rates were increased in the presence of bupivacaine in 7.6 mM K(+) medium, eventually degenerating into a loss of oscillations after several minutes of bupivacaine exposure. Irregular oscillations did not occur with lidocaine until the K(+) concentration was increased to 10 mM. Increasing the Mg(2+) and Ca(2+) concentrations by 2 mM each recovered oscillation that had been suppressed by bupivacaine in high K(+) buffer. Evaluation of intracellular Ca(2+) oscillations in neonatal rat suggests that increased extracellular K(+) may be an important component of bupivacaine cardiotoxicity. IMPLICATIONS: Evaluation of intracellular Ca(2+) oscillations in neonatal rat myocytes suggests that increased extracellular K(+) may be an important component of bupivacaine cardiotoxicity.

Synergistic effect between intrathecal non-NMDA antagonist and gabapentin on allodynia induced by spinal nerve ligation in rats.

BACKGROUND: Glutamate and non-N-methyl-D-aspartate (NMDA) receptors have been implicated in the development of neuroplasticity in the spinal cord in neuropathic pain. The spinal cord has been identified as one of the sites of the analgesic action of gabapentin. In the current study, the authors determined the antiallodynic effect of intrathecal 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in a rat model of neuropathic pain. Also tested was a hypothesis that intrathecal injection of CNQX and gabapentin produces a synergistic effect on allodynia in neuropathic rats. METHODS: Allodynia was produced in rats by ligation of the left L5 and L6 spinal nerves. Allodynia was determined by application of von Frey filaments to the left hind paw. Through an implanted intrathecal catheter, 10-100 microg gabapentin or 0.5-8 microg CNQX disodium (a water-soluble formulation of CNQX) was injected in conscious rats. Isobolographic analysis was performed comparing the interaction of intrathecal gabapentin and CNQX using the ED50 dose ratio of 15:1. RESULTS: Intrathecal treatment with gabapentin or CNQX produced a dose-dependent increase in the withdrawal threshold to mechanical stimulation. The ED50 for gabapentin and CNQX was 45.9+/-4.65 and 3.4+/-0.22 microg, respectively. Intrathecal injection of a combination of CNQX and gabapentin produced a strong synergistic antiallodynic effect in neuropathic rats. CONCLUSIONS: This study shows that intrathecal administration of CNQX exhibits an antiallodynic effect in this rat model of neuropathic pain. Furthermore, CNQX and gabapentin, when combined intrathecally, produce a potent synergistic antiallodynic effect on neuropathic pain in spinal nerve-ligated rats.

Nitric oxide synthase inhibitor decreases NMDA-induced elevations of extracellular glutamate and intracellular Ca2+ levels via a cGMP-independent mechanism in cerebellar granule neurons.

These studies were designed to examine the differential effect of nitric oxide (NO) and cGMP on glutamate neurotransmission. In primary cultures of rat cerebellar granule cells, the glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulates the elevation of intracellular calcium concentration ([Ca2+]i), the release of glutamate, the synthesis of NO and an increase of cGMP. Although NO has been shown to stimulate guanylyl cyclase, it is unclear yet whether NO alters the NMDA-induced glutamate release and [Ca2+]i elevation. We showed that the NO synthase inhibitor, N(G)-monomethyl-L-arginine (NMMA), partially prevented the NMDA-induced release of glutamate and elevation of [Ca2+]i and completely blocked the elevation of cGMP. These effects of NO on glutamate release and [Ca2+]i elevation were unlikely to be secondary to cGMP as the cGMP analogue, dibutyryl cGMP (dBcGMP), did not suppress the effects of NMDA. Rather, dBcGMP slightly augmented the NMDA-induced elevation of [Ca2+]i with no change in the basal level of glutamate or [Ca2+]i. The extracellular NO scavenger hydroxocobalamine prevented the NMDA-induced release of glutamate providing indirect evidence that the effect of NO may act on the NMDA receptor. These results suggest that low concentration of NO has a role in maintaining the NMDA receptor activation in a cGMP-independent manner.

Effects of L-trans-pyrrolidine-2,4-dicarboxylate, a glutamate uptake inhibitor, on NMDA receptor-mediated calcium influx and extracellular glutamate accumulation in cultured cerebellar granule neurons.

Glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylate (PDC, 20 muM) elevated basal and N-methyl-D-aspartate (NMDA, 100 muM)-induced extracellular glutamate accumulation, while it did not augment kainate (100 muM)-induced glutamate accumulation in cultured cerebellar granule neurons. However, pretreatment with PDC for 1 h significantly reduced NMDA-induced glutamate accumulation, but did not affect kainate-induced response. Pretreatment with glutamate (5 muM) for 1 h also reduced NMDA-induced glutamate accumulation, but did not kainate-induced response. Upon a brief application (3-10 min), PDC did neither induce elevation of intracellular calcium concentration ([Ca(2+)](i)) nor modulate NMDA-induced [Ca(2+)](i) elevation. Pretreatment with PDC for 1 h reduced NMDA-induced [Ca(2+)](i) elevation, but it did not reduce kainate-induced [Ca(2+)](i) elevation. These results suggest that glutamate concentration in synaptic clefts of neuronal cells is increased by prolonged exposure (1 h) of the cells to PDC, and the accumulated glutamate subsequently induces selective desensitization of NMDA receptor.

Dual effects of NMDA-induced intracellular Ca2+ elevations on cGMP levels in cultured cerebellar granule neurons.

1. Cyclic GMP (cGMP) levels were markedly elevated by N-methyl-D-aspartate (NMDA) within 1-3 min of incubation, then gradually decreased with incubation time. 2. The NMDA-induced intracellular Ca2+ elevations showed maximal levels just after adding NMDA and were maintained for 60 min. 3. NMDA did not show augmentation of cGMP elevation with sodium nitroprusside (SNP), rather it decreased the SNP-induced cGMP elevation after exposure for 60 min. 4. The NMDA-induced elevation of cGMP was remarkably augmented with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 1mM), after 60 min of incubation.

Kainate produces concentration-dependent elevation of glutamate release but not cGMP levels in cultured neuron.

1. Treatment of cultured cerebellar granule cells for 3 min with N-methyl-D-aspartate (NMDA) resulted in a concentration-dependent elevation of cyclic GMP. However, neither kainate (KA) nor NMDA produced a concentration-dependent elevation of this nucleotide after exposing cells to the agonist for 60 min. 2. Unlike the case for cGMP, both KA and NMDA produced concentration-dependent elevations of glutamate for 60 min incubation. 3. The NMDA-induced elevations of cGMP and glutamate were blocked by selective NMDA receptor antagonists. 4. The selective KA/alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist, 6,7-nitroquinoxaline-2,3-dione (DNQX), blocked the KA-induced elevations of cGMP with 3-min exposures, but it augmented the response with 60-min exposures. However, the KA-induced release of glutamate was prevented by DNQX. 5. The KA/AMPA receptor antagonist, GYKI 52466, blocked all KA-induced responses regardless of the incubation times.

The iron component of sodium nitroprusside blocks NMDA-induced glutamate accumulation and intracellular Ca2+ elevation.

These studies were designed to compare the effects of nitric oxide (NO) generating compounds with those of several iron containing compounds which do not generate NO on glutamate receptor function. Stimulation of primary cultures of cerebellar granule cells with N-methyl-D-aspartate (NMDA) or kainate results in the elevation of intracellular calcium ([Ca2+]i) and cGMP and the release of glutamate. The iron containing compounds, sodium nitroprusside (SNP), potassium ferrocyanide (K4Fe(CN)6) and potassium ferricyanide (K3Fe(CN)6) decrease the NMDA-induced release of glutamate. SNP is the only compound of the above 3 agents which generates NO. A non-iron, NO generating compound, S-nitroso-N-acetylpenicillamin (SNAP), has no effect on the NMDA-induced glutamate release. Potassium ferrocyanide (Fe II), but not potassium ferricyanide (Fe III), blocks NMDA-induced cGMP elevations after 3 min exposure times. This contrasts with the NO generating compounds (both SNP and SNAP) which elevate cGMP levels. Furthermore, both potassium ferrocyanide (Fe II) and SNP (Fe II) suppress the elevation of [Ca2+]i induced by NMDA but neither potassium ferricyanide (Fe III) nor SNAP are effective in this regard. These effects are also independent of cyanide as another Fe II compound, ferrous sulfate (FeSO4) is also able to suppress NMDA-induced elevations of [Ca2+]i. SNP was unable to suppress kainate receptor functions. Collectively, these results indicate that Fe II, independently of NO, has effects on NMDA receptor function.

Acute, chronic and differential effects of several anesthetic barbiturates on glutamate receptor activation in neuronal culture.

The acute and chronic effects of several anesthetic barbiturates, in therapeutic concentrations, on the excitatory amino acid (EAA)-induced elevation of intracellular calcium levels ([Ca2+]i) were examined in neuronal tissue culture. The ultrashort-acting barbiturate, thiamylal, was effective in blocking elevations of [Ca2+]i induced by kainate, N-methyl-D-aspartate (NMDA), and quisqualate or by membrane depolarization with 40 mM KCl. The structurally similar barbiturate, secobarbital which differs from thiamylal only by having an oxygen in place of a sulfur, was able to block elevations induced by the above EAAs but was less effective than thiamylal and did not significantly reduce [Ca2+]i that resulted from membrane depolarization with KCl. Pentobarbital, while differing from secobarbital by only a methyl group, was without effect on either the NMDA- or 40 mM KCl-induced elevations of [Ca2+]i. By contrast, cyproheptadine, a compound that has been shown to block Ca2+ channels, has a different profile from the above barbiturates in that cyproheptadine is more effective in blocking elevation of [Ca2+]i induced by membrane depolarization with KCl while the barbiturates are more effective in reducing [Ca2+]i induced by EAAs. An anticonvulsant barbiturate, phenobarbital, did not reduced elevations of [Ca2+]i induced by any EAA tested or by membrane depolarization with KCl. When cells were treated chronically with thiamylal for 4 days, 2-6 h after the abrupt drug withdrawal there was a hyperresponsiveness to the elevations of [Ca2+]i induced by both kainate and NMDA but not by quisqualate. A similar hyperresponsiveness was not seen after the chronic treatment with phenobarbital.(ABSTRACT TRUNCATED AT 250 WORDS)

Amitriptyline, desipramine, cyproheptadine and carbamazepine, in concentrations used therapeutically, reduce kainate- and N-methyl-D-aspartate-induced intracellular Ca2+ levels in neuronal culture.

The glutamate receptor agonists, kainate and N-methyl-D-aspartate (NMDA) result in the elevation of intracellular calcium levels ([Ca2+]i) in primary cultures of cerebellar granule neurons. Several tricyclic antidepressants (TCAs), amitriptyline (0.5-1 microM), desipramine (1 microM) and doxepine (1 microM) partially prevent this elevation induced by both of these excitatory amino acids (EAAs), but not elevations of [Ca2+]i induced by another EAA, quisqualate. Evidence suggests that this EAA-tricyclic interaction may involve voltage-dependent Ca2+ channels since amitriptyline also partially blocks the elevation of [Ca2+]i induced by membrane depolarization with 40 mM KCl. However, the blockade is not reversed in high concentrations of extracellular Ca2+ ([Ca2+]o) as would be predicted by a direct interaction with Ca2+ channels. Cyproheptadine (0.5-1 microM), a serotonin antagonist that is structurally similar to amitriptyline, causes similar effects as reported above for the TCAs; however, ketanserine (10 microM), also a serotonin antagonist but without the tricyclic nucleus, is less effective in this regard. Carbamazepine, an anticonvulsant with a tricyclic nucleus, produces similar effects as the above three compounds only in higher, yet therapeutic, concentrations (50 microM). Neither 5-hydroxytryptamine nor norepinephrine (100 microM, each) had effects on the EAA-induced elevation of [Ca2+]i. This is the first report to show an interaction of tricyclic antidepressants with the function of glutamate receptors in concentrations which are consistent with therapeutic dosages.

Selective effects of cyanide (100 microM) on the excitatory amino acid-induced elevation of intracellular calcium levels in neuronal culture.

The effect of low concentrations of cyanide on the excitatory amino acid-induced elevations of intracellular calcium levels ([Ca2+]i) was studied in cerebellar granule cells using ratio fluorometry with fura-2. Glutamate, kainate, N-methyl-D-aspartate (NMDA), quisqualate (50 microM, each) and membrane depolarization by 40 mM KCl caused elevations of [Ca2+]i which were 10-, 10-, 3-, 2.3-, 10-fold over baseline levels, respectively. Cyanide, 100 microM, greatly augmented the increases in [Ca2+]i induced by glutamate, kainate and NMDA but not those induced by quisqualate or KCl. In the absence of these excitatory amino acids, cyanide had no significant effect in concentrations up to 400 microM. Elevations of [Ca2+]i induced by quisqualate and KCl were not significantly augmented by higher concentrations of cyanide (400 microM). Selective antagonists could block the effect of cyanide+the respective agonist; however, the calcium channel blockers, lanthanum and diltiazem lowered both NMDA- and kainate-induced elevations of [Ca2+]i, yet neither blocked increases in calcium when 100 microM cyanide was added. Collectively, these data support an interaction of cyanide with the excitatory amino acid receptor.

Amitriptyline prevents N-methyl-D-aspartate (NMDA)-induced toxicity, does not prevent NMDA-induced elevations of extracellular glutamate, but augments kainate-induced elevations of glutamate.

The effect of amitriptyline on kainate- and N-methyl-D-aspartate (NMDA)-induced toxicity and release of amino acids from cerebellar granule neurons was studied. The ED50 for amitriptyline, imipramine, and nortriptyline protection against NMDA-induced toxicity was 6.9, 6.5, and 1.3 microM, respectively. None of these compounds protected against kainate-induced toxicity. Even though amitriptyline was protective against NMDA-induced toxicity, it had no effect on the NMDA-induced increase in extracellular levels of glutamate or aspartate from these cells, indicating a dissociation between NMDA receptor activation (as indicated by glutamate content elevations) and NMDA-induced toxicity. However, kainate and quisqualate treatment resulted in elevations of glutamate and taurine levels that were further augmented in the presence of 25 microM amitriptyline. These findings confirm the reports of others that tricyclic antidepressants have neuroprotective effects related to the NMDA receptor and expand on these reports by showing that even though there is protection against toxicity, the NMDA receptor is nevertheless activated, suggesting an involvement of these compounds at sites removed from the receptor. Furthermore, this is the first report showing an interaction of tricyclic antidepressants with the function of non-NMDA receptors.

Cyanide selectively augments kainate- but not NMDA-induced release of glutamate and taurine.

The effect of cyanide on the kainate-, quisqualate- and N-methyl-D-aspartate (NMDA)-induced release of several amino acids from cerebellar granule neurons was studied. Cyanide, 100 microM, augmented the kainate- and quisqualate-induced release of glutamate and taurine in neurons but had no effect on the NMDA-induced release of these excitatory amino acids. In addition to the interaction with the above excitatory amino acids, cyanide had effects on several amino acids independent of excitatory amino acid stimulation; cyanide treatment resulted in a significant elevation over saline controls of arginine and taurine, but not alanine, aspartate+asparagine or glycine. With the exception of taurine, this pattern was not apparent in cells treated with any of the above excitatory amino acid.

Low calcium-induced release of glutamate results in autotoxicity of cerebellar granule cells.

Primary cultures of cerebellar rat granule neurons were grown for 18-22 days in vitro in the absence of antibiotics. When the cultures were placed in a low calcium (no EGTA) balanced salt solution at room temperature, rapid cell death occurred usually within 30 min of placing cells in the buffer. Changes in the cells were evident within 10 min and included an apparent cellular granulation with a partial loss of cell body birefringence at 10 x magnification which was complete by 30 min. This rapid death was prevented by (1) replacing chloride in the buffer with acetate; (2) increasing the osmolarity of the buffer by 30% with sucrose; (3) the addition of the selective excitatory amino acid (EAA) antagonist, 2-amino-7-phosphonoheptanoic acid (APH, 200 microM) but not by the selective kainate-quisqualate antagonist, glutamylaminomethylsulfonic acid (GAMS, 400 microM); or (4) the addition of one of the following calcium channel antagonists, verapamil (400 microM) diltiazem (150 microM) or lanthanum (5 microM). Placing cells in low calcium buffer resulted in a 3.7- and 3.2-fold increase in the non-selective secretion of aspartate and glutamate (as well as other amino acids) over baseline secretion (same buffer except containing 2.5 mM calcium). This increase was partially prevented by verapamil, but not by APH or chloride deletion. Verapamil only partially prevented the efflux of glutamate in buffer containing 1 mM EGTA. These results indicate that placing cells in low calcium buffer results in neurotoxicity secondary to both the influx of chloride and water in conjunction with the efflux of amino acids, some of which stimulate an excitatory amino acid receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased response of cerebellar cGMP to kainate but not NMDA or quisqualate following barbital withdrawal from dependent rats.

Female Sprague-Dawley rats were maintained on a diet of powdered food containing barbital for 8 weeks before the drug was abruptly withdrawn. Twenty-four hours later both barbital-dependent and control rats were injected intracerebroventricular (i.c.v.) with saline or one of four doses of kainic acid (KA) or in a separate experiment with saline or one of three doses of N-methyl-D-aspartic acid (NMDA) or of quisqualic acid (QA). After 4.5 min, the animals were killed by focused microwave irradiation, and the cerebella were collected. The levels of cyclic guanosine 3',5' monophosphate (cGMP) were markedly elevated in the cerebella of barbital-withdrawn rats when compared to controls. When compared to saline treatment, KA, at all dosages, resulted in a significantly greater elevation of cerebellar cGMP in the barbital-withdrawn rats than was induced by drug withdrawal alone. Only the two higher dosages of KA produced a significant elevation of this parameter in the control rats. Unlike KA, neither QA or NMDA produced any greater elevations of cGMP in barbital withdrawn rats than were induced by drug withdrawal alone. These collective results suggest that there is an increase in the response to KA but not QA or NMDA following the withdrawal of barbital from dependent rats.

Quisqualate, high calcium concentration and zero-chloride prevent kainate-induced toxicity of cerebellar granule cells.

Kainic acid (KA; 100 microM), results in the death of all cultured rat cerebellar granule cells (18-22 days in vitro) within 30 min. Changes in the cells are evident within 2 min of applying the excitatory amino acid (EAA) and include an apparent cellular granulation with a loss of cell body birefringence at 10 X magnification. Quisqualic acid (QA; 25 microM) completely prevents this KA-induced neurotoxicity. In addition, cells are protected from toxicity by increasing calcium concentrations to 10 mM. Moreover, following a 30 min exposure and after washing the cells free of these compounds, cells placed in culture media remain alive 24 h later. Interestingly, neurons die when placed in a balanced salt solution which lacks calcium even when no KA is present. This death is also dependent on the presence of chloride and is prevented with the non-selective EAA antagonist, kynurenic acid, but is not prevented by QA. Collectively, these data suggest that the activation of the EAA receptor by KA in cerebellar granule cells is at least partially regulated by calcium and chloride and is suppressed by QA. Furthermore, placing granule cells in zero-calcium results in neuronal death which appears to be mediated by EAA mechanisms.

Anticonvulsive activity of several excitatory amino acid antagonists against barbital withdrawal-induced spontaneous convulsions.

Several excitatory amino acid antagonists were tested for an ability to prevent spontaneous convulsions seen during the barbital abstinence syndrome in rats. Barbital-dependent animals were continuously infused intracerebroventricularly (i.c.v.) for the first 48 h following barbital withdrawal with either saline, 2-amino-7-phosphonoheptanoic acid (APH), magnesium sulfate, glutamyldiethyl ester (GDEE) or cis-2,3-piperidine dicarboxylic acid (PDA) using the highest dosages which did not affect normal behavior of the rats. All animals were observed continuously from 12 to 48 h postwithdrawal and the number of spontaneous convulsions observed in each animal was recorded. After this time, animals were killed by focused microwave irradiation and the cerebellas were collected for determination of cyclic guanosine monophosphate (cGMP) levels. While both APH and MgSO4 dramatically prevented convulsions, only APH prevented the withdrawal-induced elevation of cerebellar cGMP. PDA and GDEE had no statistically significant effect on either cerebellar cGMP levels or on convulsive activity. Although the effect of GDEE was not statistically significant, the number of convulsions was reduced to 1/3 those seen in control animals. These data implicate N-methyl-d-aspartate (NMDA) receptor-mediated pathways in seizure activity associated with the barbital abstinence syndrome and show that the withdrawal-induced elevation of cerebellar cGMP can occur without the induction of convulsions.

Cultured cerebellar cells as an in vitro model of excitatory amino acid receptor function.

Primary cultures of neurons from 8-day-old rat pups were grown for 10 days in vitro in antibiotic-free media and then analysed for changes in cyclic guanosine monophosphate (cGMP) in response to several excitatory amino acid (EAA) agonists or related antagonists. Kainic acid (KA), N-methyl-D-aspartic acid (NMDA) and quisqualic acid (QA) produced dose- and calcium-dependent increases in cGMP with KA producing the largest and QA the least increase in this cyclic nucleotide. The increase induced by NMDA was additive with both KA and QA; however, KA and QA were not additive with each other. In fact, QA completely antagonized the effects of KA and to a much greater degree than did the EAA antagonists, glutamylaminomethylsulfonic acid (GAMS) or cis-2,3-piperidine dicarboxylic acid (PDA). 2-Amino-7-phosphonoheptanoic acid completely prevented the NMDA-induced elevations of cGMP yet had little effect on either the KA- or QA-induced elevations of this parameter. GAMS and PDA, on the other hand, were more effective in blocking the effects of KA and QA than of NMDA. These data show that cGMP levels in cerebellar granule cells provide an excellent model for studying the subtypes of EAA receptors.

2-Amino-7-phosphonoheptanoic acid, a selective antagonist of N-methyl-D-aspartate, prevents barbital withdrawal-induced convulsions and the elevation of cerebellar cyclic GMP in dependent rats.

Female Sprague-Dawley rats were maintained on a diet of barbital for 8 weeks, a period of time previously shown to result in tolerance to and dependence on the drug. After completing this course, the barbital was abruptly withdrawn and the selective antagonist of N-methyl-d-aspartate (NMDA), 2-amino-7-phosphonoheptanoic acid (APH), or saline was infused intracerebroventricularly over 48 hr. Control rats which had not received barbital, were similarly infused with either saline or APH. All animals were observed for 12-48 hr following the withdrawal of the barbital; spontaneous convulsions, previously reported to be numerous and severe after withdrawal of the drug, were counted and graded according to severity. Forty-eight hr after withdrawal of barbital, the rats were killed by focussed microwave irradiation and cerebellae were collected for later determination of levels of cGMP. Nine convulsions occurred in 29 rats withdrawn from barbital and infused intracerebroventricularly with APH, this contrasted markedly with 61 convulsions seen in 29 animals withdrawn from the drug and infused with saline. There was a 3-fold elevation of levels of cGMP in the saline-infused, barbital-withdrawn rats when compared to control rats infused with saline. This evaluation was markedly, although not completely, prevented by the intracerebroventricular infusion of APH. These data provide evidence that dicarboxylic amino acid pathways, specifically those acting through NMDA receptors, are involved in seizure activity seen following abrupt abstinence from barbital.

Activity-induced elevation of cerebellar cyclic GMP occurs in the absence of climbing fiber pathways.

The inferior olivary nuclei (ION) of Sprague-Dawley rats were chemically lesioned with 3-acetylpyridine (3-AP), and the completeness verified by the lack of retrograde labeling of the ION following the injection of horseradish peroxidase (HRP) into the cerebellum. The effects of locomotor activity or of immobilization on cerebellar cyclic guanosine monophosphate (cGMP) levels were determined in control saline-treated and experimental, 3-AP-treated rats. Three subgroups of rats from both the control and the experimental groups of rats were required to swim for 60 s, immobilized for 60 s or unmanipulated before being killed by microwave irradiation, and the cerebella were collected for cGMP determination. There was no statistically significant difference in cGMP levels between immobilized and unmanipulated rats in either the experimental or control groups. Pretreatment with 3-AP reduced cerebellar cGMP levels in both the immobilized and the unmanipulated rats to 50% of those observed in the comparably treated groups of saline-treated controls. When compared to the corresponding group of unmanipulated rats, locomotor activity induced a significantly greater elevation of cerebellar cGMP in the experimental animals than in the controls (P less than 0.05). These results indicate that while inputs from the ION to the cerebellar Purkinje cells are probably important in maintaining the normal levels of cGMP seen in inactive rats, the locomotor-induced elevation of this parameter occurs in the absence of climbing fibers (from ION). The mechanisms responsible for the greater activity-induced elevation of cGMP levels seen in rats receiving 3-AP over control rats are discussed.

2-Amino-7-phosphonoheptanoic acid, a selective N-methyl-D-aspartate antagonist, blocks swim-induced elevation of cerebellar cyclic guanosine monophosphate.

In order to explore how rapidly locomotor activity induces an elevation in cerebellar cyclic guanosine monophosphate (cGMP) content, Sprague-Dawley rats, pretrained to swim a 2.5-m course, were required to swim from one to 5 laps representing from 7 to 40s of strenuous activity. Immediately after completing the swimming task, each animal was killed by microwave irradiation and the cerebellum was collected for subsequent determination of the cGMP content. There was no difference in the cerebellar cGMP content between rats swimming one lap, i.e. for 7 s, and control rats that did not swim. However, there was a linear increase in the cGMP over control values from 1.8- to 2.4-fold in rats swimming 3 and 5 times, respectively. The first significant elevation of the cerebellar cGMP was seen at 24 s (3 laps). To determine if acidic amino acid pathways were involved in this elevation, a low dosage of a selective NMDA antagonist, 2-amino-7-phosphonoheptanoic acid (APH) was injected intracerebroventricularly 4 min before having rats swim 4 laps. This low dosage of APH, which alone had no effect on the cerebellar cGMP content, completely blocked the swim-induced elevation of this parameter. These data provide the first report of how quickly locomotor activity elevates the cerebellar cGMP content and further suggest that an NMDA receptor-mediated pathway is involved in the activity-induced elevation of this parameter.