Trevor Dennis

Desensitization of the neuronal 5-HT carrier following its long-term blockade

In vivo extracellular unitary recordings, in vitro 3H-5-hydroxy- tryptamine (5-HT) uptake, and 3H-paroxetine binding assays were used to assess the effect of acute and long-term administration of the 5-HT reuptake inhibitor paroxetine on the neuronal 5-HT transporter in the rat dorsal hippocampus. Recovery time of the firing activity of CA3 hippocampus pyramidal neurons following microiontophoretic application of 5-HT was used as an index of in vivo reuptake activity. In a first series of experiments, the acute intravenous administration of paroxetine and 5-HT denervation with the neurotoxin 5,7- dihydroxytryptamine produced a marked prolongation of the suppressant effect of 5-HT, indicating that reuptake into 5-HT terminals plays a significant role in terminating the action of microiontophoretically applied 5-HT. In a second series of experiments, rats were treated with paroxetine (10 mg/kg/d, s.c.) for 2 or 21 d. In both treatment groups, there was a marked prolongation of the effect of microiontophoretically applied 5-HT; however, in rats treated for 2 d, the prolongation was significantly greater than that observed in rats treated for 21 d. After the 21 d treatment with paroxetine and a 48 hr washout, the prolongation of the effect of microiontophoretically applied 5-HT by acute intravenous paroxetine was significantly reduced, suggesting a decrease in the number of 5-HT carriers. In keeping with this interpretation, following the same treatment regimen, there was a 50% and 60% reduction of the in vitro 3H-5-HT uptake in hippocampal and dorsal raphe slices, respectively, and a reduced effectiveness of paroxetine in blocking 3H-5-HT uptake in both regions. The determination of the binding parameters of 3H-paroxetine revealed that, in rats treated for 21 d with paroxetine (10 mg/kg/d, s.c.), following a 48 hr washout Kd values were unchanged but Bmax values were reduced by 70% and 60% in hippocampal and cortical membranes, respectively.

Characterization of 5-hydroxytryptamine1A properties of flesinoxan: In Vivo electrophysiology and hypothermia study

Flesinoxan is a high affinity and selective 5-hydroxytryptamine1A (5-HT1A) ligand which, unlike the 5-HT1A agonists of the azapirone class, does not generate 1-(2-pyrimidinyl)piperazine, an alpha 2-adrenoreceptor antagonist. In view of potential antidepressant effects of flesinoxan, this study was undertaken to characterize its 5-HT1A properties in the rat brain using in vivo electrophysiology and hypothermia paradigms. The suppressant effect of microiontophoretic applications of flesinoxan on the firing activity of CA3 pyramidal neurons was blocked by concomitant application of the 5-HT1A antagonist BMY 7378. Compared to gepirone, the efficacy of flesinoxan to suppress the firing activity of CA3 pyramidal neurons was significantly greater. While the coapplication of flesinoxan antagonized the suppressant effect of 5-HT on CA3 pyramidal neurons, it failed to do so on dorsal raphe 5-HT neurons, indicating that flesinoxan acts as a partial agonist at postsynaptic and as a full agonist at presynaptic 5-HT1A receptors. The capacity of flesinoxan to antagonize the effect of 5-HT on CA3 pyramidal neurons was similar to that of 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and significantly greater than that of gepirone. The intravenous administration of flesinoxan suppressed the firing activity of both CA3 pyramidal neurons and dorsal raphe 5-HT neurons. However, when compared to 8-OH-DPAT, significantly higher doses of flesinoxan were required. The acute brain penetration of [3H]flesinoxan and [3H]8-OH-DPAT was, therefore, determined. Nine minutes after intravenous administration, [3H]8-OH-DPAT reached significantly greater brain concentration than [3H]flesinoxan. Subcutaneous administration of flesinoxan and 8-OH-DPAT produced a dose-dependent hypothermia. The flesinoxan-induced hypothermia was significantly attenuated by prior administration of the non-selective 5-HT1A antagonist pindolol and the 5-HT1/2 antagonist methysergide. Similar degrees of hypothermia were achieved with 3 mg/kg of flesinoxan and 0.5 mg/kg of 8-OH-DPAT. The maximal effect of flesinoxan occurred 30 min later than that of 8-OH-DPAT and faded more slowly. The 5-HT1A properties of flesinoxan suggest that it may be an effective anxiolytic/antidepressant agent.

Calcitonin gene-related peptide (hCGRPα) binding sites in the nucleus accumbens. Atypical structural requirements and marked phylogenic differences

The distribution of [125I]hCGRP alpha binding sites was studied in tissue sections from rat brain and, at the level of the nucleus accumbens in the brains of 6 other species. In the rat, very high levels of binding were found in the nucleus accumbens, the amygdaloid complex and mammillary body while high amounts were localized to the superficial layers of the superior colliculus, temporal cortex, cerebellum (molecular layer), frontal cortex and inferior olive. Moderate densities of [125I]hCGRP alpha binding were observed in the medial geniculate nucleus, inferior colliculus and substantia nigra. Regional competition studies in rat brain showed that salmon calcitonin was almost as effective as hCGRP alpha in competing for [125I]hCGRP alpha binding sites in the nucleus accumbens but was mostly inactive in other regions such as the mesolimbic cortex and the striatum. On the basis of their atypical sensitivity to salmon calcitonin, [125I]hCGRP alpha binding sites in the rat nucleus accumbens, which appear between postnatal days 4 and 7, do not seem to correspond to either the CGRP1 or CGRP2 receptor subtypes. Marked species differences were observed in the distribution of [125I]hCGRP alpha binding sites, especially in the nucleus accumbens. In the mouse, low densities of hCGRP alpha sites were observed in striatum and fronto-parietal cortex while low to moderate levels were found in the medial and posterior aspects of the nucleus accumbens. A similar distribution was seen in the guinea pig brain albeit of generally higher density. In the rat, very high amounts of [125I]hCGRP alpha binding were seen in the nucleus accumbens while lower levels were found in the striatum and certain cortical areas.(ABSTRACT TRUNCATED AT 250 WORDS)

Antidepressant-induced modulation of GABAA receptors and β-adrenoceptors but not GABAB receptors in the frontal cortex of olfactory bulbectomised rats

The effects of prolonged administration of antidepressant drugs, belonging to three different classes, on high-affinity GABAA receptor, GABAB receptor and beta-adrenoceptor binding parameters were determined in the frontal cortex of olfactory bulbectomised rats. Clorgyline (1 mg/kg/day), paroxetine (10 mg/kg/day) or desipramine (10 mg/kg/day) were administered for 21 days via subcutaneous osmotic minipumps implanted in the scapular region 7 days after bulbectomy. Cortical GABAA receptor densities, defined with [3H]gamma-aminobutyric acid ([3H]GABA), were significantly increased following bulbectomy. This effect on Bmax values was reversed by all three antidepressant drugs. GABAB receptor densities decreased slightly after bulbectomy. Chronic antidepressant administration had no effect on GABAB receptor binding parameters. Olfactory bulbectomy did not induce any changes in cortical beta-adrenoceptor binding parameters determined with [3H]CGP-12177 ((-)-4-(3-t- butylamino-2-hydroxypropxy)- [5,7-3H]benzimidazol-2-one). However, prolonged administration of all three antidepressant drugs induced a downregulation of beta-adrenoceptors. The results of the present study confirm the involvement of cortical GABAA rather than GABAB receptors in the olfactory bulbectomy animal model of human depression. Moreover, the data further support the hypothesis that a decrease in function of the GABAA receptor complex could play a role in the therapeutic effects of antidepressant treatments.

Effect of acute and prolonged tianeptine administration on the 5-HT transporter: electrophysiological, biochemical and radioligand binding studies in the rat brain

In the present study, in vivo extracellular unitary recordings, in vitro [3H]5-HT uptake and [3H]cyanoimipramine binding assays were used to assess the effect of acute and prolonged administration of the putative antidepressant tianeptine, on the 5-hydroxytryptamine (5-HT) transporter. Microiontophoretic application of tianeptine onto dorsal hippocampus CA3 pyramidal neurons, as well as its intravenous administration (2 mg/kg), increased their firing frequency. Following intracerebroventricular administration of 5,7-dihydroxytryptamine, the activation induced by the microiontophoretic application of tianeptine remained unchanged, thus suggesting that the 5-HT carrier is not involved in this effect. Furthermore, in spite of its activating effect on CA3 pyramidal neuron firing frequency, the intravenous administration of tianeptine did not alter the time of recovery of these neurons from microiontophoretic applications of 5-HT, an index of 5-HT uptake activity. In keeping with this observation, the acute administration of tianeptine did not change the effectiveness of the 5-HT reuptake blocker paroxetine (1 mg/kg, i.v.) in prolonging the suppressant effect of microiontophoretically-applied 5-HT. However, in rats that had received tianeptine for 14 days (20 mg/kg/day, s.c.), the recovery time from the suppressant effect of microiontophoretic applications of 5-HT was reduced by 40% and the effectiveness of paroxetine (1 mg/kg, i.v.) was decreased. These effects were no longer observed following a 48 h washout period. In a second series of experiments, the ability of tianeptine to interfere with the uptake blocking capacity of paroxetine was assessed in vitro, using hippocampal slices obtained from rats that had been treated with tianeptine for 14 days (20 mg/kg/day, s.c.; by minipump). The effectiveness of paroxetine to block [3H]5-HT uptake was unchanged in slices obtained from rats still bearing the osmotic minipump at the time of the sacrifice, as well as from those which had undergone a 48 h washout period. To assess whether prolonged administration of tianeptine would induce adaptive changes on 5-HT uptake sites, [3H]cyanoimipramine-binding parameters were measured following a 48 h washout period. Affinity values remained unchanged while density values were significantly increased in cortex (+22%) but not in hippocampus (+12%). It is concluded that, i) the activation of CA3 pyramidal neurons observed following acute tianeptine administration cannot be attributed to its 5-HT uptake enhancing properties and ii) the prolonged administration of tianeptine induces adaptive changes on cortical but not on hippocampal 5-HT transporters.

Differential effects of olfactory bulbectomy on GABAA and GABAB receptors in the rat brain.

GABAergic mechanisms have been implicated in the bilateral olfactory bulbectomy (OBX) animal model of depression, where GABAB receptor binding sites have been shown to decrease markedly at specific time points after OBX. However, as no detailed time course of events has been determined, the present study investigated the effects of OBX on high-affinity GABAA, GABAB, beta-adrenergic, and benzodiazepine receptor binding parameters in membrane preparations from rat brain regions at weekly intervals (1-4 weeks) after OBX. Persistent significant increases (40-60%) in Bmax values of high affinity GABAA receptors were observed in the frontal cortex throughout the period investigated following OBX. Bmax values in the hippocampus increased significantly after 1 week (53%) but were not statistically significant thereafter. No changes in GABAA binding parameters were observed in the hypothalamus or cerebellum. Conversely, GABAB receptor densities were significantly decreased in the frontal cortex after 1 (-38%) and 2 (-41%) weeks and moderately decreased 3 and 4 weeks (-27 and -23%, respectively) after OBX, while in the cerebellum they were significantly increased after 1 week (96%) and returned to sham-operated levels by 3 weeks. No changes in GABAB receptor binding parameters were observed in the hippocampus or hypothalamus. Binding parameters for benzodiazepine receptor binding sites or beta-adrenoceptors were not modified throughout the time course. GABAergic transmission, reflected by changes in GABAA and GABAB receptor density in the frontal cortex, may be altered in OBX rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute effects of γ-vinyl GABA (vigabatrin) on hippocampal GABAergic inhibition in vitro

The acute effects of γ-vinyl-GABA (GVG) on GABAergic inhibition were investigated in the hippocampal slice preparation using the paired-pulse test of inhibition during extracellular recordings. Superfusion of GVG (100–500 μM) for 60 min resulted in a concentration-dependent decrease in GABAergic inhibition. Slices superfused with higher concentrations of GVG (0.5–1 mM) were hyperexcitable as demonstrated by the appearance of multiple spikes. Binding studies showed that GVG (1 mM) had no effect on the binding of [3H]flunitrazepam or [3H]TBOB and displaced no more than 15% of specific [3H]GABA binding, which indicates that GVG-induced disinhibition is not mediated through an action at the GABAA receptor complex. Consistent with this suggestion is the finding that GVG (500 μM) had little effect on the inhibition of the orthodromically evoked CA1 population spike produced by the GABAA receptor agonist muscimol (10 μM), whereas this inhibition was considerably attenuated by the GABAA receptor antagonist, bicuculline methiodide (5 μM). The results of this study suggest that the acute actions of GVG on the GABAergic neurotransmitter system are not involved in its anticonvulsant effect.

Quantitative autoradiographic studies of the effects of bilateral olfactory bulbectomy in the rat brain: central- and peripheral-type benzodiazepine receptors.

We investigated the discrete regional effects of bilateral olfactory bulbectomy on central- and peripheral-type benzodiazepine receptors in rat brains at weekly intervals until one month after bulb ablation. Persistent increases in [3H]flunitrazepam binding to central benzodiazepine receptors were observed in the cingulum (27%) and in the frontal (15%) and parietal (14%) cortices. Progressive increases in central benzodiazepine receptors, reaching statistical significance four weeks after olfactory bulbectomy, were observed in the ventromedial thalamic nucleus (35%), the lateral hypothalamic region (22%), the basolateral amygdaloid nucleus (23%) and substantia nigra (25%). Persistent major increases (between four- and six-fold) in [3H]PK-11195 [eH]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide binding to peripheral-type benzodiazepine receptors were observed in all anterior olfactory nuclei. Similarly, throughout the time period studied, [3H]PK-11195 binding densities were increased two- to three-fold in the piriform cortex and lateral olfactory tract. These observations confirm the usefulness of [3H]PK-11195 binding as a marker of neuronal insult in the brain. Moreover, the persistent regional increases in [3H]flunitrazepam binding to central-type benzodiazepine receptors suggest that GABAergic transmission is altered following olfactory bulb ablation.

Antidepressants reverse the olfactory bulbectomy-induced decreases in splenic peripheral-type benzodiazepine receptors in rats

The present study investigated the effects of 21-day administration of clorgyline (1 mg/kg/day), desipramine (10 mg/kg/day) or paroxetine (10 mg/kg/day) on peripheral-type benzodiazepine receptors in rat peripheral tissues following bilateral olfactory bulbectomy. Thymus and spleen weights decreased as a result of bulbectomy. Subsequent antidepressant drug administration had no further effects on the weights of thymus glands but increased those of spleens. In thymus glands, higher densities of peripheral-type benzodiazepine receptors were observed in medulla than in cortex; no significant variations were observed following bulbectomy or antidepressant drug administration. In spleen, higher densities were observed in white pulp than in red pulp. The bulbectomy-induced decreases in binding densities observed in both regions were reversed following administration of antidepressants. Adrenal peripheral-type benzodiazepine receptors were not altered by bulbectomy or subsequent treatment with clorgyline or desipramine while paroxetine upregulated these receptors. No changes in kidney peripheral-type benzodiazepine receptors were observed. The present study confirms that cell lines of the rat immune system possess high densities of peripheral-type benzodiazepine receptor binding sites and further support the contention that, following olfactory bulbectomy, rats may present an antidepressant-reversible immunitary dysfunction.

Electrophysiology of Central Serotonin Neurotransmission

Several classes and subtypes of serotonin (5-HT) binding sites have been identified using radioligand binding techniques. The existence of these multiple 5-HT receptors has recently been confirmed by cloning techniques [1 2]. There now also existsin vivoandin vitroelectrophysiological evidence for distinct functional roles of these different 5-HT receptors. The present review will principally bear on 5-HTAreceptors but will also provide a brief review of the single-cell recording experiments on the other types of 5-HT receptors, and on the 5-HT uptake carrier, emphasizing whenever possible the clinical relevance of the data.