Inhibition of sympathetic preganglionic discharges by epinephrine and and alpha-methylepinephrine

The present study was undertaken to determine the effect of iontophoretic applications of epinephrine (E) and its derivative alpha-methylepinephrine (mE) on the discharges of sympathetic preganglionic neurons (SPNs). Spontaneously active SPNs located in thoracic segment T2 were antidromically identified in White Carneaux pigeons anesthetized with urethane and immobilized with purified alpha-cobratoxin. All SPNs tested were inhibited by E, mE, several other catecholamines, clonidine, GABA, glycine and morphine. The inhibitory effects of E and mE but not those of GABA were antagonized by iontophoretic applications of the preferential alpha 2-antagonists piperoxane and yohimbine, but not by the alpha 1-antagonist prazosin or the beta-antagonist sotalol when similarly applied. The inhibitory effects of GABA, glycine and morphine were respectively antagonized by bicuculline methiodide, strychnine and naloxone, but these antagonists failed to alter the action of E. It is concluded that (1) epinephrine and its alpha-methyl derivative inhibit the discharges of SPNs via the activation of alpha 2-receptors and (2) the epinephrine-induced inhibition does not result from the secondary release of GABA, glycine or opioid peptides from afferent terminals or interneurons.     

Opiate antagonism of glycine-evoked membrane polarizations in cultured mouse spinal cord neurons

The effect of several opiate receptor agonists on the responses of spinal cord neurons to putative inhibitory and excitatory amino acids was studied using an in vitro model system, cultured fetal mouse spinal cord neurons, and bath application of opiates. Intracellular recordings were made from the cultured neurons with conventional voltage recording techniques or under voltage clamp conditions. The putative amino acid neurotransmitters were applied by iontophoresis or micropressure ejection.Our main finding is that the opiate agonists, morphine and levorphanol (5–100 μM), consistently depressed the responses evoked by the putative inhibitory amino acid neurotransmitters glycine and β-alanine but not GABA. Dextrorphan, the inactive isomer of levorphanol, also depressed the glycine and β-alanine responses, but higher concentrations were required. The excitatory glutamate response was unaltered by these opiates. Leucine enkephalin, an opioid peptide, had no effect on the amino acid responses in the neurons where it was also tested. The opiate antagonist naloxone (10–100 μM) did not reverse the morphine or levorphanol depression of the amino acid responses making it unlikely that opiate receptors mediate this effect.Strychnine was considerably more effective than morphine as a glycine antagonist, producing depressions at nM concentrations compared to the μM concentrations required for morphine. Preliminary studies indicate that both morphine and strychnine act in a non-competitive manner. However, additional studies will be required before the sites of action for these agents can be identified.The possible pharmacological or toxicological significance of the present work remains to be determined. Considering the high doses of opiates (μM concentrations) required to depress the glycine and β-alanine responses, it is unlikely that this action is relevant to normal therapeutic situations. However, such concentrations of opiates are often utilized in pharmacological studies and may be achieved when opiates are applied by iontophoresis. Our data indicate that consideration of the present opiate action should be made when μM concentrations or iontophoretic application of opiates are used for pharmacological studies of CNS tissue.     

Inhibitory effect of intrathecal glycine on the micturition reflex in normal and spinal cord injury rats

We examined the influence of lumbosacral glycinergic neurons on the spinobulbospinal and spinal micturition reflexes. Female rats were divided into intact rats, rats with acute injury to the lower thoracic spinal cord (SCI), and rats with chronic SCI. Under urethane anesthesia, isovolumetric cystometry was performed in each group before and after intrathecal (IT) injection of glycine or strychnine into the lumbosacral cord level. The glutamate and glycine levels of the lumbosacral cord were measured after injection of glycine or strychnine in intact and chronic SCI rats. Expression of strychnine-sensitive glycine receptor alpha-1 (GlyR alpha1) mRNA in the lumbosacral cord was also assessed in both rats. In chronic SCI rats, the interval and amplitude of bladder contractions were shorter and smaller when compared with intact rats. IT glycine (0.1-100 microg) prolonged the interval and decreased the amplitude of bladder contractions in both intact rats and chronic SCI rats. IT strychnine (0.01-10 microg) elevated the baseline pressure in intact rats and induced bladder contraction in acute SCI rats. On amino acid analysis, IT glycine (0.01-100 microg) decreased the glutamate level of the lumbosacral cord in intact rats, but not in chronic SCI rats. The glycine level of the lumbosacral cord was 54% lower in chronic SCI rats when compared with intact rats, while the GlyR alpha1 mRNA level did not change after SCI. These results suggest that glycinergic neurons may have an important inhibitory effect on the spinobulbospinal and spinal micturition reflexes at the level of the lumbosacral cord.     

Contribution of spinal inhibitory receptors in heterosegmental antinociception induced by noxious stimulation

Noxious (i.e. painful) stimulation in the rat induces profound heterosegmental antinociception as demonstrated by the ability of either thermal stimulation (50 degrees C water) or subdermal capsaicin injection in the hindpaw to attenuate the nociceptive trigeminal jaw-opening reflex. Importantly, noxious stimulus-induced antinociception (NSIA) is mediated by endogenous opioids (as well as other neurotransmitters) in nucleus accumbens, as indicated by the ability of intra-accumbens administration of mu- or delta-opioid receptor antagonists to block NSIA. Although noxious peripheral stimulation is known to release excitatory neurotransmitters such as glutamate at the level of the spinal cord, the present study was designed to test the hypothesis that NSIA depends on further activation of spinal inhibitory receptors. This hypothesis was based on findings that inhibition of spinal processing (e.g. intrathecal local anaesthetic administration) also produces heterosegmental antinociception mediated by endogenous opioids in nucleus accumbens. Thus, to reconcile the paradoxical findings that both spinal excitation and inhibition appear to activate the same nucleus accumbens opioid-mediated antinociceptive mechanism, we investigated whether spinal administration of antagonists for inhibitory receptors would block the antinociceptive effect of subdermal capsaicin. We report that spinal administration of selective antagonists for mu-opioid (Cys2, Tyr3, Orn5, Pen7amide), kappa-opioid (nor-binaltorphimine), GABA-A (bicuculline), GABA-B (CGP 35348) and glycine (strychnine) receptors significantly reduced NSIA. The selective delta-opioid receptor antagonist naltrindole had no significant effect. These results, together with our previous findings, suggest that peripheral noxious stimuli release endogenous opioids, GABA and glycine in the spinal cord which, in turn, inhibit tonic pronociceptive spinal activity to produce heterosegmental antinociception mediated in nucleus accumbens.     

Early development of glycine- and GABA-mediated synapses in rat spinal cord.

Motoneuron responses to the inhibitory amino acids glycine and GABA, and the contribution of inhibitory synapses to developing sensorimotor synapses were studied in rat spinal cords during the last week in utero. In differentiating motoneurons, glycine and GABA induced Cl(-)-dependent membrane depolarizations and large decreases in membrane resistance. These responses gradually decreased during embryonic development, and at birth they were significantly smaller than in embryos. In motoneurons of embryos and neonates, dorsal root stimulation produced only depolarizing potentials, some of which reversed at -50 mV membrane potential. Reduction of extracellular Cl- concentrations increased the amplitude of these potentials, suggesting that they are generated by Cl- current. Contribution of Cl(-)-dependent potentials to compound dorsal root-evoked potentials was studied by determining the effects of glycine and GABA antagonists on them. In motoneurons of embryos at days 16-17 of gestation (D16-D17), strychnine or bicuculline blocked dorsal root-evoked potentials. This suppression was neither the result of a decrease in neuronal excitability nor the inhibition of glutamate receptors. Strychnine-evoked depression was not blocked by atropine, indicating that it was not due to disinhibition of muscarinic synapses. By D19, strychnine and bicuculline significantly increased dorsal root-evoked potentials rather than blocking them. This reversed function did not result from an increase in neuronal excitability or changes in the specificity of strychnine and bicuculline antagonism. The number of glycine- and GABA-immunoreactive cells increased 20% between D17 and D19. The number of immunoreactive cells and fibers significantly increased in the motor nuclei and dorsal horn laminae. These morphological changes may contribute to establishment of new synaptic contacts on motoneurons, thus changing the actions of strychnine and bicuculline on dorsal root-evoked potentials.     

Ethanol alters synaptic activity in cultured spinal cord neurons

The acute effects of ethyl alcohol on mammalian central neurons were investigated using electrophysiological techniques and an in vitro model system, cultured fetal mouse spinal cord neurons. Intracellular recordings were made from the cultured neurons to evaluate the effect of alcohol (10-100 mM) on membrane potential, membrane permeability, amplitude of the action potential, sensitivity of the neurons to putative neurotransmitters and the process of synaptic transmission. Alcohol was applied by superfusion; putative amino acid neurotransmitters were applied by micropressure ejection. The most dramatic effect of alcohol on the spinal cord neurons was a reduction in the spontaneous activity (excitatory and inhibitory synaptic potentials and action potentials) and the glutamate evoked synaptic activity. Alcohol doses as low as 20-30 mM, concentrations which reflect blood levels during intoxication, were effective. Membrane potential, membrane permeability, and amplitude of the action potential were relatively resistant to these low doses of alcohol; at the higher alcohol doses, no effect or only modest alterations of these characteristics were observed. The responses of the neurons to the putative excitatory neuro-transmitter glutamate, and inhibitory transmitters GABA and glycine were also relatively resistant to alcohol exposure. These data indicate that acute exposure to alcohol has a predominantly inhibitory action on the activity of the cultured mammalian CNS neurons, and that this inhibition is most likely due to an alteration in the process of synaptic transmission.     

GABA, not glycine, mediates inhibition of latent respiratory motor pathways after spinal cord injury

Previous work has shown that latent respiratory motor pathways known as crossed phrenic pathways are inhibited via a spinal inhibitory process; however, the underlying mechanisms remain unknown. The present study investigated whether spinal GABA-A and/or glycine receptors are involved in the inhibition of the crossed phrenic pathways after a C2 spinal cord hemisection injury. Under ketamine/xylazine anesthesia, adult, female, Sprague-Dawley rats were hemisected at the C2 spinal cord level. Following 1 week post injury, rats were anesthetized with urethane, vagotomized, paralyzed and ventilated. GABA-A receptor (bicuculline and Gabazine) and glycine receptor (strychnine) antagonists were applied directly to the cervical spinal cord (C3-C7), while bilateral phrenic nerve motor output was recorded. GABA-A receptor antagonists significantly increased peak phrenic amplitude bilaterally and induced crossed phrenic activity in spinal-injured rats. Muscimol, a specific GABA-A receptor agonist, blocked these effects. Glycine receptor antagonists applied directly to the spinal cord had no significant effect on phrenic motor output. These results indicate that phrenic motor neurons are inhibited via a GABA-A mediated receptor mechanism located within the spinal cord to inhibit the expression of crossed phrenic pathways.     

Involvement of opioid receptors in the CGRP8-37-induced inhibition of the activity of wide-dynamic-range neurons in the spinal dorsal horn of rats

The present study was performed to explore the involvement of opioid receptors in the calcitonin gene-related peptide 8-37 (CGRP8-37, an antagonist of CGRP receptor)-induced inhibition of the activity of wide-dynamic-range (WDR) neurons in the spinal dorsal horn of rats. Extracellular recording was performed with a multibarrelled glass micropipette, and the chemicals were delivered by micro-iontophoresis. The discharge frequency of WDR neurons was evoked by subcutaneous electrical stimulation applied to the ipsilateral hindpaw. Iontophoretic application of CGRP8-37 by an ejection current of 160 nA induced significant inhibition of the discharge frequency of WDR neurons. The inhibitory effect of CGRP8-37 on the activity of WDR neurons was attenuated by later iontophoretic application of the opioid antagonist naloxone. Furthermore, the effect of CGRP8-37 was attenuated by either iontophoretic application of the kappa-receptor antagonist nor-binaltorphimine (nor-BNI) or the mu-receptor antagonist beta-funaltrexamine (beta-FNA) but not by the delta-receptor antagonist naltrindole. The results indicate that kappa- and mu-opioid receptors on the membrane of WDR neurons are involved in the modulation of CGRP8-37-induced antinociception in dorsal horn of the spinal cord in rats.     

Inhibition of sympathetic preganglionic discharges by epinephrine and α-methylepinephrine

The present study was undertaken to determine the effect of iontophoretic applications of epinephrine (E) and its derivative α-methylepinephrine (mE) on the discharges of sympathetic preganglionic neurons (SPNs).Spontaneously active SPNs located in thoracic segment T2 were antidromically identified in White Carneaux pigeons anesthetized with urethane and immobilized with purified α-cobratoxin.All SPNs tested were inhibited by E, mE, several other catecholamines, clonidine, GABA, glycine and morphine.The inhibitory effects of E and mE but not those of GABA were antagonized by iontophoretic applications of the preferential α₂-antagonists piperoxane and yohimbine, but not by the α₁-antogonist praxosin or the β-antagonist sotalol when similarly applied.The inhibitory effects of GABA, glycine and morphine were respectively antagonized by bicuculline methiodide, strychnine and naloxone, but these antagonists failed to alter the action of E.It is concluded that (1) epinephrine and its α-methyl derivative inhibit the discharges of SPNs via the activation of α₂-receptors and(2) the epinephrine-induced inhibition does not result from the secondary release of GABA, glycine or opioid peptides from afferent terminals or interneurons.     

Inhibitory postsynaptic actions of taurine, GABA and other amino acids on motoneurons of the isolated frog spinal cord.

The actions of glycine, GABA, alpha-alanine, beta-alanine and taurine were studied by intracellular recordings from lumbar motoneurons of the isolated spinal cord of the frog. All amino acids tested produced a reduction in the amplitude of postsynaptic potentials, a blockade of the antidromic action potential and an increase of membrane conductance. Furthermore, membrane polarizations occurred, which were always in the same direction as the IPSP. All these effects indicate a postsynaptic inhibitory action of these amino acids. When the relative strength of different amino acids was compared, taurine had the strongest inhibitory potency, followed by beta-alanine, alpha-alanine, GABA and glycine. Topically applied strychnine and picrotoxin induced different changes of post-synaptic potentials, indicating that distinct inhibitory systems might be influenced by these two convulsants. Interactions with amino acids showed that picrotoxin seletively diminished the postsymaptic actions of GABA, while strychnine reduced the effects of taurine, glycine, alpha- and beta-alanine. But differences in the susceptibility of these amino acid actions to strychnine could be detected: the action of taurine was more sensitively blocked by strychnine compared with glycine, alpha- and beta-alanine. With regard to these results the importance of taurine and GABA as transmitters of postsynaptic inhibition on motoneurons in the spinal cord of the frog is discussed.     

Putative glycine receptors in Hydra: a biochemical and behavioural study

Glycine acts as an inhibitory transmitter in the lower brain stem and spinal cord of vertebrate species, while very few data are yet available to support a similar role in invertebrate nervous systems. Here we report the identification and characterization of glycine receptors in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) by biochemical and behavioural studies. Saturation experiments revealed the occurrence of one population of binding sites of nanomolar affinity (KD = 33 nm) and low capacity (Bmax = 79 fmol/mg protein) for [(3)H]strychnine. The addition of glycine or taurine (0.1 microm-1 mm) produced a dose-dependent inhibition of [(3)H]strychnine binding. Beta-alanine (0.1-1 mm) did not significantly affect [(3)H]strychnine binding. The pharmacological properties of these receptors compare with those of vertebrate glycine receptors. Stimulation of Hydra polyps by reduced glutathione resulted in a significant increase in the duration of mouth opening in the presence of glycine, taurine or beta-alanine. The enhancement of the response was related both to amino acid (10-100 microm) and to glutathione concentration (1-10 microm). The effects of glycine or its agonists were suppressed by strychnine (1-10 microm). D-serine, a glycine agonist at the vertebrate NMDA receptor, produced opposite effects to those of glycine. The effects of d-serine were suppressed by 5,7-dichlorokynurenic acid but not by strychnine. In vitro, [(3)H]strychnine binding was not displaced by d-serine. These results indicate a dual action of glycine in Hydra tissues. The hypothesis that NMDA receptors may also be present in this elementary nervous system is proposed.     

Opioid peptides decrease calcium-dependent action potential duration of mouse dorsal root ganglion neurons in cell culture

Opiate alkaloid and opioid peptide actions on spontaneous neuronal activity and postsynaptic amino acid responsiveness were assessed using intracellular recording techniques applied to murine spinal cord neurons in primary dissociated cell culture. Application of opiates was by superfusion and amino acids by iontophoresis. Glycine and GABA but not glutamate responses were antagonized by the opiate alkaloids. Since opiate effects on glycine and GABA responses were not naloxone-reversible, only weakly stereospecific, and not produced by the opioid peptide [d-Ala²]-Met-enkephalinamide, it is unlikely that these effects were mediated by opiate receptors. Opiate depression of glycine inhibition was correlated with the induction of paroxysmal depolarizations in cultured spinal cord neurons, suggesting that antagonism of inhibitory amino acid transmission may underlie the convulsant actions of high concentrations of the opiate alkaloids.     

Glycine receptors in the caudal pontine reticular formation: are they important for the inhibition of the acoustic startle response?

The present paper sought to test the hypothesis that inhibitory glycine receptors (GlyRs) on giant neurons of the caudal pontine reticular formation (PnC) are involved in the inhibition of the acoustic startle response (ASR) in rats. First we provided evidence for the presence of the strychnine-sensitive inhibitory GlyR on PnC neurons by immunocytochemical labeling using an antibody against the α₁ subunit of the GlyR. We then measured the ASR as well as two ASR inhibiting phenomena, short-term habituation and prepulse inhibition, after microinjections of the glycine antagonist strychnine (0, 5 or 10 nmol) or the glycine agonist β-alanine (0, 50 or 100 nmol) into the PnC. Neither strychnine nor β-alanine had a measurable influence on any of the parameters of the ASR investigated (amplitude, short-term habituation, prepulse inhibition). In contrast, systemic injection of strychnine (1 mg/kg) markedly increased the ASR amplitude. The systemic administration of strychnine did not impair prepulse inhibition. The human ‘startle disease’ (hyperekplexia), an exaggerated startle response, is caused by a defect of the α₁ subunit of the inhibitory GlyR, but it is unclear at which site in the central nervous system this defect ultimately leads to the symptoms of hyperekplexia. Our data indicate that a blockade of the inhibitory GlyRs in the PnC does not affect the ASR of rats, suggesting that deficient GlyRs in the PnC might not be involved in the etiology of the human ‘startle disease’. We conclude that the inhibitory GlyRs on PnC neurons are not necessary for the inhibition of the ASR and believe that they are involved in another behavioral context.     

Enkephalin Expression in Spinal Cord Neurons is Modulated by Drugs Related to Classical and Peptidergic Transmitters

The effects of various neurotransmitter agonists and antagonists on the synthesis and release of methionine enkephalin (mENK) in neuronal cultures of mouse spinal cord and dorsal root ganglia have been measured. Blockade of electrical activity with tetrodotoxin between days 9 and 13 in culture caused a > 95% decrease in the number of mENK-immunoreactive neurons. This effect was also seen upon the blockade of glycine and beta-adrenergic receptors with strychnine and propranolol, respectively, and stimulation of GABA receptors with muscimol. Stimulation of beta-adrenergic receptors with isoproterenol, or blockade of glutamate and GABA receptors with 2-aminophosphonovalerate and strychnine, respectively, had a qualitatively opposite action on both the number of mENK-immunoreactive neurons and enkephalin peptide levels measured by radioimmunoassay. Application of substance P also enhanced the mENK cell number. These data suggest that, at least in the spinal cord, characteristics other than the average level of impulse activity in the afferent input may be critical to the regulation of expression of mENK.     

Evidence in favor of a neurotransmitter role of glycine in the rat cerebral cortex

In the present study we analyze whether glycine satisfies some electrophysiological and biochemical criteria to consider it as a putative transmitter in the rat cerebral cortex. Intracellular recordings from rat sensory-motor cortex showed that in 15-20% of the tested neurons glycine hyperpolarized the cell membrane, decreased the firing rate and flattened the evoked EPSP-IPSP sequence by increasing the membrane conductance. The iontophoretic application of strychnine antagonized the block of 'spontaneous' firing and the membrane hyperpolarization induced by glycine. Moreover, in a group of neurons, strychnine decreased the amplitude and duration of the IPSP and brought back the membrane potential to resting values. Previously accumulated [3H]glycine and endogenous glycine were released from cortical synaptosomal preparations by depolarizing stimuli in a Ca2+-dependent way. The release pattern of glycine was qualitatively similar in cortical and in spinal synaptosomes. [14C]Glycine was rapidly synthetized from [14C]serine in cortical synaptosomal preparations, and the newly formed [14C]glycine was released by depolarizing stimuli in a Ca2+-dependent way. It is concluded that glycine, which is generally considered as an inhibitory neurotransmitter in the spinal cord, medulla and pons, may also have a transmitter role in a discrete number of cortical neurons of some mammalian species.U     

Specific antagonism of GABA-mediated postsynaptic inhibition in cultured mammalian spinal cord neurons: a common mode of convulsant action

Mammalian spinal neurons grown in tissue culture were used to study the effects of the four convulsants-penicillin, pentylenetetrazol, picrotoxin, and bicuculline-on these neurons' responses to amino acids. Bath application of all four convulsants produced paroxysmal depolarizing events in the neurons; iontophoresis of the four convulsants selectively depressed responses produced by iontophoresis of the putative inhibitory transmitter GABA, and effected this depression without altering either inhibitory responses to beta-alanine or glycine, or excitation mediated by glutamate. These results support the hypothesis that the convulsant activity of these agents comes in part from selective antagonism of GABA-mediated postsynaptic inhibition.     

Cholinergic, noradrenergic, and serotonergic inhibition of fast synaptic transmission in spinal lumbar dorsal horn of rat

It is known that spinal nociceptive sensory transmission receives descending inhibitory and facilitatory modulation from supraspinal structures. Glutamate is the major fast excitatory transmitter between primary afferent fibers and spinal dorsal horn neurons. In whole-cell patch clamp recordings from dorsal horn neurons in spinal slices, we investigated synaptic mechanisms for inhibitory modulation at the lumbar level of the spinal cord. Application of the cholinergic receptor agonist carbachol produced a dose-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSCs) (IC(50) 13 microM). Postsynaptic injection of two different types of G-protein inhibitors, guanosine 5'-O-2-thiophosphate or guanosine 5'-O-3-thiotriphosphate, blocked the inhibition produced by carbachol. Clonidine, a selective alpha-adrenergic receptor agonist, also produced a dose-dependent inhibition of EPSCs (IC(50) 7 microM) that was reduced by postsynaptic inhibition of G-proteins. The inhibitory effect of serotonin was likewise mediated by postsynaptic G-proteins. Our results suggest that activation of postsynaptic neurotransmitter receptors plays a critical role in inhibition of glutamate mediated sensory responses by acetylcholine, norepinephrine, and serotonin. Our results support the hypothesis that descending sensory modulation may be mediated by multiple neurotransmitter receptors in the spinal cord.     

Sarcophine and (7S, 8R)-dihydroxydeepoxysarcophine from the Red Sea soft coral Sarcophyton glaucum as in vitro and in vivo modulators of glycine receptors

The inhibitory glycine receptor (GlyR) is a key mediator of synaptic signalling in spinal cord, brain stem, and higher centres of the central nervous system. We examined the glycinergic activity of sarcophine (SN), a marine terpenoid known for its various biological activities, and its trans-diol derivative (7S, 8R)-dihydroxy-deepoxysarcophine (DSN). SN was isolated from the Red Sea soft coral Sacrophyton glaucum, DSN was semisynthesized by hydrolysis of the epoxide ring. In cytotoxicity tests against HEK293 cells, SN and DSN had LD₅₀ values of 29.3 ± 3.0 mM and 123.5 ± 13.0 mM, respectively. Both compounds were tested against recombinant human α1 glycine receptors in HEK293 cells using whole-cell recording techniques. Both, SN and DSN were shown for the first time to be inhibitors of recombinant glycine receptors, with K_Ivalues of 2.1 ± 0.3 μM for SN, and 109 ± 9 μM for DSN. Receptor inhibition was also studied in vivo in a mouse model of strychnine toxicity. Surprisingly, in mouse experiments strychnine inhibition was not augmented by either terpenoid. While DSN had no significant effect on strychnine toxicity, SN even delayed strychnine effects. This could be accounted for by assuming that strychnine and sarcophine derivatives compete for the same binding site on the receptor, so the less toxic sarcophine can prevent strychnine from binding. The combination of modulatory activity and low level of toxicity makes sarcophines attractive structures for novel glycinergic drugs.     

Naloxone antagonism of GABA-evoked membrane polarizations in cultured mouse spinal cord neurons

Pharmacological studies using an in vitro model system were carried out to determine if naloxone, an opiate receptor antagonist, could have effects on neuronal membranes which were unrelated to its action as an opiate receptor antagonist. Intracellular recordings were made from cultured mammalian spinal cord neurons. Putative amino acid neurotransmitters and naloxone were applied by iontophoresis or superfusion. When naloxone was co-iontophoresed with the amino acids a depression of the GABA response resulted. This depression was dose-dependent and reversible. At the lower doses of naloxone tested, the depression was specific since the glycine and glutamate responses were unaffected. At the higher doses of naloxone tested, alterations in the glycine and/or glutamate responses and membrane input resistance were frequently observed. The naloxone depression of the GABA response did not appear to involve opiate receptors since (+)-naloxone, the inactive isomer, equally depressed the GABA response. Analysis of the effect of naloxone on GABA dose-response curves indicates that naloxone acts as a competitive antagonist at the neuronal GABA receptors. Similar results were obtained when naloxone was applied by superfusion. However, high concentrations of naloxone (0.1-1 mM) were required, suggesting that naloxone has a low affinity for the GABA receptor. These data indicate that under some experimental conditions naloxone could not be considered a specific opiate antagonist.     

Strychnine interactions with acetylcholine, dopamine and serotonin receptors in Aplysia neurons

The effects of strychnine on synaptic transmission in the abdominal ganglion ofAplysia californica and on the responses of individual neurons to iontophoretic application of acetylcholine, dopamine and serotonin were studied using conventional techniques of intracellular recording. Strychnine inhibited all classical (relatively short-lasting) excitatory and inhibitory postsynaptic potentials as well as the various sodium- and chloride-dependent phoresis responses. Only the potassium-dependent inhbitions of prolonged duration, activated in some cells transsynaptically or by dopamine or acetylcholine application, were not antagonized; sometimes these inhibitions were enhanced. Log-dose-response curves indicated that for each of the 3 drugs the depolarizing responses were more sensitive to strychnine than were the hyperpolarizing responses. Also, a given strychnine concentration generally inhibited the serotonin and dopamine responses to a greater extent than the acetylcholine responses.The antagonism by strychnine of the Na⁺- and Cl^?-dependent phoresis responses — similar to the action of curare on these receptors — is apparently due to selective interactions with some membrane receptors for the applied drugs.Finally, the limitations to the analysis of the dose-response curves generated by the iontophoretic technique are briefly discussed. It is suggested that the strychnine inhibitions of the acetylcholine and dopamine depolarizing responses might be due to a competitive inhibitory action, whereas in respect to the changes in their dose-response curves, the chloride-dependent ACh-H response and the 5-HT-D and H responses of these receptors seem to be blocked by a non-competitive action of the drug.