Chronic depolarization induced by veratridine increases the survival of rat retinal ganglion cells ‘in vitro’

During the last decades it has been shown that trophic molecules released by target, afferent and glial cells play a pivotal role controlling neuronal cell death. Trophic molecules are able to inhibit this regressive event during development as well as during degenerative diseases. One of the mechanisms involved in the control of neuronal survival by afferent cells requires the release of trophic molecules stimulated by electrical activity. It has been demonstrated that veratridine (a depolarizing agent that keeps the Na⁺ channels opened) induces an increase in neuronal survival. In the present work we show that 3 μM veratridine induced a two-fold increase on the survival of retinal ganglion cells after 48 h in culture. The veratridine effect was inhibited by 50 μM amiloride (an inhibitor of Ca²⁺ channels), 25 μM benzamil (an inhibitor of Na⁺ channels), 30 μM dantrolene and 7.5 μM caffeine (both inhibitors of Ca²⁺ release from the endoplasmatic reticulum) and 10 μM BAPTA-AM (an intracellular Ca²⁺ chelator). However, 5 μM nifedipine (a selective inhibitor of voltage-dependent -type Ca²⁺ channels) and 100 μM MK 801 (an inhibitor of NMDA receptors) did not block the veratridine effect. On the other hand, treatment with 10 μM genistein (an inhibitor of tyrosine kinase enzymes), 20 μM fluorodeoxyuridine (an inhibitor of cell proliferation) or 10 μM atropine (an antagonist of muscarinic receptors) completely abolished the effect of veratridine. Taken together, our results indicate that veratridine increases the survival of rat retinal ganglion cells through mechanisms involving Na⁺ influx, intracellular Ca²⁺ release, activation of tyrosine kinase enzymes and cellular proliferation. They also indicate that cholinergic activity plays an important role in the veratridine effect.     

GABA, acting at both GABAA and GABAB receptors, inhibits the release of cholecystokinin-like material from the rat spinal cord in vitro.

Superfusion of slices of the dorsal zone of the lumbar enlargement of the rat spinal cord with an artificial cerebrospinal fluid allowed the collection of cholecystokinin-like material (CCKLM) whose Ca²⁺-dependent release could be evoked by tissue depolarization with 30 mM K⁺. Studies on the possible influence of GABA and related agonists on this process showed that the amino acid, the GABA_A agonist, muscimol, and the GABA_B agonist, baclofen, inhibited the K⁺-evoked release of CCKLM from the rat spinal cord in a concentration-dependent manner. Maximal inhibition did not exceed −40% with either agonist. Furthermore, the effects of GABA_A and GABA_B receptor stimulation were not additive. Whereas the effects of muscimol (10 μM) and baclofen (1 μM) could be completely antagonized by bicuculline (1 μM) and phaclofen (10 μM), respectively, complete blockade of the inhibition by GABA (1 μM) could only be achieved in the presence of both antagonists. These data indicate that both GABA_A and GABA_B receptors are involved in the negative influence of GABA onto CCK-containing neurones within the dorsal horn of the rat spinal cord. Apparently, these receptors are not located on CCK-containing neurones themselves, since the inhibitory effect of GABA on the K⁺-evoked release of CCKLM could be completely prevented by tetrodotoxin (1 μM). As CCK acts centrally as an endogenous opioid antagonist, such a GABA-inhibitory control of spinal CCK-containing neurones might participate in the analgesic action of the amino acid via the intrathecal route.     

Effects of thiopental, halothane and isoflurane on the calcium-dependent and -independent release of GABA from striatal synaptosomes in the rat

The effects of the anesthetic agents thiopental, halothane and isoflurane on the release of GABA induced by depolarization and/or reversal of the GABA carrier were investigated in a synaptosomal preparation obtained from the rat striatum. Veratridine (1 μM) and KCl (9 mM) elicited a significant Ca²⁺-dependent release of [³H]GABA. The KCl-evoked release was not significantly modified in the presence of nipecotic acid (10⁻⁵ M), a selective blocker of the neuronal GABA carrier. The [³H]GABA release was significantly decreased by ω-conotoxin (10⁻⁷ M, a blocker of the N voltage-dependent Ca²⁺ channels, but was affected by neither nifedipine (10⁻⁴ M) nor ω-Aga-IVA (10⁻⁷ M) which block the L and Ca²⁺ channels, respectively. Thiopental application (10⁻⁵ to 10⁻³ M) was followed by a dose-related, significant, decrease in both the veratridine and KCl-induced releases, whether nipecotic acid was present or not. In contrast, halothane and isoflurane (1–3%) failed to alter [³H]GABA release. Altogether, these results suggest that reduction of the depolarization-evoked GABA release might contribute to thiopental anesthesia, but this seems unlikely for volatile anesthetics.     

Restoration of cholinomimetic activity by clonidine in cholinergic plus noradrenergic lesioned rats

Lactate production (J_lac), oxygen consumption rate (Q_O2), plasma membrane potentials (E_m) and cytosolic free calcium levels [Ca²⁺]_i were studied on symaptosomes isolated from rat brains, incubated in presence of high doses of nicardipine (90 μM), diltiazem (0.5 mM) and verapamil (0.25 mM), and submitted to depolarizing stimulation or inhibition of mitochondrial respiration. Nicardipine was able to completely prevent the veratridine-induced stimulation ofJ_lac, Q_O2andE_m depolarization, whereas diltiazem and verapamil were less effective, although the concentrations used were 5 and 3 times higher, respectively, than nicardipine. Diltiazem, verapamil and nicardipine (9 μM) also prevented the veratridine-induced increase in [Ca²⁺]_i, this effect being much less pronounced if the drugs were added after veratridine. Monensin (20 μM) was also able to increase [Ca²⁺]_i but this effect was not affected by verapamil. Synaptosomes were also submitted to an inhibition of respiration of intrasynaptic mitochondria by incubation with rotenone (5 μM); in this condition of mimicked hypoxiaE_m was more positive of about 11 mV; none of the drugs utilized modified this situation. The rotenone-induced 3-fold increase inJ_lac was barely modified by diltiazem and verapamil but it was completely abolished by nicardipine. The possible mechanism of the counteracting action of the drugs towards veratridine stimulation and rotenone inhibition and the involvement of Na⁺/Ca²⁺ exchanger in affecting [Ca²⁺]_i are discussed.     

In botulinum type A-poisoned frog motor endings ouabain induces phasic transmitter release through Na-Ca exchange

Ouabain (100 μM) applied for 60 min to botulinum A (BoTx) poisoned motor junctions increases, in a time-dependent manner, the mean number of acetylcholine quanta released by nerve stimulation and enhances the delayed transmitter release. The drug does not affect spontaneous quantal release. The observed effects on evoked transmitter release cannot be explained by changes in the configuration of presynaptic currents recorded from motor terminals. They suggest that in BoTx-poisoned motor endings the level of intraterminal Ca²⁺, lower than that required for the activation of quantal transmitter release, can be effectively increased through the reversed operation of an Na⁺-Ca²⁺ exchange system that normally uses the Na⁺ gradient to extrude Ca²⁺.     

Endogenous bursting due to altered sodium channel function in rat hippocampal CA1 neurons

Intracellular recordings were obtained from pyramidal neurons in the rat hippocampal CA1 area in order to investigate membrane mechanisms involved in veratridine-induced epileptiform activity. Veratridine (0.03−0.2 μM) caused no changes in the passive membrane parameters including the resting potential, input resistance, and time constant. In the presence of small doses (0.03−0.1 μM) of veratridine, a single stimulus caused a relatively slow, large, synaptic-independent potential called the slow depolarizing after-potential (SDAP). When the hippocampal slice was treated with higher doses of veratridine (over 0.1 μM), bursting, or seizure-like activity (SLA) occurred in response to a brief super threshold intracellular stimulation. The duration of SLA bursting could be as long as ten seconds depending on the amplitude of SDAP, and was independent of the stimulus strength or duration. The frequency and configuration of SLA were sensitive to changes in membrane potential caused by applied DC current. At 0.3 μM or higher, veratridine induced spontaneous rhythmic bursting that was also sensitive to membrane potential changes. The evoked or spontaneous bursting is characterized by being: (1) independent of synaptic transmission in that it persisted after complete blockade of evoked synaptic potential with kynurenic acid (0.5 mM), (2) sensitive to selective inhibition by low doses of the specific sodium channel blockers tetrodotoxin (TTX) or cocaine with no apparent influence on the evoked action potential. These results indicate that endogenous SLA bursting can be induced in hippocampal CA1 pyramidal neurons when certain properties of sodium channels are altered by veratridine.     

Inhibition of the rat brain sodium channel Nav1.2 after prolonged exposure to gabapentin

Prolonged exposure of neurons to gabapentin inhibits repetitive firing of Na⁺-dependent action potentials. Here, we studied the effect of such prolonged exposure to gabapentin on a rat sodium channel, Nav1.2. After 3 days of continuous incubation with gabapentin (10–1000 μM), Nav1.2 current density was decreased dose-dependently relative to untreated cells. The reduction was 57% at 30 μM gabapentin, while higher concentrations (100–1000 μM) did not result in greater effects. Prolonged treatment with gabapentin also caused the channel to inactivate at more hyperpolarized potentials. These effects provide a mechanistic basis for the inhibition of Na⁺-dependent repetitive firing upon prolonged exposure to gabapentin and may contribute to its anticonvulsant activity.     

Cannabinoid CB1 receptor facilitation of substance P release in the rat spinal cord,measured as neurokinin 1 receptor internalization

The contribution of CB1 receptors in the spinal cord to cannabinoid analgesia is still unclear. The objective of this study was to investigate the effect of CB1 receptors on substance P release from primary afferent terminals in the spinal cord. Substance P release was measured as neurokinin 1 (NK1) receptor internalization in lamina I neurons. It was induced in spinal cord slices by dorsal root stimulation and in live rats by a noxious stimulus. In spinal cord slices, the CB1 receptor antagonists AM251, AM281 and rimonabant partially but potently inhibited NK1 receptor internalization induced by electrical stimulation of the dorsal root. This was due to an inhibition of substance P release and not of NK1 receptor internalization itself, because AM251 and AM281 did not inhibit NK1 receptor internalization induced by exogenous substance P. The CB1 receptor agonist ACEA increased NK1 receptor internalization evoked by dorsal root stimulation. The effects of AM251 and ACEA cancelled each other. In vivo, AM251 injected intrathecally decreased NK1 receptor internalization in spinal segments L5 and L6 induced by noxious hind paw clamp. Intrathecal AM251 also produced analgesia to radiant heat stimulation of the paw. The inhibition by AM251 of NK1 receptor internalization was reversed by antagonists of μ‐opioid and GABA_B receptors. This indicates that CB1 receptors facilitate substance P release by inhibiting the release of GABA and opioids next to primary afferent terminals, producing disinhibition. This results in a pronociceptive effect of CB1 receptors in the spinal cord.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [³H]cyclohexyladenosine (CHA) to the cellular fractions and P₂ subfractions of the goldfish brain was studied. The A₁ receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P₂), 30 mM K⁺ stimulated glutamate, taurine and GABA release in a Ca²⁺-dependent fashion, whereas the aspartate release was Ca²⁺-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 μM) inhibited K⁺-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A₁ receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K⁺ (30 mM) stimulated Ca²⁺ influx (46.8±6.8%) and this increase was completely abolished by pretreatment with 100 nM ω-conotoxin. Pretreatment with 100 μM R-PIA or 100 μM CHA, reduced the evoked increase of intra-synaptosomal Ca²⁺ concentration, respectively by 37.7±4.3% and 39.7±9.0%. A possible correlation between presynaptic A₁ receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Brain cell membrane Na,K-ATPase activity following severe hypoxic injury in the newborn piglet

This study tests the hypothesis that severe brain hypoxia causes decreased Na⁺,K⁺-ATPase activity, resulting in permanent alterations in the neuronal cell membranes. Seventeen anesthetized piglets (normoxic control (NC), no recovery after hypoxia (Group 1), 6 h normoxic recovery (Group 2), and 48 h normoxic recovery (Group 3) were studied. Hypoxia was induced by lowering the FiO₂ to maintain PCr/P_i ratio at 25% of baseline for 1 h as monitored by ³¹P-NMR spectroscopy. PCr/P_i returned to 57% of baseline by 6 h and was normal by 48 h. At termination, cortical tissue Na⁺,K⁺-ATPase activity was determined. Na⁺,K⁺-ATPase activity was measured in cortical membrane preparations by determining the rate of ATP hydrolysis. NC membranes had Na⁺,K⁺-ATPase activity of 58.3 ± 1.3 μM P_i/mg protein/h (mean ± S.E.M.). Na⁺,K⁺-ATPase activity was reduced in Groups 1, 2, and 3 (45.8 ± 1.3, 47.4 ± 3.6, 48.7 ± 2.9 μM P_i/mg protein/h) (P < 0.05 compared to NC). There was no differene in enzyme activity among Groups 1, 2, or 3. The data show that in spite of recovery of neuronal oxiditive phosphorylation (PCr/P_i) by 48 h, there is a permanent decrease in Na⁺,K⁺-ATPase activity in cells that have undergone severe hypoxic injury. The persistent decrease in Na⁺,K⁺-ATPase activity indicates ongoing cell injury following severe cerebral hypoxia, and that recovery of oxidative phosphorylation as indicated by PCr/P_i values cannot be used as an index of recovery of cell function.     

Concurrent folate treatment prevents Na,K-ATPase activity inhibition and memory impairments caused by chronic hyperhomocysteinemia during rat development

We investigated the hypothesis that folate administration would prevent hyperhomocysteinemia-induced memory deficits and Na⁺,K⁺-ATPase activity inhibition. Chronic hyperhomocysteinemia was induced from the 6th to the 28th day of life by subcutaneous injection of homocysteine (0.3–0.6 μmol/g), twice a day; control Wistar rats received the same volume of saline solution (0.9% NaCl). Half of the homocysteine- and saline-treated groups also received intraperitoneal administration of folate (0.011 μmol/g) from the 6th to the 28th day of life. A group of animals was killed 12 h after the last injection, plasma and parietal cortex were collected for biochemical analysis. Another group stayed at Central Animal House until 60th day of life, when the rats were submitted to behavioral testing in water maze or were killed for evaluation of cortical Na⁺,K⁺-ATPase activity. Results showed that hyperhomocysteinemia impaired reference memory for platform location, as assessed by fewer crossings to the platform place and increased latency for the first crossing, when compared to controls. In the working memory task homocysteine-treated animals also needed more time to find the platform. We also observed that Na⁺,K⁺-ATPase activity was reduced in parietal cortex of hyperhomocysteinemic rats sacrificed 12 h after the last injection of homocysteine (29-day-old rats). In contrast, this enzyme was not altered when the rats were sacrificed 31 days after the treatment (60-day-old rats). Hyperhomocysteinemic rats treated with folate had all those impairments prevented, an effect probably related to folate antioxidant properties.     

Uptake of leucine and alanine by cultured cerebral capillary endothelial cells

Pure cultures of rat cerebral capillary endothelium have been used to study the A- and L-systems of amino acid transport. Leucine is taken up by a non-concentrative mechanism that can be saturated, and competivively inhibited by phenylalanine. Uptake is rapid, with equilibiration apparent 3–min (al experiments performed at 37 °C). The K_M for transport was 83 μM ± 26(mean ±S.E.M.., n = 3 which is in good agreement with recent vi vivo report using unanesthtissed rats. Alanine was transported by a saturable, concentrative mechanism. Dependence on Na⁺ -ions was demonstrated by lack of specific uptake in Na⁺ -ffree buffer and reduced uptake after preincubation in ouabain — Na⁺, K⁺ -ATPase inhibitor. The K_M was 325 μM ±88 (mean ± S.E.M., n = 3). The finding ac an active A-system transporter in vitro suggests that the cells may have lost the polarity they demonstrate in vivo. The relevance of these findings to transport of nutrients nd drugs across the blood-brain barrier is discussed.     

Pain, nociception and spinal opioid receptors

1. Opioid peptides derived from proenkephalin and prodynorphin are differentially distributed in the spinal cord. Proenkephalin peptides are preferentially located in the sacral portion of the cord while prodynorphin peptides are concentrated in the cervical spinal cord.

2. μ opioid receptor are highly concentrated in superficial layers of the dorsal horn in all the spinal cord.

3. δ opioid receptor are more diffusely distributed in the gray matter of the spinal cord. These sites are principally located in cervical and thoracic portions of the spinal cord.

4. κ opioid receptors are highly concentrated in the superficial layers of the lumbo-sacral spinal cord. Its density decreased in the upper levels of the spinal cord.

5. It appears that μ opioid receptors are indifferentially activated by thermal, pressure and visceral nociceptive inputs. δ receptors are more likely to be involved in thermal nociception while κ opioid binding sites are associated to visceral pain nociceptive inputs.     

Different spatial distributions of sodium channels in the slowly and rapidly adapting stretch receptor neuron of the crayfish

Inward Na⁺ currents were studied, using a two-microelectrode intracellular voltage-clamp technique, in the slowly adapting (SA) and rapidly adapting (RA) stretch receptor neurons of the crayfish after the axons were cut at different distances from the soma. In the SA neuron, inward Na⁺ currents were recorded in the soma even when the axon was cut as close as 100 μm from the center of the soma, indicating the presence of Na⁺ channels in these parts. Also, two populations of Na⁺ channels seem to exist in the SA neuron. In the RA neuron, only minute Na⁺ currents were observed if the axon was shorter than 250 μm. The results strongly indicate that the voltage-gated Na⁺ channels in the SA and RA neurons have different distributions and that the difference in the spatial distribution of Na⁺ channel types may be important for the difference in firing properties in the two types of neurons.     

μ-Opioid receptor-induced Ca mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca-dependent mechanism

Morphine, a preferential μ-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca²⁺-dependent mechanism, few studies have examined whether Ca²⁺ mediates the effect of opioids on cell proliferation, or whether a particular Ca²⁺ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ-opioid receptors and Ca²⁺ mobilization in morphine-induced astrocyte development. Morphine (1 gmM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca²⁺-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca²⁺ ([Ca²⁺]_o), or in unmodified media containing Ca²⁺ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or l-glutamate (100 μM) for 0-72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca²⁺ ([Ca²⁺]ⁱ) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphine's actions were mimicked by the selective μ, agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca²⁺]_i in developing astroglia. At normal [Ca²⁺]_o, morphine attenuated DNA synthesis by increasing [Ca²⁺]_i; low [Ca²⁺]_o (0.3 mM) blocked this effect, while treatment with Ca²⁺ ionophore or glutamate mimicked morphine's actions. At extremely low [Ca²⁺]_o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca²⁺]_i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca²⁺ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP₃-sensitive Ca²⁺ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca²⁺-dependent Ca²⁺ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca²⁺-dependent Ca²⁺ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.     

Estrogen receptor-alpha is required for estrogen-induced mu-opioid receptor internalization

Endogenous opioid circuits are pivotal for the regulation of sexual receptivity. Treatment of mice with morphine, a preferential mu-opioid receptor (MOR) agonist, severely attenuates lordosis. Estrogen induces internalization of MOR in cell groups of the limbic-hypothalamic lordosis-regulating circuit. Because rapid MOR internalization is mediated by estrogen release of endogenous opioid peptides, internalization has been used as a neurochemical signature of estrogen action in the central nervous system. Together these results indicate that estrogen induces a MOR mediated inhibition of sexual receptivity. To determine which estrogen receptor, estrogen receptor-alpha (ERalpha) or estrogen receptor-beta (ERbeta), mediates MOR internalization, ERalpha knockout (ERalphaKO), ERbeta knockout (ERbetaKO) and wild-type (WT) mice were used in the present study. WT, ERalphaKO and ERbetaKO mice had similar MOR distributions in the limbic-hypothalamic lordosis-regulating circuit. Estrogen treatment internalized MOR in the medial preoptic nucleus of ovariectomized WT and ERbetaKO, but not ERalphaKO mice. Treatment of ERalphaKO mice with the selective endogenous MOR ligand, endomorphin-1, induced levels of MOR internalization similar to WT mice suggesting that MOR in ERalphaKO mice could be activated and were probably functional. The results of the present experiments indicate that ERalpha is required for estrogen-induced MOR internalization and suggest that ERalpha can mediate rapid actions of estrogen.     

Spatiotemporal pH dynamics following insertion of neural microelectrode arrays

Insertion trauma is a critical issue when assessing intracortical electrophysiological and neurochemical recordings. Previous reports document a wide variety of insertion techniques with speeds ranging from 10 μm/s to 10 m/s. We hypothesize that insertion speed has an effect on tissue trauma induced by implantation of a neural probe. In order to monitor the neural interface during and after probe insertion, we have developed a silicon-substrate array with hydrous iridium oxide microelectrodes for potentiometric recording of extracellular pH (pH_e), a measure of brain homeostasis. Microelectrode sites were sensitive to pH in the super-Nernstian range (−85.9 mV/pH unit) and selective over other analytes including ascorbic acid, Na⁺, K⁺, Ca²⁺, and Mg²⁺. Following insertion, arrays recorded either triphasic or biphasic pH_e responses, with a greater degree of prolonged acidosis for insertions at 50 μm/s than at 0.5 mm/s or 1.0 mm/s (p < 0.05). Spatiotemporal analysis of the recordings also revealed micro-scale variability in the pH_e response along the array, even when using the same insertion technique. Implants with more intense acidosis were often associated histologically with blood along the probe tract. The potentiometric microsensor array has implications not only as a useful tool to measure extracellular pH, but also as a feedback tool for delivery of pharmacological agents to treat surgical brain trauma.     

Modulation by muscarine and opioid receptors of acetylcholine release in slices from striato-striatal grafts in the rat

The accumulation of tritium during incubation with [³H]choline and the subsequent efflux of tritium were studied in striatal slices from non-operated rats, in striatal slices from animals which had received a contralateral striatal ibotenic acid lesion, and in slices from striato-striatal suspension grafts, 16–31 weeks after implantation into previously lesioned striata. In graft slices, the accumulation of tritium as well as the overflow of tritium evoked by electrical stimulation (360 pulses, 3 Hz) was much smaller than in slices from non-operated controls. The muscarine receptor agonist oxotremorine (0.1–1 μmol/l) inhibited the stimulation-evoked overflow, and this effect was blocked by the muscarine receptor antagonists atropine (0.1 μmol/l) and pirenzepine (1 μmol/l) in all experimental groups to the same extent. The δ-receptor selective opioid peptide [d-Pen², d-Pen⁵]enkephalin (0.3 μmol/l) inhibited [³H]acetylcholine release in all groups, although its effect was smaller in grafts than in normal tissue. The preferential μ-receptor agonist [d-Ala², N-methyl-Phe⁴,Gly-ol⁵]enkephalin also reduced [³H]acetylcholine release in all groups, but only at the high concentration of 10 μmol/l. The effect of both drugs was antagonized by naloxone (1 μmol/l). The preferential к-receptor agonist ethylketocyclazocine enhanced the stimulation-evoked overflow in non-operated animals, an effect abolished by naloxone and also by sulpiride. In grafts, ethylketocyclazocine caused no change. It is concluded that acetylcholine release in striato-striatal grafts can be modulated by muscarine autoreceptors and by opioid δ receptors. The enhancement by к-receptor activation of [³H]acetylcholine release in non-operated striata depends on a dopaminergic input to the cholinergic cells which does not exist in grafts.     

Tetrodotoxin-dependent glutamate release in the rat nucleus accumbens during concurrent presentation of appetitive and conditioned aversive stimuli

In vivo microdialysis combined with a high-performance liquid chromatography was used to monitor extracellular glutamate (GLU) levels in the nucleus accumbens (N.Acc) of Sprague-Dawley rats during their behavioral responses to the concurrent presentation of appetitive and conditioned aversive stimuli. The presentation of a highly palatable diet followed by a tone previously paired with footshock to rats trained to take a pellet of the diet under these experimental conditions resulted in a marked and short lasting increase in extracellular glutamate, whereas the tone alone had no effect. A similar increase of the glutamate release was observed during the presentation of a piece of rubber instead of the diet. In both cases, the increase in extracellular glutamate was completely prevented by intra-accumbal infusions through the dialysis probe of 1 μM tetrodotoxin (a voltage-dependent Na⁺ channel blocker), whereas (S)-4-carboxyphenylglycine (a cystine/glutamate exchange blocker, 5 μM) had no effect.

The data obtained suggest that behavioral responses to unpredicted change in motivational value of expected reward appear to be associated with an increase of the extracellular glutamate level in the nucleus accumbens, and impulse-dependent synaptic release, rather than non-vesicular glutamate release via cystine/glutamate exchange, is responsible for this phenomenon.     

Ammonia stimulates the release of taurine from cultured astrocytes

The effect of ammonia on the release of the neuroactive amino acids taurine (TAU), γ-aminobutyric acid (GABA) and -aspartate ( -ASP), an analog of -glutamate ( -GLU), from cultured rat cortical astrocytes was studied. NH₄Cl (1 and 5 mM) induced the release of TAU. TAU release was reduced when Na⁺ was removed, and was almost completely abolished when Cl⁻ was omitted. In contrast, TAU basal release was enhanced upon removal of Na⁺ or Cl⁻. Ammonia inhibited the release of GABA and -ASP. Ammonia-induced release of astroglial TAU may modify the neuronal excitability accompanying hyperammonemic conditions.