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.     

An increase in opiate receptor-sites is associated with enhanced cardiovascular depressant, but not respiratory depressant action of morphine

Rats were treated for 4 weeks with a constant infusion of 2 mg/kg/h of the opiate antagonist naloxone. This treatment increased μ-, σ- and ξ-binding sites by 60–180% in several brain regions, suggesting effective blockade of the 3 types of opiate sites. The significance of changes in opiate binding sites for opiate receptor mediated physiological responses were examined using cardiovascular and respiratory responses to morphine (assessed after elimination of naloxone) as physiological parameters. Chronically naloxone-treated rats showed no alteration in respiratory responses to morphine, whereas there was a marked supersensitivity to depressor and bradycardic effects and a loss of pressor and tachycardic effects of morphine. These data are the first indication that cardiovascular effects of opiates vary with changes in central opiate receptor levels. Our observations, moreover, show that there are complex relationships between receptor number and receptor-mediated effects of opiates.     

Electrophysiological correlates of presynaptic opiate receptor activation: reduction in norepinephrine-mediated inhibition from the locus coeruleus

Inhibitory responses of rat cerebellar Purkinje cells to locus coeruleus (LC) stimulation and iontophoresis of norepinephrine (NE) were examined before and after administration of morphine to determine whether the inhibitory modulation of NE release by opiates results in a functional impairment in noradrenergic synaptic action. Administration of morphine systemically (0.2-1.2 mg/kg, i.v.) or by iontophoresis reduced inhibitions in Purkinje firing elicited by LC stimulation without affecting depressions in activity induced by postsynaptic applications of NE. This antagonistic effect of morphine on LC-induced inhibition was reversed or prevented by naloxone and mimicked by administration of levorphanol but not dextrorphan. Morphine increased the excitatory response of Purkinje cells to monosynaptic input from the parallel fibers, whereas it blocked gamma-aminobutyric acid-induced inhibitions in firing via a non-opiate receptor-mediated mechanism. These results demonstrate that morphine interferes with synaptic inhibition derived from the LC and suggest that this may occur via activation of presynaptic opiate receptors residing on noradrenergic nerve terminals.     

GABA and bicuculline actions on mouse spinal cord and cortical neurons in cell culture

The neutral amino acid γ-aminobutyric acid (GABA) produced membrane hyperpolarization and increased membrane chloride ion conductance of spinal cord (SC) and cortical (CTX) neurons in cell culture. GABA dose-response curves were obtained for SC neurons by pressure applying known concentrations of GABA from micropipettes with large tips (miniperfusion pipettes). GABA response threshold was about 2 μM and large responses were elicited at GABA concentrations greater than 10 μM. Bicuculline (BICUC) (0.1–10 μM) reversibly antagonized GABA responses on both SC and CTX neurons with a half maximal inhibitory concentration of about 1 μM. BICUC antagonism of GABA responses was competitive (Lineweaver-Burke analysis). These results are compared with data on GABA and BICUC displacement of [³H]GABA binding to membranes of SC and CTX neurons in cell culture. It is suggested that high affinity GABA receptors are likely to be relevant for postsynaptic GABA responses while low affinity GABA receptors may be presynaptic.     

The spinal antinociceptive actions of morphine metabolites morphine-6-glucuronide and normorphine in the rat

The profound and prolonged effects of morphine in patients with renal dysfunction have been associated with high plasma levels of the opiate metabolites morphine-6-glucuronide (M6G) and morphine-3-glucuronide (M3G) rather than an increased concentration of morphine. We present here electrophysiological evidence to suggest that potent spinal antinociception can be produced by both M6G and normorphine, another metabolite of morphine. Extracellular recordings of Aβ- and C-fibre-evoked responses of convergent dorsal horn neuroneswere made in the halothane anaesthetised rat. M6G elicited dose-dependent, naloxone-reversible inhibitions of C-fibre-evoked responses which were completely suppressed (8% of control) by 2 μg M6G whereas Aβ-fibre-evoked responses were only reduced to 57% of controls. The ED₅₀ for the effects of M6G on C-fibre-evoked activity was calculated to be 0.53 μg. Systematic administration of M6G (2 mg/kg) also profoundly reduced noxious evoked neuronal activity. intrathecal normorphine was less potent than M6G but complete selective inhibitions of C-fibre-evoked responses could be elicited by 25 μg and the ED₅₀ was calculated to be 2.68 μg. No such inhibitions were observed following administration of M3G. A comparison with intrathecal morphine in the same preparation reveals that normorphine is equipotent with morphine whereas M6G is 13-fold more potent. These results therefore confirm that M6g and normorphine might be significant contributers to opiate analgesia after administration of morphine.     

Sensitivity of identified medial hypothalamic neurons to GABA, glycine and related amino acids; influence of bicuculline, picrotoxin and strychnine on synaptic inhibition

Medial hypothalamic neurons in pentobarbital anesthetized rats were identified by location and response to electrical stimulation of the amygdala, medial preoptic area, midbrain periaqueductal gray and median eminence. Cells were then examined for their sensitivity to microiontophoretic applications of GABA, glycine and 3 related amino acids, i.e. β-guanidinopropionic acid, δ-aminovaleric acid and β-alanine.Application of all agents decreased the spontaneous and glutamate or aspartate evoked activity of the majority of neurons in all identified categories.The majority of neurons were more sensitive to β-guanidinopropionate, δ-amminovalerate and GABA than to glycine and β-alanine.Bicuculline and picrotoxin produced a selective and reversible antagonism of depressions evoked by GABA and GABA-like amino acids whereas strychnine produced a selective and reversible antagonism of depression evoked by glycine and β-alanine.Bicuculline and picrotoxin, but not strychnine, were observed to diminish synaptic inhibition evoked by electrical stimulation of several sites when these agents were administered by microiontophoresis and by i.v. injections in convulsive doses.These observations suggest that many medial hypothalamic neurons have both GABA and glycine receptors but that GABA may have the more important role as a neurotransmitter common to afferent or recurrent inhibitory hypothalamic pathways.     

Characterization of mechanical withdrawal responses and effects of μ-, δ- and κ-opioid agonists in normal and μ-opioid receptor knockout mice

Clinical and experimental observations suggest that opiates can exert different influences on the perception of stimuli from distinct sensory modalities. Thermally-induced nociception is classically responsive to opiate agonists. μ-Opioid receptor-deficient transgenic mice are more sensitive to thermal nociceptive stimuli and morphine fails to attenuate the nociceptive responses to thermal stimuli in these animals. To enhance our understanding of opiate influences on mechanical sensitivity, we have examined withdrawal responses to a sequence of ascending forces of mechanical stimuli in mice with normal (wild type), half-normal (heterozygous) and absent (homozygous) μ-opioid receptor levels. We report data from mice examined without drug pretreatment or following pretreatment with morphine, the selective κ-opioid agonist, U50488H, and the selective δ-opioid agonist, DPDPE. Saline-pretreated mice of each genotype displayed similar, monotonically increasing frequency of withdrawal responses to the graded stimuli. Subcutaneously administered morphine produced a dose-dependent reduction in withdrawal responses in wild type and heterozygous mice, but had no significant effect in homozygous mice. Intraventricular administration of DPDPE also reduced the frequency of paw withdrawal (FPW) in wild type mice, but not in homozygous mice. In contrast, systemic U50488H produced a dose-dependent attenuation of paw withdrawal in both wild type and homozygous mice. These findings suggest that (1) interactions of endogenous peptides with μ-opioid receptors may not play a significant role in the response to mechanical stimuli in drug-free animals, and (2) deficiency of μ-opioid receptors has no functional consequence on the response to the prototypical κ-opioid receptor agonist, but decreases responses to the prototypical μ- and δ-opioid receptor agonists.     

Systemic morphine reduces GABA release in the lateral but not the medial portion of the midbrain periaqueductal gray of the rat

Neuroanatomical, electrophysiological and pharmacological studies have provided indirect evidence indicating that GABAergic neurons play a key role in opiate analgesia mediated by the midbrain periaqueductal gray (PAG) and ventromedial medulla. Although these studies suggest that systemic administration of opiates inhibits GABA release in the PAG, there have been no investigations to date that have directly examined this issue. The present study was thus designed to determine whether systemic morphine injection inhibits GABA release in the PAG of awake, freely moving rats using in vivo microdialysis and subsequent HPLC analysis. Extracellular levels of GABA, glutamate, aspartate, glycine, homocysteic acid and taurine were monitored with the microdialysis technique in either the lateral or medial portion of the ventrocaudal PAG in unanesthetized, unrestrained rats. Amino acid release was induced by infusing veratridine (75 microM, a sodium channel activator) directly through the dialysis probe. The effect of veratridine alone and the effect of veratridine in the presence of systemic morphine on the concentrations of amino acids in the PAG dialysate were determined. There were no significant differences in the basal concentrations of GABA, taurine, aspartate, glutamate, homocysteic acid and glycine between dialysates collected from the medial versus the lateral ventrocaudal PAG. Glycine, taurine and glutamate were present in the highest concentrations in dialysis samples both before and after treatment with veratridine, whereas GABA, homocysteic acid and aspartate were present in the lowest concentrations. Perfusion of veratridine into the ventrocaudal PAG resulted in significant elevation of all amino acids investigated. Except for taurine, no significant difference in veratridine-induced release between the lateral and medial PAG was observed. Tetrodotoxin (TTX) significantly blocked veratridine-induced release of GABA, aspartate, glutamate, glycine and taurine but not homocysteic acid. When rats were injected with morphine (10 mg/kg i.p.), veratridine-induced release of GABA was selectively and significantly decreased in the lateral but not the medial PAG as compared to control rats injected with saline followed by veratridine perfusion. Systemic injection of morphine or saline caused no significant change in the basal concentration of amino acids in PAG dialysate samples. These findings are consistent with the proposed mechanism of action of morphine in the lateral ventrocaudal PAG and offer the first direct evidence that systemic opiates decrease GABA release in this midbrain region.     

A quantitative description of excitatory amino acid neurotransmitter responses on cultured embryonic Xenopus spinal neurons

We have performed a quantitative analysis of excitatory amino acid neurotransmitter receptors on cultured embryonic Xenopus spinal neurons using the whole-cell patch-clamp technique. Neuroblasts and underlying mesodermal cells isolated from spinal regions of neural plate-stage embryos were placed into dissociated cell culture, and responses were studied soon after the appearance of neurites on embryonic neurons. Glutamate (Glu) receptors were separated into two general classes based on responses to the characteristic agonists quisqualate (Quis), kainate (Ka) and N-methyl-d-aspartate (NMDA); these were NMDA receptors (those activated by NMDA) and non-NMDA receptors (those activated by Ka and Quis). Half-maximal responses to Glu and other agonists on NMDA and non-NMDA receptors were determined from Hill analysis of dose response relations. The order of sensitivities observed was: Glu_NMDA(ED₅₀ = 5.1 μM) >Glu_non-NMDA(ED₅₀ = 28 μM), and for Glu receptor agonists, Quis (ED₅₀ = 1.5 μM) >NMDA(ED₅₀ = 41 μM) >Ka(ED₅₀ = 58 μM). The order of response amplitudes recorded at concentrations near the appropriate ED₅₀s was Glu_NMDA > Glu_non-NMDA, and Ka > NMDA > Quis. A 10-fold decrease in external [Na⁺] shifted the reversal potentials for Glu_non-NMDA, Ka, and Quis to more negative voltages. Increasing external [Ca²⁺] shifted the reversal potential for NMDA responses to more positive potentials, an observation consistent with Ca²⁺ permeation of the embryonic NMDA-activated channel. NMDA-evoked currents could not be recorded in nominally glycine (Gly)-free media. Addition of Gly to external solutions potentiated NMDA responses (ED₅₀ = 644nM). NMDA responses were blocked by dl-2-amino-5-phosphonovaleric acid (APV;ED₅₀ = 1.9 μM) and by Mg²⁺ at negative potentials. In their sensitivities to agonists and antagonists, and ionic dependences, amino acid neurotransmitter responses on embryonic Xenopus neurons closely resembled those previously observed for mature Xenopus and mammalian central neurons. The Glu_NMDA receptors present on these immature neurons were sufficiently sensitive to be activated by endogenous concentrations of extracellular Glu, suggesting a possible role for receptor activation in modulating early neural development.     

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.     

Polyamine effects uponN-methyl-D-aspartate receptor functioning: differential alteration by glutamate and glycine site antagonists

Polyamines such as spermidine potentiate activation of theN-methyl-D-aspartate (NMDA)-type excitatory amino acid receptor. The goal of the present study was to investigate interactions between the putative polyamine binding site and previously described sites for glutamate and glycine. Binding of the high-potency PCP receptor ligand [³H]MK-801 to well-washed rat brain membranes was used as an in vitro probe of NMDA receptor activation. Spermidine concentration-response studies were performed in the absence and presence of both glutamate and glycine, with and withoutD-(−)-2-amino-5-phosphonovaleric acid (D(−)AP-5) or 7-chlorokynurenic acid (7Cl-KYN). Incubation in the presence of spermidine alone induced a 20.4-fold increase in [³H]MK-801 binding with an EC₅₀ value of 13.3 μM. The mean concentration of spermidine which induced maximal stimulation of binding was 130 μM (n = 10,S.E.M.= 24.66,range= 25–250 μM). Glutamate (10 μM) decreased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding to 3.4 μM. Glycine (10 μM) did not significantly alter either maximum spermidine-induced [³H]MK-801 binding or the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding. Incubation in the presence of the specific glutamate antagonistD(−)AP-5 attenuated [³H]MK-801 binding in a glutamate-reversible fashion. The competitive glycine antagonist 7Cl-KYN decreased maximum spermidine-induced [³H]MK-801 binding in a glycine-reversible fashion. In addition, 7Cl-KYN increased the EC₅₀ value for spermidine-induced stimulation of [³H]MK-801 binding whileD(−)AP-5 was without effect. These findings suggest that glutamate and glycine regulate the polyamine binding site differentially. PCP-like agents induce a psychotomimetic state closely resembling schizophrenia by inhibiting NMDA receptor-mediated neurotransmission. The ability of polyamines to modulate NMDA receptor functioning suggests a potential site for pharmacological intervention.     

Local and distal effects induced by unilateral striatal application of opiates in the absence or in the presence of naloxone on the release of dopamine in both caudate nuclei and substantiae nigrae of the cat

Halothane-anesthetized cats implanted with push-pull cannulae in both caudate nuclei (CN) and substantiae nigrae (SN) were used to study the effects of naloxone and various opiates when applied into the left CN on the release of newly synthetized tritiated dopamine (DA) from nerve terminals and dendrites of the two nigro-striatal dopaminergic pathways. In all cases, the drugs (naloxone, opiates alone or in the presence of naloxone) were applied for 30 min into the left CN. When applied alone, naloxone (10⁻⁶ M) induced a delayed reduction in tritiated DA release both in the ipsilateral and contralateral CN. These effects were seen after removal of the drug from the superfusion fluid. Complementary experiments made with tritiated naloxone (10⁻⁶ M) revealed that the contralateral effect on DA release was not due to a diffusion of the opiate antagonist from its application site. Locally,d-Ala₂, Met-enkephalinamide (d-Ala₂, Met-Enk, 10⁻⁶ M) and the potent δ agonist Tyr-d-Ser-Gly-Phe-Leu-Thr (DSThr, 5 × 10⁻⁸ M) induced a biphasic increase in tritiated DA release. The local changes in tritiated DA release evoked by morphine (10⁻⁶ M) and μ agonists such as Tyr-d-Ala-Gly-NH-C₆H₁₃ (10⁻⁸ M) and fentanyl (10⁻⁸ M) differed from those of δ agonists and furthermore differed from each other. For instance, morphine induced a delayed increase in tritiated DA release whereas a biphasic increase followed by a delayed inhibition occurred with fentanyl. Among all the opiates testedd-Ala₂-Met-Enk was the only one which elicited a distal effect, that is a reduction of tritiated DA release in the ipsilateral SN. Marked differences in these opiates' effects on tritiated DA release occurred both locally and in distal structures when opiates were applied simultaneously with naloxone (10⁻⁶ M). Locally, the changes induced by μ agonists were particularly altered since during morphine's application with naloxone a reduction of tritiated DA release occurred. In addition, the opiate antagonist prevented the second increase and the delayed inhibition of tritiated DA release evoked by fentanyl (10⁻⁸ M). Interestingly, the combined application of naloxone with eitherd-Ala₂,Met-Enk (10⁻⁶ M) DSThr (5 × 10⁻⁸ M) or morphine (10⁻⁶ M) resulted in the appearance of changes in tritiated DA release in contralateral structures. The most striking effect was seen withd-Ala₂,Met-Enk which enhanced tritiated DA release in the contralateral CN and SN. These results are discussed in the light of the involvement of several types of opiate receptors and of the polysynaptic pathways responsible for the distal changes in dopaminergic transmission.     

Increased brain norepinephrine metabolism correlated with analgesia produced by the periaqueductal gray injection of opiates

Dose-dependent increases in in 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MOPEG-SO₄), a major metabolite of norepinephrine, were produced in the limbic forebrain and cerebral cortex 30 min after the bilateral injection of morphine into the periaqueductal gray (PAG). These effects were also elicited by similar injections of levorphanol. Highly significant correlations were obtained between the concentrations of MOPEG-SO₄ and the analgesic effect of the opiates and opiate actions were antagonized by systemic naloxone. These results indicate that activation of opiate receptors in the PAG may elicit the involvement of noradrenergic systems in distant brain regions in the mediation of analgesia.     

Excitatory Amino Acid-Induced Degeneration of Dendrites of Catecholamine Neurons in Rat Substantia Nigra

We have recently established a rat substantia nigra (SN) slice preparation in which a sensitive index of excitatory amino acid (EAA) toxicity was degeneration of the dendritic arbor of catecholamine neurons labelled by immunostaining for tyrosine hydroxylase (TH). The present study examined the pharmacological characteristics of EAA-induced neurotoxicity. Rats were anesthetised by halothane inhalation and killed, the brain was rapidly removed, and 400-μm-thick SN slices cut in the horizontal plane on a vibratome. Slices were incubated in saline buffer at 35°C for 15 min to 6 h in the presence or absence or absence of kainic acid (KA) orN-methyl- -aspartate (NMDA) in concentrations ranging from 10 to 500 μM. The slices were then fixed and resectioned into 40-μm sections that were coplanar with the parent slice. Dopaminergic SN neurons were labeled using antibody to tyrosine hydroxylase (TH) coupled to diaminobenzidine. A feature of the immunostaining was that it labeled not only the cell body but also the prolific dendritic arborization of SN neurons. Dendritic damage was quantified by counting the proportion of neurons with intact dendrites after treatment with EAA. KA and NMDA caused loss of dendrites that was prevented by CNQX (20 μM) and MK-801 (20 μM), respectively, indicating that activation of either NMDA or non-NMDA receptors produces neurotoxicity. EAA-induced dendritic damage was observed within 2 h of treatment with a low concentration (10 μM) of KA and within 15 min if the concentration was increased to 500 μM. Thus the loss of dendrites occurs rapidly and precedes disintegration of the cell bodies. Furthermore, brief (15 min) exposure to EAA initiated damage in the dendrites which progressed after the EAA was removed from its receptor. The observations are consistent with the postulated role of EAAs in neurodegenerative diseases. Labeling the dendritic arbor provides a sensitive approach to investigating the cellular mechanisms of neurodegeneration of catecholamine neurons.     

Evidence for taurine as an inhibitory neurotransmitter in cerebellar stellate interneurons: selective antagonism by TAG (6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide)

Using a novel amino acid antagonist, TAG (6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide), intradendritic electrophysiological investigations were carried out to obtain evidence for taurine as a neurotransmitter in the cerebellum. The hyperpolarizing action of taurine on Purkinje cell dendrites in guinea pig cerebellar slices was selectively antagonized by TAG (200 μM), while the actions of GABA, glycine and β-alanine were virtually unaffected. TAG shifted the log dose-response curve of the taurine action to the right in parallel, indicating a competitive antagonism. A hyperpolarizing synaptic potential which was evoked by electrical stimulation of the upper region of the cerebellar molecular layer and recorded from a Purkinje cell dendrite. was reversed to a depolarizing one at a membrane potential of -70 mV. The hyperpolarization induced by exogenously applied taurine was also reversed at the same potential. Moreover, TAG (200 μM) completely and reversibly blocked the synaptic potential. These results suggest that taurine may be an inhibitory neurotransmitter in stellate neuronal synapses on Purkinje cell dendrites.     

Intranigral GR-113808, a selective 5-HT4 receptor antagonist, attenuates morphine-stimulated dopamine release in the rat striatum

GR-113808, a potent and selective 5-HT₄ receptor antagonist, was infused through a microdialysis probe into the striatum and nucleus accumbens of awake rats, and basal and morphine-stimulated extracellular concentrations of dopamine (DA) were measured in these regions. At 1 and 10 μM GR-113808 did not effect the extracellular concentrations of DA in either region and 100 μM significantly reduced dialysate DA only in the striatum. A subcutaneous dose of 5 mg/kg morphine significantly raised extracellular concentrations of DA in the striatum and nucleus accumbens from 60 to 120 min after injection and the effect was not modified by 10 μM GR-113808 infused through the probe 20 min before and for 60 min after morphine. Bilateral injections of GR-113808 (1, 2.5 and 10 μg/0.5 μl) in the substantia nigra pars compacta did not affect dialysate DA in the striatum, except for a significant increase 120 min after the injection of 10 μg but the highest dose of GR-113808 prevented the increase of striatal DA caused by 5 mg/kg morphine s.c. The results suggest that 5-HT₄ receptors in the substantia nigra modulate the activity of the dopaminergic nigrostriatal system only when the neurons are activated.     

Interaction of lontophoretically applied morphine with responses of interneurons in cat spinal cord

A previous study reported that morphine blocked excitation by glutamate and depression by glycine. The present study examines the interaction of naloxone, an antagonist of morphine and dextrorphan, an inactive stereoisomer, with the responses elicited by these two amino acid putative transmitters. Drugs were applied by microiontophoresis from multibarreled pipettes onto sensory interneurons in the lumbar spinal cord of spinal cats. Both naloxone and dextrorphan were found to reduce the excitation produced by glutamate in a manner similar to morphine, thereby ruling out this interaction as relevant to analgesia. Neither naloxone nor dextrorphan were capable of reducing glycine-induced depression. However, naloxone did not antagonize the morhine blockade of glycine depression and it is therefore unlikely that morphine's interaction with glycine depression is relevant to morphine analgesia. The effects of iontophoretically applied morphine were also tested on the excitation of interneurons by cutaneous stimuli and were found to be small and varied. Neurons were excited by natural light and noxious tactile stimuli and by electrical cutaneous stimulation. Depression of naturally evoked responses was observed in 16% of neurons (13% were enhanced) and depression of electrically evoked responses in 9% of neurons (38% enhanced). Depression of lamina IV neurons was not observed. Although the results demonstrate a depression of responses of some spinal cord sensory interneurons by morphine, it is not thought that they support a direct action of morphine on these interneurons as the major site of its action in the production of analgesia.     

Effect of prolonged exposure to morphine on responsiveness of human and invertebrate immunocytes to stimulatory molecules

This study deals with a novel role of morphine in the modulation of cellular responsiveness to immunostimulatory substances that, at first glance, appears to be in contrast to the well documented immunoinhibitory short-term effects of opiate alkaloids on cells simultaneously exposed to stimulatory molecules. Vertebrate and invertebrate immunocytes pre-exposed to morphine (10⁻ M) in vitro for at least 24 h prior to the administration of lipopolysaccharide (LPS; 1.0 μ/ml) or other immunoactivating substances have revealed a distinct enhancement of their responsiveness to these signals e.g. monocytes exposed to LPS alone resulted in 21% activation, whereas the morphine pretreated level was at 40% (P < 0.01). Prolonged pretreatment with morphine of naive human monocytes had the same effect on their sensitivity to plasma from patients having undergone cardiopulmonary bypass (CPB) operations followed by a diffuse inflammatory response. These results suggest that endogenous opiates may participate, in more than one way, in re-establishing an organism's readiness to meet a new demand on its immune system. Additional support for the concept of a role of endogenous opiates in immunomodulation was obtained by the results of in vivo tests with experimentally induced stress in Mytilus. Following their stress-induced stimulation, these animals' immunocytes could be shown to become exposed for some time to a measurable rise in endogenous morphine-like material (9 pmol/ml increasing to 59). These immunocytes, like those preincubated with exogenous morphine, displayed a heightened sensitivity to stimulation by LPS (control 21.3 ± 3.1% activation compared to 47.2 ± 5.1) when the morphine levels dropped. The mechanism of this enhancement of responsiveness to immunostimulation following the prolonged exposure of immunocytes to morphine, and its relationship with the known short-term immunoinhibitory opiate effects on the immune system, remains to be ascertained.     

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.     

Effects of morphine and morphine withdrawal on adrenergic neurons of the rat rostral ventrolateral medulla

In urethane anesthetized rats, iontophoretic application of morphine or α-methylnoradrenaline (α-MNE) inhibited (80–100%) the discharges of all putative adrenergic (C1) cells of the rostral ventrolateral medulla (RVLM). The effect of morphine was blocked selectively by naloxone while that of α-MNE was blocked selectively by theα₂-adrenergic antagonist idazoxan. Putative C1 cells were inhibited (75–100%) by low i.v. doses of clonidine (10–15 μg/kg). Most cells (7/10) were also inhibited by morphine i.v. (81% at 7 mg/kg). Two cells were slightly excited at doses below 2 mg/kg and inhibited at higher doses. Three cells were excited only. All effects of morphine i.v. were reversed by naloxone (1 mg/kg, i.v.). Intravenous administration of naloxone to morphine-dependent rats increased significantly the firing rate of all putative C1 adrenergic cells (from 5.8 ± 0.9 spikes/s to 12.3 ± 1.5 spikes/s;n = 8). During withdrawal these cells could still be inhibited (80–100%) by i.v. injection of clonidine (15 μg/kg). C-Fos expression induced by naltrexone-precipitated withdrawal was examined in the brainstem of freely moving morphine-dependent rats pretreated with clonidine or saline before injection of the opioid antagonist. The locus coeruleus (LC) of the same rats was examined for comparison. Morphine withdrawal without clonidine treatment significantly increased the number of Fos-like-immunoreactive (Fos-LIR) cells in the RVLM and LC. Clonidine pretreatment (1 mg/kg, i.p.) reduced the number of withdrawal-activated Fos-LIR cells in LC by 81%. In the RVLM this reduction averaged 37% for all cell types and 48% for C1 adrenregic cells. Further, a very large proportion of RVLM neurons that expressed c-Fos during morphine withdrawal (83%) were immunoreactive forα₂A-adrenergic receptors. This study suggests that, like noradrenergic cells of the LC, C1 adrenergic neurons of the RVLM are: (i) inhibited by both opiate andα₂-adrenergic receptor agonists; and (ii) activated during naloxone-precipitated morphine withdrawal, Since C1 cells are considered essential to sympathetic tone generation, their inhibition by morphine may contribute to the hypotensive effects of this opioid agonist in non-dependent individuals. Their excitation during opiate withdrawal may also contribute to the autonomic activation that characterizes this syndrome. Finally, inhibition of C1 cells by clonidine may contribute to the clinically recognized efficacy of this drug to attenuate autonomic signs of opiate withdrawal.