In vivo evidence that 5-hydroxytryptamine (5-HT) neuronal firing and release are not necessarily correlated with 5-HT metabolism.

The relationship between 5-hydroxytryptamine release, metabolism and unit activity has been investigated in the anaesthetized rat. 5-Hydroxytryptamine release and metabolism were monitored in vivo by the measurement of extracellular 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the frontal cortex using in vivo voltammetry combined with nafion-coated and uncoated electrically pretreated carbon fibre electrodes. The monoamine oxidase inhibitor pargyline (100 mg/kg) increased extracellular 5-hydroxytryptamine and decreased 5-hydroxyindoleacetic acid. The 5-hydroxytryptamine releaser fenfluramine (10 mg/kg i.p.) acutely increased extracellular 5-hydroxytryptamine while having no effect on 5-hydroxyindoleacetic acid and the effect on extracellular 5-hydroxytryptamine was markedly reduced in rats pretreated (four weeks) with 5,7-dihydroxytryptamine. 8-Hydroxy-2-(di-n-propyl-amino) tetralin (10 micrograms/kg i.v.), an agonist at the 5-hydroxytryptamine1A somatodendritic autoreceptor, inhibited 5-hydroxytryptamine neuronal firing in the dorsal raphe nucleus and decreased extracellular 5-hydroxytryptamine during the period when firing was inhibited but did not alter extracellular 5-hydroxyindoleacetic acid. In contrast 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridin-4-yl) (RU 24969), which is an agonist at the terminal autoreceptor in the rat, had no effect on 5-hydroxytryptamine neuronal firing but decreased 5-hydroxytryptamine and 5-hydroxyindoleacetic acid. The results support the view that extracellular 5-hydroxyindoleacetic acid is not a good index of 5-hydroxytryptamine release and that under specific circumstances 5-hydroxytryptamine neuronal firing, release and metabolism are independent of one another.

Effects of a selective 5-HT2 agonist, DOI, on 5-HT neuronal firing in the dorsal raphe nucleus and 5-HT release and metabolism in the frontal cortex.

Systemic administration of the 5-HT2 agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (50 and 100 micrograms kg-1, i.v.) inhibited dorsal raphe neuronal firing. DOI (100 micrograms kg-1, i.v.) also produced a decrease in extracellular 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the frontal cortex measured by microdialysis. However, local administration of DOI into the frontal cortex produced no change in extracellular 5-HT and 5-HIAA at any dose given (1, 10 and 100ng). The results demonstrate that DOI is a potent inhibitor of 5-HT neuronal firing and terminal release and that the effects on release are not mediated by an action within the terminal region. The site of action and the receptor involved in the inhibition remains to be determined.

Effects of repeated treatment with 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on the autoregulatory control of dorsal raphe 5-HT neuronal firing and cortical 5-HT release.

These experiments were designed to examine the effects of repeated 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) treatment on the autoregulatory control of cortical 5-HT release and dorsal raphe nucleus (DRN) 5-HT neuronal cell firing. Repeated DOI treatment decreased the behavioural responsiveness (wet-dog shakes) of 5-HT2 receptors and attenuated the inhibitory effects of the 5-HT1A receptor agonist, 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), on both cortical 5-HT release and DRN 5-HT neuronal firing. In contrast, the inhibitory effect of acute DOI on cortical 5-HT release and DRN 5-HT neuronal firing was unaffected by repeated DOI treatment. The results demonstrate that changes in the responsiveness of 5-HT2 receptor function may influence the responsiveness of presynaptic 5-HT1A receptors regulating 5-HT neuronal function. The results also provide further evidence that the inhibition of cortical 5-HT release and DRN 5-HT neuronal firing produced by DOI is not mediated by 5-HT2 receptor activation.

Can benzodiazepines be classified by characterising their anticonvulsant tolerance-inducing potential?

The development of anticonvulsant tolerance with three benzodiazepines was assessed in mice using a slow intravenous infusion of pentylenetetrazol as the convulsive stimulus. Chlordiazepoxide (12.5 mg/kg b.d.) and midazolam (0.75 mg/kg b.d.) induced a slowly evolving tolerance over 15 days whereas nitrazepam (0.6 mg/kg b.d.) induced a very marked rapid tolerance which developed no further during 6 days treatment. Tolerance appeared to be incomplete with all three benzodiazepines. Possible explanations for the differences in tolerance profile are discussed and an alternative basis for the classification of benzodiazepines is suggested.

Blockade of two voltage-dependent potassium channels, mKv1.1 and mKv1.2, by docosahexaenoic acid.

The effects of the polyunsaturated fatty acid, docosahexaenoic acid, were examined on two single cloned potassium channels, mKv1.1 and mKv1.2, stably expressed in Chinese hamster ovary cells using whole-cell patch clamp techniques. Docosahexaenoic acid produced a time- and dose-dependent, reversible block of mKv1.1 and mKv1.2. Interestingly, docosahexaenoic acid increased the rate of activation of mKv1.2 leading to an enhancement of current amplitude at short intervals following activating the voltage step. This phenomenon was not seen in the case of mKv1.1. Intracellular administration of docosahexaenoic acid did not block either type of channel. These findings suggest that docosahexaenoic acid inhibits mKv1.1 and mKv1.2 channels by acting at an extracellular site and by an open-channel blocking mechanism.

Effects of idazoxan on dorsal raphe 5-hydroxytryptamine neuronal function.

The effects of the alpha 2-adrenoceptor antagonist idazoxan on 5-hydroxytryptamine (5-HT) neuronal firing and release have been investigated. Idazoxan, administered i.v. (10 micrograms/kg and 0.5 mg/kg) increased dorsal raphe nucleus (DRN)-5-HT neuronal firing rate in a dose-dependent fashion. At the higher dose, a voltammetric study revealed increases in extracellular 5-HT and 5-hydroxyindole acetic acid (5-HIAA) levels, there was no effect with the lower dose. Intra-raphe administration of idazoxan (1 ng) also elevated the firing rate of 5-HT neurones in the dorsal raphe, suggesting that idazoxan may produce the increase in firing by a direct effect in the DRN. However, microiontophoretic application of idazoxan did not increase the firing rate of 5-HT neurones in the DRN. Thus the increase in the firing rate of 5-HT neurones in the DRN observed with systemic and local administration of idazoxan is probably not due to a direct action of idazoxan on the 5-HT neurone. Possibly the idazoxan acted at alpha 2-adrenoceptors located on noradrenergic terminals thus stimulating noradrenaline release and consequently increased 5-HT activity. Chronic administration of idazoxan (0.8 mg/kg per h for 14 days), using osmotic mini-pumps, caused an elevation in basal firing rate and an attenuation of the inhibitory response of DRN 5-HT neurones to the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT) (10 micrograms/kg i.v.). This finding suggests that chronic infusion with idazoxan leads to desensitisation of the 5-HT1A somatodendritic autoreceptor.

Inhibition of 5-hydroxytryptamine neuronal activity by the 5-HT agonist, DOI.

Systemic, intra-raphe and microiontophoretic administration of the 5-hydroxytryptamine (5-HT)1C/5-HT2 agonist (1-(2,5-dimethoxy-4-iodophenyl)-2- aminopropane (DOI) inhibited the firing of 5-HT neurones in the dorsal raphe. DOI administered systemically and directly into the raphe also decreased the extracellular concentration of 5-hydroxytryptamine (5-HT) in the frontal cortex. In contrast, the administration of DOI directly into the frontal cortex did not significantly alter the concentration of frontal cortical extracellular 5-HT. The reduction of the firing rate of 5-HT neurons in the dorsal raphe and extracellular 5-HT concentration in the frontal cortex induced by systemic administration of DOI could not be blocked by the 5-HT2 antagonist ketanserin, ritanserin (5-HT2/5-HT1C antagonist) or the putative 5-HT1A antagonist, pindolol. These results suggest that the inhibition of 5-HT neuronal firing seen with administration of DOI is mediated via an action within the dorsal raphe and at least in close proximity to the 5-HT neurone cell bodies. The decrease in frontal cortical extracellular concentration of 5-HT release was not due to a direct action in the frontal cortex itself and may possibly be as a result of the decrease in the firing rate of the 5-HT neurones in the dorsal raphe. The mechanism of action of DOI to produce these effects is, however, unclear and warrants further investigation.

LSD has high efficacy relative to serotonin in enhancing the cationic current Ih: intracellular studies in rat facial motoneurons.

The effects of LSD (d-lysergic acid diethylamide) on rat facial motoneurons were compared to those of 5-hydroxytryptamine (5-HT) in brain slices by means of current clamp and single-electrode voltage-clamp recordings. As previously reported, 5-HT, in part by decreasing a resting potassium conductance, produced a reversible depolarization (approximately 5 mV), an increase in input resistance, and an enhancement in electrical excitability. LSD also produced an increase in electrical excitability, although with a much slower onset and longer duration. However, in contrast to 5-HT, LSD produced only a slight depolarization (1-2 mV). Moreover, in the presence of LSD the depolarizing effect of 5-HT was markedly attenuated. The 5-HT2/5-HT1C agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) produced effects intermediate between LSD and 5-HT. The LSD-induced increase in electrical excitability was completely reversed by spiperone, a 5-HT2/5-HT1A antagonist, and by ritanserin, a 5-HT2/5-HT1C antagonist; the effects of 5-HT were also reduced by these 2 antagonists, but complete blockade did not occur at the concentrations and durations tested. Surprisingly, LSD was found to enhance the hyperpolarization-activated nonspecific cation current Ih to a greater extent than did 5-HT; this enhancement was blocked by both spiperone and ritanserin. These results indicate that, despite having low efficacy relative to 5-HT in decreasing resting potassium conductance, LSD has high efficacy in enhancing the Ih current in rat facial motoneurons; possible mechanisms for this difference are discussed.

On the mechanism of 4-aminopyridine action on the cloned mouse brain potassium channel mKv1.1.

1. This study used the whole-cell patch clamp technique to investigate the mechanism of action of the K+ channel blocker 4-aminopyridine (4-AP) on the cloned K+ channel mouse Kv1.1 (mKv1.1) expressed in Chinese hamster ovary cells. 2. Cells transfected with mKv1.1 expressed a non-inactivating, delayed rectifier-type K+ current. 4-AP induced a dose-, voltage- and use-dependent block of mKv1.1. 3. 4-AP blockade of mKv1.1 was similar whether 4-AP was administered extracellularly (IC50 = 147 microM) or intracellularly (IC50 = 117 microM). 4. Inclusion of the first twenty amino acids of the N-terminus sequence of the Shaker B K+ channel ('inactivation peptide') in the patch electrode transformed mKv1.1 into a rapidly inactivating current. The time constant of decay for the modified current was dependent on the concentration of inactivation peptide, and under these conditions extracellular 4-AP had a reduced potency (IC50 values of 471 and 537 microM for 0.5 and 2 mg ml-1 inactivation peptide, respectively). 5. A permanently charged analogue of 4-AP, 4-aminopyridine methiodide (4-APMI), was found to block mKv1.1 when applied inside the cell, but was without effect when administered externally. 6. Decreasing the intracellular pH (pHi) to 6.4 caused an increase in 4-AP potency (IC50 = 76 microM), whereas at pHi 9.0, the 4-AP potency fell (IC50 = 295 microM). Conversely, increasing extracellular pH (pHo) to 9.0 caused an increase in 4-AP potency (IC50 = 93 microM), whereas at pHo 6.4, 4-AP potency decreased (IC50 = 398 microM). 7. Taken together, these findings support the hypotheses that the uncharged form of 4-AP crosses the membrane, and that it is predominantly the cationic form which acts on mKv1.1 channels intracellularly, possibly at or near to the binding site for the inactivation peptide.