A longevity study with enhancer substances (selegiline, BPAP) detected an unknown tumor-manifestation-suppressing regulation in rat brain.

AIMS: First proof to show that (-)-deprenyl/selegiline (DEP), the first selective inhibitor of MAO-B, later identified as the first ß-phenylethylamine (PEA)-derived synthetic catecholaminergic activity enhancer (CAE) substance and (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP), the tryptamine-derived presently known most potent, selective, synthetic enhancer substance, are specific markers of unknown enhancer-sensitive brain regulations. MAIN METHODS: Longevity study disclosing the operation of tumor-manifestation-suppressing (TMS) regulation in rat brain. Immonohistochemical identification of a fibromyxosarcoma in rats. Experiments with human medulloblastoma cell lines. Analysis of the mechanism of action of enhancer substances. KEY FINDINGS: Whereas 20/40 saline-treated rats manifested a fibromyxosarcoma, in groups of rats treated with 0.001mg/kg DEP: 15/40 rats; with 0.1mg/kg DEP: 11/40 rats (P<0.01); with 0.0001mg/kg BPAP: 8/40 rats (P<0.001); with 0.05mg/kg BPAP: 7/40 rats (P<0.01) manifested the tumor. Experiments with human medulloblastoma cell lines, HTB-186 (Daoy); UW-228-2, showed that BPAP was devoid of direct cytotoxic effect on tumor cells, and did not alter the direct cytotoxic effectiveness of temozolomide, cisplatin, etoposide, or vincristine. Interaction with distinct sites on vesicular monoamine-transporter-2 (VMAT2) is the main mechanism of action of the enhancer substances which clarifies the highly characteristic bi-modal, bell-shaped concentration-effect curves of DEP and BPAP. SIGNIFICANCE: Considering of the safeness of the enhancer substances and the finding that DEP and BPAP, specific markers of unknown enhancer sensitive brain regulations, detected the operation of an enhancer-sensitive TMS-regulation in rat brain, it seems reasonable to test in humans low dose DEP or BPAP treatment against the spreading of a malignant tumor.

Neuroprotective and anticonvulsant effects of EGIS-8332, a non-competitive AMPA receptor antagonist, in a range of animal models.

BACKGROUND AND PURPOSE: Blockade of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors is a good treatment option for a variety of central nervous system disorders. The present study evaluated the neuroprotective and anticonvulsant effects of EGIS-8332, a non-competitive AMPA receptor antagonist, as a potential drug candidate. EXPERIMENTAL APPROACH: AMPA antagonist effects of EGIS-8332 were measured using patch-clamp techniques. Neuroprotective and anticonvulsant effects of EGIS-8332 were evaluated in various experimental models, relative to those of GYKI 53405. KEY RESULTS: EGIS-8332 inhibited AMPA currents in rat cerebellar Purkinje cells and inhibited the AMPA- and quisqualate-induced excitotoxicity in primary cultures of telencephalon neurons (IC(50)=5.1-9.0 microM), in vitro. Good anticonvulsant actions were obtained in maximal electroshock-, sound- and chemically-induced seizures (range of ED(50)=1.4-14.0 mg kg(-1) i.p.) in mice. Four days after transient global cerebral ischaemia, EGIS-8332 decreased neuronal loss in the hippocampal CA1 area in gerbils and rats. EGIS-8332 dose-dependently reduced cerebral infarct size after permanent middle cerebral artery occlusion in mice and rats (minimum effective dose=3 mg kg(-1) i.p.). Side effects of EGIS-8332 emerged much above its pharmacologically active doses. A tendency for better efficacy of GYKI 53405 than that of EGIS-8332 was observed in anticonvulsant tests that reached statistical significance in few cases, while the contrary was perceived in cerebral ischaemia tests. CONCLUSIONS AND IMPLICATIONS: EGIS-8332 seems suitable for further development for the treatment of epilepsy, ischaemia and stroke based on its efficacy in a variety of experimental disease models, and on its low side effect potential.

Mechanisms underlying the long-term behavioral effects of traumatic experience in rats: the role of serotonin/noradrenaline balance and NMDA receptors.

Traumatic stressors induce long-lasting changes in behavior. It is believed that all three glutamatergic, serotonergic and noradrenergic neurotransmission play a role in the development of such behavioral changes, but their relative importance and relationship is poorly understood. We have shown previously that a single exposure of rats to electric shocks induces social avoidance for about 10 days. Here we assessed social avoidance 24 h after shock exposure in rats with chemically lesioned serotonergic and noradrenergic neurons. The effects of the NMDA receptor blocker MK-801 were also studied. When the serotonin/noradrenaline balance was shifted towards serotonergic dominance via chemical lesions, the behavioral dysfunction was markedly attenuated. The disruption of serotonergic neurotransmission (that lead to noradrenergic dominance) significantly increased the behavioral deficit. Shock responding was not secondary to lesion-induced differences in social behavior. Noteworthy, the brain noradrenaline/serotonin ratio correlated negatively with shock-induced social avoidance, suggesting that the ratio rather than absolute levels are important in this respect. In line with this assumption, double lesions had minor effects on social avoidance, suggesting that these monoaminergic systems modulate, but do not mediate the behavioral deficit. The blockade of NMDA receptors abolished the development of stress-induced social avoidance both when applied before shocks and when applied before behavioral testing. We confirmed that the long-term behavioral effects of traumatic experience result from glutamatergic activation, the effects of which are mediated by NMDA receptors. The development of the behavioral deficit is modulated by the balance between serotonergic and noradrenergic neurotransmission, possibly via effects on shock-induced glutamatergic activation.

Glycine transporter type-1 and its inhibitors.

The ionotropic glutamate receptor NMDA is allosterically modulated by glycine, a coagonist, its presence is an absolute requirement for receptor activation. The transport of glycine in glutamatergic synapse is carried out by glycine transporter-1 (GlyT1), a Na+/Cl(-)-dependent carrier molecule. The primary role of GlyT1 is to maintain glycine concentrations below saturation level at postsynaptic NMDA receptors. Several isoforms of GlyT1 (a-e) have been identified, which are expressed both in glial and neuronal cell membranes. GlyT1 operates bidirectionally: it decreases synaptic glycine concentration when operates in normal mode and releases glycine from glial cells as operates in a reverse mode. It is expected that non-transportable, non-competitive inhibitors of GlyT1 may have therapeutic value in CNS disorders characterized by hypofunctional NMDA receptor-mediated glutamatergic neurotransmission. Accordingly, GlyT1 inhibitors exhibited antipsychotic profile in a number of animal tests. The first promising in vitro and in vivo experiments with glycine itself, and its N-methyl analogue, sarcosine, had initiated the syntheses of potential GlyT1 inhibitors with more complex structures, in which, however, the glycine or sarcosine moiety had always been incorporated. Those attempts led to the development of two compounds, ALX-5407 and Org-24461 with high inhibitory potency; however, none of which is now considered as a drug candidate due, most probably, to safety and/or pharmacokinetic issues. More recently, several structurally new series of highly potent inhibitors with no aminomethylcarboxy group have also been discovered. Some of them might be expected to fulfill all requirements for clinical development. The new generation of GlyT1 inhibitors may represent a novel treatment of patients suffering from schizophrenia and/or other neuropathological conditions.

The pharmacology of the neurochemical transmission in the midbrain raphe nuclei of the rat.

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with [(3)H]serotonin ([(3)H]5-HT), superfused and the release of [(3)H]5-HT was determined at rest and in response to electrical stimulation. Compartmental analysis of [(3)H]5-HT taken up by raphe tissue indicated various pools where the neurotransmitter release may originate from these stores differed both in size and rate constant. 5-HT release originates not only from vesicles but also from cytoplasmic stores via a transporter-dependent exchange process establishing synaptic and non-synaptic neurochemical transmission in the serotonergic somatodendritic area. Manipulation of 5-HT transporter function modulates extracellular 5-HT concentrations in the raphe nuclei: of the SSRIs, fluoxetine was found 5-HT releaser, whereas citalopram did not exhibit this effect. Serotonergic projection neurons in the raphe nuclei possess inhibitory 5-HT(1A) and 5-HT(1B/1D) receptors and facilitatory 5-HT(3) receptors, which regulate 5-HT release in an opposing fashion. This observation indicates that somatodendritic 5-HT release in the raphe nuclei is under the control of several 5-HT homoreceptors. 5-HT(7) receptors located on glutamatergic axon terminals indirectly inhibit 5-HT release by reducing glutamatergic facilitation of serotonergic projection neurons. An opposite regulation of glutamatergic axon terminals was also found by involvement of the inhibitory 5-HT(7) and the stimulatory 5-HT(2) receptors as these receptors inhibit and stimulate glutamate release in raphe slice preparation, respectively, Furthermore, postsynaptic 5-HT(1B/1D) heteroreceptors interact with release of GABA in inhibitory fashion in raphe GABAergic interneurons. Serotonergic projection neurons also possess glutamate and GABA heteroreceptors; NMDA and AMPA receptors release 5-HT, whereas both GABAA and GABAB receptors inhibit somatodendritic 5-HT release. Evidence was found for reciprocal interactions between serotonergic and glutamatergic as well as serotonergic and GABAergic innervations in the raphe nuclei. Serotonergic neurons in the raphe nuclei also receive noradrenergic innervation arising from the locus coeruleus and alpha-1 and alpha-2 adrenoceptors inhibited [(3)H]5-HT release in our experimental conditions. The close relation between 5-HT transporter and release-mediating 5-HT autoreceptors was also shown by addition of L-deprenyl, a drug possessing inhibition of type B monoamine oxidase and 5-HT reuptake. L-Deprenyl selectively desensitizes 5-HT(1B) but not 5-HT(1A) receptors and these effects are not related to inhibition of 5-HT metabolism but rather to inhibition of 5-HT transporter.

The role of glycineB binding site and glycine transporter (GlyT1) in the regulation of [3H]GABA and [3H]glycine release in the rat brain.

The effect of N-methyl-D-aspartic acid (NMDA), a selective glutamate receptor agonist, on the release of previously incorporated [3H]gamma-aminobutyric acid(GABA) was examined in superfused striatal slices of the rat. NMDA (0.01 to 1.0 mM) increased [3H]GABA overflow with an EC50 value of 0.09 mM. The [3H]GABA releasing effect of NMDA was an external Ca2+-dependent process and the GABA uptake inhibitor nipecotic acid (0.1 mM) potentiated this effect. These findings support the view that NMDA evokes GABA release from vesicular pool in striatal GABAergic neurons. Addition of glycine (1 mM), a cotransmitter for NMDA receptor, did not influence the NMDA-induced [3H]GABA overflow. Kynurenic acid (1 mM), an antagonist of glycineB site, decreased the [3H]GABA-releasing effect of NMDA and this reduction was suspended by addition of 1 mM glycine. Neither glycine nor kynurenic acid exerted effects on resting [3H]GABA outflow. These data suggest that glycineB binding site at NMDA receptor may be saturated by glycine released from neighboring cells. Glycyldodecylamide (GDA) and N-dodecylsarcosine, inhibitors of glycineT1 transporter, inhibited the uptake of [3H]glycine (IC50 33 and 16 microM) in synaptosomes prepared from rat hippocampus. When hippocampal slices were loaded with [3H]glycine, resting efflux was detected whereas electrical stimulation failed to evoke [3H]glycine overflow. Neither GDA (0.1 mM) nor N-dodecylsarcosine (0.3 mM) influenced [3H]glycine efflux. Using Krebs-bicarbonate buffer with reduced Na+ for superfusion of hippocampal slices produced an increased [3H]glycine outflow and electrical stimulation further enhanced this release. These experiments speak for glial and neuronal [3H]glycine release in hippocampus with a dominant role of the former one. GDA, however, did not influence resting or stimulated [3H]glycine efflux even when buffer with low Na+ concentration was applied.

Binding characteristics of GYKI-46 903, a non-competitive ligand at 5-HT3 receptors.

GYKI-46903 [(+)-(5S,6R)-4-(4-fluorophenyl)-6-propionyloxy-1-aza-bicyclo[3.3.1]-non-3-ene-hydrochloride], a cognition enhancer identified as a non-competitive antagonist of 5-HT3receptors in isolated guinea-pig ileum, was investigated for allosteric action at 5-HT3 receptors in rat cortical membranes by using [3H]granisetron. Equilibrium and kinetic protocols were applied and the competitive antagonist granisetron was included as a negative control. In competition studies, both granisetron and GYKI-46 903 displaced the radioligand with K(i) values of 0.20 +/- 0.02 and 79.84 +/- 0.28 nM, respectively. The inhibition curve for GYKI-46 903 resulted in a Hill slope significantly greater than unity ( 1.37 +/- 0.11), whereas the slope for granisetron was 0.88 +/- 0.08, not different from unity. These results indicate non-competitive and competitive interactions, respectively. Scatchard analysis yielded a linear plot, suggesting a single population of binding sites with a Kd of 0.13 +/- 0.01 nM and a Bmax) of 13.15 +/- 0.34 fmol per mg of protein. Scatchard plots obtained in the absence and presence of granisetron (0.1-3 nM) or GYKI-46 903 (30-1000 nM) revealed a concentration-dependent increase in Kd values by either of these compounds. Granisetron left the Bmax unchanged, but there was a significant increase in the Bmax by GYKI-46 903, which could point to an atypical allosteric interaction. The Schild plot derived from the Kd shifts induced by granisetron was linear with a slope of 1.02, not different from unity, as expected from a competitive interaction. The Schild regression for GYKI-46 903 was linear with a slope of 1.20, deviating significantly from unity, which may also indicate an allosteric interaction. Both the association and dissociation curves of [3H]granisetron were monoexponential. The dissociation rate constant (K(-1)) and the association rate constant (K(+1)) were 0.32 +/- 0.01 min(-1) and 1.15 min(-1) x nM(-1), respectively. The dissociation driven by an excess concentration of ondansetron ( 1 microM) in the absence and presence of granisetron (0.1-3 nM) or GYKI-46 903 (30-10 000 nM) was not influenced by the compounds under study, as compared with the control, indicating the lack of an allosteric effect on the dissociation. Summing up, the binding profile of GYKI-46 903 may reflect a mixed type of action, including a negative allosteric interaction.

Reciprocal innervation between serotonergic and GABAergic neurons in raphe nuclei of the rat.

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain in order to study serotonergic-GABAergic interaction. The slices were loaded with either [3H] serotonin or [3H]GABA, superfused and the electrically induced efflux of radioactivity was determined. The GABA(A) receptor agonist muscimol (3 to 30 microM) and the GABA(B) receptor agonist baclofen (30 and 100 microM) inhibited [3H]serotonin and [3H]GABA release. These effects of muscimol were reversed by the GABA(A) antagonists bicuculline (100 microM). The GABA(B) antagonist phaclofen (100 microM) also antagonized the baclofen-induced inhibition of [3H]serotonin and [3H]GABA release. Phaclofen by itself increased [3H]serotonin release but it did not alter [3H]GABA overflow. Muscimol (10 microM) and baclofen (100 microM) also inhibited [3H]serotonin release after depletion of GABAergic neurons by isoniazid pretreatment. These findings indicate the presence of postsynaptic GABA(A) and GABA(B) receptors located on serotonergic neurons. The 5-HT1A receptor agonist 8-OH-DPAT (0.01 to 1 microM) and the 5-HT1B receptor agonist CGS-12066A (0.01 to 1 microM) inhibited the electrically stimulated [3H]serotonin and [3H]GABA release. The 5-HT1A antagonist WAY-100135 (1 microM) was without effect on [3H]serotonin and [3H]GABA efflux by itself but it reversed the 8-OH-DPAT-induced transmitter release inhibition. During KCl (22 mM)-induced depolarization, tetrodotoxin (1 microM) did not alter the inhibitory effect of CGS-12066A (1 microM) on [3H]GABA release, it did blocked, however, the ability of 8-OH-DPAT (1 microM) to reduce [3H]GABA efflux. After depletion of raphe serotonin neurons by p-chlorophenylalanine pretreatment, CGS-12066A (1 microM) still inhibited [3H]GABA release whereas in serotonin-depleted slices, 8-OH-DPAT (1 microM) was without effect on the release. We conclude that reciprocal influence exists between serotonergic projection neurons and the GABAergic interneurons or afferents in the raphe nuclei and these interactions may be mediated by 5-HT1A/B and GABA(A/B) receptors. Both synaptic and non-synaptic neurotransmission may be operative in the 5-HTergic-GABAergic reciprocal interaction which may serve as a local tuning in the neural connection between cerebral cortex and midbrain raphe nuclei.

New non competitive AMPA antagonists.

New halogen atom substituted 2,3-benzodiazepine derivatives condensed with an azole ring on the seven membered part of the ring system of type 3 and 4 as well as 5 and 6 were synthesized. It was found that chloro-, dichloro- and bromo-substitutions in the benzene ring and additionally imidazole ring condensation on the diazepine ring can successfully substitute the methylenedioxy group in the well known molecules GYKI 52466 (1) and GYKI 53773 (2) and the 3-acetyl-4-methyl structural feature in 2, respectively, preserving the highly active AMPA antagonist characteristic of the original molecules. From the most active compounds (3b,i) 3b (GYKI 47261) was chosen for detailed investigations. 3b revealed an excellent, broad spectrum anticonvulsant activity against seizures evoked by electroshock and different chemoconvulsive agents indicating a possible antiepileptic efficacy. 3b was found to be highly active in a transient model of focal ischemia predictive of a therapeutic value in human stroke. 3b also reversed the dopamine depleting effect of MPTP and antagonized the oxotremorine induced tremor in mice indicating a potential antiparkinson activity.

Neurochemical and electrophysiological studies on the functional significance of burst firing in serotonergic neurons.

We have previously described a population of 5-hydroxytryptamine neurons which repetitively fires bursts of usually two (but occasionally three or four) action potentials, with a short (<20 ms) interspike interval within a regular low-frequency firing pattern. Here we used a paradigm of electrical stimulation comprising twin pulses (with 7- or 10-ms inter-pulse intervals) to mimic this burst firing pattern, and compared the effects of single- and twin-pulse electrical stimulations in models of pre- and postsynaptic 5-hydroxytryptamine function. Firstly, we measured the effect of direct electrical stimulation (2 Hz for 2 min) of rat brain slices on efflux of preloaded [3H]5-hydroxytryptamine. In this in vitro model, twin-pulse stimulation increased the efflux of tritium by about twice as much as did single-pulse stimulation. This effect was evident in the medial prefrontal cortex (area under the curve: 2. 59+/-0.34 vs 1.28+/-0.22% relative fractional release), as well as in the caudate-putamen (3.93+/-0.65 vs 2.17+/-0.51%) and midbrain raphe nuclei (5.42+/-1.05 vs 2.51+/-0.75%). Secondly, we used in vivo microdialysis to monitor changes in endogenous extracellular 5-hydroxytryptamine in rat medial prefrontal cortex in response to electrical stimulation (3 Hz for 10 min) of the dorsal raphe nucleus. In this model, twin-pulse stimulation of the dorsal raphe nucleus increased 5-hydroxytryptamine by approximately twice as much as did single-pulse stimulation at the same frequency (area under the curve: 50.4+/-9.0 vs 24.2+/-4.4 fmol). Finally, we used in vivo extracellular recording to follow the response of postsynaptic neurons in the rat medial prefrontal cortex to 5-hydroxytryptamine released by dorsal raphe stimulation. Electrical stimulation of the dorsal raphe nucleus (1 Hz) induced a clear-cut poststimulus inhibition in the majority of cortical neurons tested. In these experiments, the duration of poststimulus inhibition following twin-pulse stimulation was markedly longer than that induced by single-pulse stimulation (200+/-21 vs 77+/-18.5 ms). Taken together, the present in vitro and in vivo data suggest that in 5-hydroxytryptamine neurons, short bursts of action potentials will propagate along the axon to the nerve terminal and will enhance both the release of 5-hydroxytryptamine and its postsynaptic effect.

Negative allosteric modulators of AMPA-preferring receptors inhibit [(3)H]GABA release in rat striatum.

The effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a selective glutamate receptor agonist, on the release of previously incorporated [(3)H]GABA was examined in superfused striatal slices of the rat. The slices were loaded with [(3)H]GABA in the presence of beta-alanine (1 mM) and superfused with Krebs-bicarbonate buffer containing nipecotic acid (0.1 mM) and aminooxyacetic acid (0.1 mM) to inhibit GABA uptake and metabolism. AMPA (0.01 to 3 mM) increased basal [(3)H]GABA outflow and nipecotic acid potentiated this effect. The [(3)H]GABA releasing effect of AMPA was an external Ca(2+)-dependent process in the absence but not in the presence of nipecotic acid. Cyclothiazide (0.03 mM), a positive modulator of AMPA receptors, failed to evoke [(3)H]GABA release by itself, but it dose-dependently potentiated the [(3)H]GABA releasing effect of AMPA. The AMPA (0.3 mM)-induced [(3)H]GABA release was antagonized by NBQX (0.01 mM) in a competitive fashion (pA(2) 5.08). The negative modulator of AMPA receptors, GYKI-53784 (0.01 mM) reversed the AMPA-induced [(3)H]GABA release by a non-competitive manner (pD'(2) 5.44). GYKI-53784 (0. 01-0.1 mM) also decreased striatal [(3)H]GABA outflow on its own right, this effect was stereoselective and was not influenced by concomitant administration of 0.03 mM cyclothiazide. GYKI-52466 (0. 03-0.3 mM), another negative modulator at AMPA receptors, also inhibited basal [(3)H]GABA efflux whereas NBQX (0.1 mM) by itself was ineffective in alteration of [(3)H]GABA outflow. The present data indicate that AMPA evokes GABA release from the vesicular pool in neostriatal GABAergic neurons. They also confirm that multiple interactions may exist between the agonist binding sites and the positive and negative modulatory sites but no such interaction was detected between the positive and negative allosteric modulators. Since GYKI-53784, but not NBQX, inhibited [(3)H]GABA release by itself, AMPA receptors located on striatal GABAergic neurons may be in sensitized state and phasically controlled by endogenous glutamate. It is also postulated that these AMPA receptors are located extrasynaptically on GABAergic striatal neurons.

Postsynaptic integration of cholinergic and dopaminergic signals on medium-sized GABAergic projection neurons in the neostriatum.

The effects of cholinergic drugs and the interaction between cholinergic and dopaminergic compounds were studied on electrically evoked [3H]gamma-aminobutyric acid (GABA) overflow in slices of the rat neostriatum. Slices were prepared and loaded with [3H]GABA in the presence of beta-alanine and then superfused with Krebs-bicarbonate buffer containing aminooxyacetic acid and nipecotic acid to inhibit GABA uptake and metabolism, respectively. The nonselective muscarinic agonist oxotremorine (0.1-10 microM) increased the release of [3H]GABA and the selective M1 receptor agonist McN-A-343 (0.1-10 microM) exerted similar effect. The stimulatory effect of oxotremorine (10 microM) on [3H][GABA overflow was antagonized by the nonselective muscarinic antagonist atropine (1 microM) and the selective M1 receptor antagonist pirenzepine (0.1-1.0 microM). The M2 receptor antagonist methoctramine (1.0 microM) did not alter the stimulatory effect of oxotremorine. Of the muscarinic receptor antagonists atropine, pirenzepine, and methoctramine (1.0 microM) failed to affect [3H]GABA overflow. The M3 receptor antagonist p-F-HHSiD (1 microM) increased [3H]GABA overflow and p-F-HHSiD and oxotremorine were found to be additive in increasing this effect. The D2 dopamine receptor antagonist sulpiride (10 microM) increased the electrical stimulation-induced [3H]GABA overflow, and this stimulation was counteracted by concomitant administration of atropine (1 microM). McN-A-343 and sulpiride also increased the KCl-induced [3H]GABA overflow from superfused neostriatal slices and tetrodotoxin (1 microM) did not affect these stimulations. These data indicate that the release of GABA in the neostriatum is under the control of M1 stimulatory and M3 inhibitory muscarinic receptors. Dopamine, which exerts inhibition on GABA release via D2 receptors, may counteract the M1 facilitation, and M1 and D2 receptors involved in the cholinergic-dopaminergic interaction may be located postsynaptically on medium-sized spiny GABAergic projection neurons.

Feedback stimulation of somatodendritic serotonin release: a 5-HT3 receptor-mediated effect in the raphe nuclei of the rat.

Slices from rat midbrain containing the raphe nuclei and from hippocampus were prepared, loaded with [3H]5-HT and superfused and the resting and the electrically stimulated [3H]5-HT release was measured. The 5-HT3 receptor agonist 2-methyl-5-HT (1 to 10 micromol/l) increased the resting tritium outflow in superfused raphe nuclei slices, EC50 5.3 micromol/l. The 2-methyl-5-HT-induced increase of tritium outflow was an external Ca2+-independent process and was not altered by reserpine pretreatment but it was reversed by addition of the 5-HT uptake inhibitor fluoxetine (1 micromol/l). The 5-HT3 receptor antagonists ondansetron and GYKI-46 903 (1 micromol/l) did not antagonize the stimulatory effect of 2-methyl-5-HT on resting tritium outflow. 2-Methyl-5-HT in lower concentration increased the electrically induced tritium overflow from raphe nuclei slices (EC50 0.56 micromol/l) and also from hippocampal slices preloaded with [3H]5-HT. These effects were reversed by 1 micromol/l of ondansetron and GYKI-46903. The 5-HT3 receptor antagonists (1 micromol/l) were without effects on depolarization-evoked [3H]5-HT release at 2 Hz stimulation, when 10 Hz stimulation was used, ondansetron and GYKI-46 903 reduced the tritium overflow from raphe nuclei slices. These data indicate that 5-HT3 receptors positively alter depolarization-induced somatodendritic 5-HT release in the raphe nuclei. They also show that 2-methyl-5-HT is able to evoke 5-HT release not only from vesicles but also from cytoplasmic stores via a transporter-dependent exchange process.

Influence of dopamine on GABA release in striatum: evidence for D1-D2 interactions and non-synaptic influences.

Striatal slices from the rat were preincubated with [3H]GABA and superfused in the presence of nipecotic acid and aminooxyacetic acid, inhibitors of high-affinity GABA transport and GABA aminotransferase, respectively. GABA efflux was estimated by monitoring tritium efflux, 98% of which was in the form of [3H]GABA. The following three major observations were made: (1) The overflow of GABA evoked by electrical field stimulation (8 Hz) was increased two-fold by SKF-38393 (10 microM), an agonist at the D1 family of dopamine receptors. This increase was completely blocked by the D1 receptor antagonist SCH-23390 (10 microM). However, SCH-23390 had no effect on GABA overflow when given alone. Thus, dopamine agonists appear to exert an excitatory influence on GABA release; however, this effect was not elicited by endogenous dopamine under the conditions of this experiment. (2) Electrically evoked GABA overflow was reduced 50% by quinpirole (10 microM), an agonist at the D2 family of dopamine receptors, and this effect was blocked by the D2 antagonist sulpiride (10 microM). Moreover, exposure to sulpiride alone caused a 60% increase in GABA overflow, and this effect was abolished by 3-iodotyrosine (2 mM), a dopamine synthesis inhibitor. Thus, D2 agonists appear to exert an inhibitory influence on dopamine release, an effect that can be exerted by endogenous stores of dopamine. (3) The stimulatory effect of SKF-38393 was attenuated by quinpirole, whereas the sulpiride-induced increase in GABA efflux was attenuated by SCH-23390. Sulpiride also increased [3H]GABA efflux during KCl-induced depolarization, an effect that was antagonized by SCH-23390 as in the case of electrical stimulation. However, although tetrodotoxin did not alter the stimulatory effect of sulpiride, it did block the ability of SCH-23390 to antagonize the sulpiride-induced increase in GABA overflow. These latter results suggest that there is an interaction between D1 and D2 receptors whereby the effects of dopamine mediated via D1 sites are inhibited by an action on D2 sites. In conclusion, our results suggest that (i) dopamine agonists can exert an excitatory influence on depolarization-induced GABA release within neostriatum via D1 receptors and an inhibitory influence via D2 receptors; (ii) under the conditions of these experiments, endogenous dopamine fails to act on D1 sites but does exert an inhibitory influence via D2 sites; and (iii) there is an interaction between D1 and D2 receptors such that the actions of dopamine mediated via D1 sites are inhibited as a result of the concomitant actions exerted via D2 sites.

Dopaminergic inhibition of striatal GABA release after 6-hydroxydopamine.

We have examined the regulation of striatal GABA release by endogenous dopamine in rats with partial degeneration of dopamine-containing neurons. 6-Hydroxydopamine was administered into the lateral ventricles or medial forebrain bundle. Either 3 days or 3 weeks later, slices of neostriatum were prepared, preloaded with [3H]GABA, and superfused in order to measure [3H]GABA overflow in response to electrical stimulation (8 Hz). The loss of dopaminergic terminals was estimated by measuring tissue levels of dopamine. The impact of endogenous dopamine on [3H]GABA was evaluated by measuring the ability of sulpiride, a D2 dopamine receptor antagonist, to increase the depolarization-induced [3H]GABA overflow. In non-treated or vehicle-pretreated rat neostriatum, sulpiride (10 microM) increased the depolarization-induced [3H]GABA overflow to 193% of control. Three days after lesioning, the stimulatory effect of sulpiride on [3H]GABA overflow was identical to that seen in control rats so long as the loss of tissue dopamine did not exceed 60%, although with larger lesions the sulpiride-induced response was reduced. Three weeks after lesioning, however, the stimulatory effect of sulpiride on electrically evoked [3H]GABA overflow remained at the level seen in control tissue even in cases where tissue dopamine was reduced to 13% of normal. In contrast, no sulpiride-induced increase in [3H]GABA overflow was detected 3 weeks after nearly complete lesions with reduced tissue dopamine to 20% of normal. These data suggest that short- and long-term compensatory changes maintain dopaminergic control over GABAergic projection neurons and interneurons until the loss of dopamine innervation is almost complete.

GYKI-46 903, a non-competitive antagonist for 5-HT3 receptors.

The effects of GYKI-46 903 ((+)endo-4-propionyloxy-6-(4-fluorophenyl)-1-azabicyclo [3.3.1]non-6-ene HCl), on 5-HT3 receptors have been studied and compared with ondansetron in peripheral organs in vitro and in vivo, and in a receptor binding assay in membranes prepared from rat cerebral cortex. GYKI-46 903 was found to be a non-competitive antagonist at 5-HT3 receptors present in non-stimulated longitudinal muscle strip of guinea-pig ileum (pD2' against serotonin = 5.54), and also in 5-methoxytryptamine-pretreated electrically stimulated ileal preparations (pD2' against serotonin = 5.26). On the contrary, ondansetron was found to be a competitive antagonist for 5-HT3 receptors; the pA2 value against serotonin was 7.40 in non-stimulated ileum, and it was 7.08 in electrically stimulated ileal preparation pretreated with 5-methoxytryptamine. In displacement studies, the pIC50 values of GYKI-46 903 and ondansetron against [3H]granisetron binding to rat cerebral cortex membranes were 6.91 and 8.58 respectively. GYKI-46 903, when administered by intravenous infusion, antagonized the decrease in heart rate evoked by serotonin (Bezold-Jarisch reflex) in anaesthetized rats, and the maximal reversal was less than 50%. This was in striking contrast with ondansetron, which, after intravenous injection, completely antagonized the serotonin-induced bradycardia with an ID50 value of 3.28 ug/kg. These data classify GYKI-46 903 as a non-competitive antagonist for 5-HT3 receptors.

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release.

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [3H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium efflux from raphe nuclei slices preloaded with [3H]serotonin, and this inhibition was reversed by the 5-HT1A receptor antagonist (+)WAY-100135. The 5-HT1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [3H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubated with [3H]serotonin was completely external Ca(2+)-dependent, and omega-conotoxin GVIA and Cd2+, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced tritium release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT1A and 5-HT1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca2+ channels; (3) the 5-HT1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K+ channels.

Evidence that ibogaine releases dopamine from the cytoplasmic pool in isolated mouse striatum.

We measured the effect of ibogaine on the tritium efflux from isolated mouse striatum preloaded with [3H]dopamine ([3H]DA). Ibogaine increased the basal tritium outflow in a concentration-dependent manner, but it was without effect on electrical stimulation-induced tritium overflow. Separation of the released radioactivity after ibogaine administration showed that this drug increased the release of [3H]DA and [3H]-dihydroxyphenylacetic acid ([3H]DOPAC), but the efflux of O-methylated-deaminated metabolites was not changed. The dopamine (DA)-releasing effect of ibogaine was reduced by the DA uptake inhibitors cocaine and nomifensine. The tritium efflux evoked by ibogaine was not altered by omission of Ca2+ from the perfusion buffer or by inhibition of the voltage-sensitive Na+ channels with tetrodotoxin. Ibogaine maintained its effect on release from superfused striatum prepared from reserpine-pretreated mice. The ibogaine-induced tritium release measured from mouse striatum that was preloaded with [3H]DA was not affected by the D-2 DA receptor ligands (-)-quinpirole and (+/-)-sulpiride, indicating that the ibogaine-induced release is not subject to presynaptic autoreceptor regulation. Ibogaine failed to affect [3H]DA uptake and retention in mouse striatum. These data indicate that at the nerve terminal level ibogaine releases DA, and the primary source for the release is probably the cytoplasmic pool. The DA-releasing effect of ibogaine may have importance in mediation of its hallucinogenic action, as seen in a frequent practice in African cults.

Effect of acetyl-L-carnitine on extracellular amino acid levels in vivo in rat brain regions.

Acetyl-L-carnitine (ALCAR) was found to have beneficial effects in senile patients. In recent years many of its effects on the nervous system have been examined, but its mechanism(s) of action remains to be elucidated. We previously reported that it causes release of dopamine in the striatum. In the present paper we report that ALCAR, when administered at intracerebral sites via microdialysis, stimulates the release of amino acids in a concentration-dependent and regionally heterogeneous manner. The effect was strong in the striatum and cerebellum, less so in the frontal cortex, and weak in the thalamus. Seven amino acids were measured: the increase in the level of aspartate, glutamate, and taurine was substantial, and the increase in the level of glycine, serine, threonine, alanine, and glutamine in the microdialysate was minor. The stimulatory effect of ALCAR on the release of amino acids in the striatum was inhibited by the muscarinic antagonist atropine, but was not inhibited by the nicotinic antagonist mecamylamine. The effect of ALCAR on the levels of most of the amino acids tested was independent of the presence of Ca2+ in the perfusate. These results indicate that ALCAR, when administered intracerebrally at fairly high concentrations, can affect the level and the release not only of such neurotransmitters as acetylcholine and dopamine, but also of amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl-L-carnitine releases dopamine in rat corpus striatum: an in vivo microdialysis study.

The effect of acetyl-L-carnitine, a compound reported to be beneficial for senile patients, on the release of dopamine (DA) from the striatum was studied by using in vivo brain dialysis in anesthetized rats coupled with HPLC-electrochemical detection. Striatal infusion of acetyl-L-carnitine increased the efflux of DA with no apparent changes in efflux of DA metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 4-hydroxy-3-methoxyphenylacetic acid (HVA). The DA-releasing effect of acetyl-L-carnitine was concentration- and Ca(2+)-dependent, and was abolished by omega-conotoxin fraction GVIA and tetrodotoxin, inhibitors of the voltage-dependent Ca2+ and Na+ channels, respectively. Nomifensine, an inhibitor of DA reuptake did not alter the DA-releasing property of acetyl-L-carnitine. DA released from the striatum by acetyl-L-carnitine was decreased by reserpine pretreatment whereas the d-amphetamine-evoked DA outflow was not affected. In contrast to acetyl-L-carnitine, d-amphetamine reduced the extracellular concentrations of DOPAC and HVA. We conclude from the present data that acetyl-L-carnitine evokes DA release from the vesicular pools of the nigrostriatal dopaminergic neurons by a Ca(2+)-dependent, exocytotic process.