Sidney B. Auerbach

Extracellular serotonin and 5-hydroxyindoleacetic acid in hypothalamus of the unanesthetized rat measured by in vivo dialysis coupled to high-performance liquid chromatography with electrochemical detection: dialysate serotonin reflects neuronal release

In vivo dialysis coupled to high-performance liquid chromatography with electrochemical detection (HPLC-EC) was used for measurement of extracellular serotonin (5-HT) in the hypothalamus of unanesthetized, unrestrained rats. A series of experiments was carried out to determine if 5-HT in the dialysis solution was released from nerve terminals. Fenfluramine, a 5-HT-releasing drug and fluoxetine, a 5-HT-reuptake inhibitor, both significantly increased extracellular 5-HT. Elevating potassium concentration in the dialysis solution also significantly increased 5-HT. Reciprocally, 5-HT was significantly reduced to about half of control levels with either local administration of tetrodotoxin, zero calcium dialysis solution, or systemic administration of 8-hydroxy-2-(di-n-propylamino)tetralin, a 5-HT1A agonist that suppresses 5-HT neuronal activity via activation of the somatodendritic autoreceptor. In addition, 5-HT levels were elevated during the dark portion of the light-dark cycle, a period when rats are more active. Changes in extracellular 5-hydroxyindoleacetic acid (5-HIAA) rarely followed changes in 5-HT. The results indicate that 5-HT in the dialysis solution, but not 5-HIAA, was a reliable measure of depolarization-induced release of 5-HT from nerve terminals. This is the first report establishing the reliability of in vivo dialysis coupled to HPLC-EC for measurement of synaptically released 5-HT.

Serotonin autoreceptor function and antidepressant drug action

This article briefly summarizes, within the context of a brief review of the relevant literature, the outcome of our recent rat microdialysis studies on (1) the relative importance of serotonin (5-HT)1A versus 5-HT1B autoreceptors in the mechanism of action of 5-HT reuptake blocking agents, including putative regional differences in this regard, and (2) autoreceptor responsiveness following chronic SSRI administration. First, our data are consistent with the primacy of 5-HT1A autoreceptors in restraining the elevation of 5-HT levels induced by SSRIs, whereas nerve terminal 5-HT1B autoreceptors appear to have an accessory role in this regard. Second, there is an important interplay between cell body and nerve terminal 5-HT autoreceptors, and recent findings suggest that this interplay may potentially be exploited to obtain regionally preferential effects on 5-HT neurotransmission in the central nervous system, even upon systemic drug administration. In particular, emerging data suggest that somatodendritic 5-HT1A autoreceptorand nerve terminal 5-HT1B autoreceptor-mediated feedback may be relatively more important in the control of 5-HT output in dorsal raphe-frontal cortex and median raphe-dorsal hippocampus systems, respectively. Third, 5-HT autoreceptors evidently retain the capability to limit the 5-HT transmission-promoting effect of SSRIs after chronic treatment. Thus, although the responsiveness of these sites is probably somewhat reduced, residual autoreceptor capacity still remains an effective restraint on large increases in extracellular 5-HT, even after prolonged treatment. If a further increase in extracellular 5-HT is crucial to the remission of depression in patients responding only partially to prolonged administration of antidepressants, then sustained adjunctive treatment with autoreceptor-blocking drugs may consequently prove useful in the long term.

Increase in extracellular serotonin produced by uptake inhibitors is enhanced after chronic treatment with fluoxetine

The effect of prolonged uptake inhibition with fluoxetine (10 mg/kg/day i.p. x 14 days) on extracellular serotonin (5-HT) in the rat diencephalon was monitored using in vivo microdialysis. The increase in extracellular 5-HT after repeated administration of fluoxetine was significantly greater than the increase produced by a single injection of this uptake blocker. This difference may have been due to a decrease in somatodendritic autoreceptor sensitivity, since the response to a low dose of the 5-HT1A agonist 8-OH-DPAT (25 micrograms/kg i.v.) was abolished in the chronic rats, while the response to a high dose (100 micrograms/kg i.v.) was attenuated as compared to animals injected once with fluoxetine.

Differential regulation of 5-hydroxytryptamine release by GABAA and GABAB receptors in midbrain raphe nuclei and forebrain of rats.

1 Extracellular 5‐hydroxytryptamine (5‐HT) was determined in dorsal raphe nucleus (DRN), median raphe nucleus (MRN) and nucleus accumbens by use of microdialysis in unanaesthetized rats. 2 Infusion of the γ‐aminobutyric acid (GABA)A receptor agonist muscimol into DRN and MRN resulted in decreased 5‐HT in DRN and MRN, respectively. Muscimol infusion into nucleus accumbens had no effect on 5‐HT. 3 Infusion of the GABAA receptor antgonist bicuculline into DRN resulted in increased DRN and nucleus accumbens 5‐HT. Bicuculline infusion into MRN had no effect on 5‐HT. This suggests that endogenous GABA had a tonic, GABAA receptor‐mediated inhibitory effect on 5‐HT in DRN, but not in MRN. 4 Infusion of the GABAB receptor agonist baclofen into DRN produced a decrease in DRN 5‐HT. Baclofen infusion into nucleus accumbens resulted in decreased nucleus accumbens 5‐HT. This suggests that GABAB receptors are present in the area of cell bodies and terminals of 5‐hydroxytryptaminergic neurones. 5 Infusion of the GABAB receptor antagonists phaclofen and 2‐hydroxysaclofen had no effect on midbrain raphe and forebrain 5‐HT. This suggests that GABAB receptors did not contribute to tonic inhibition of 5‐HT release. 6 In conclusion, 5‐HT release is physiologically regulated by distinct subtypes of GABA receptors in presynaptic and postsynaptic sites.

Correlation of dopamine release in the nucleus accumbens with masculine sexual behavior in rats

Extracellular dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in the nucleus accumbens (N. Acc.) were measured by in vivo microdialysis during male sexual activity. DA and DOPAC were significantly increased during copulation, but not during mild tail pinch. These results are consistent with studies showing increases in N. Acc. DA associated with positively reinforcing environmental stimuli.

Further evidence for the importance of 5-HT1A autoreceptors in the action of selective serotonin reuptake inhibitors

The clinical efficacy of antidepressants that block serotonin (5-hydroxytryptamine, 5-HT) reuptake may be restrained by indirect activation of autoreceptors. In vivo microdialysis in rat hippocampus was used to assess the release-inhibitory properties of the 5-HT reuptake inhibitors citalopram and paroxetine. When reuptake was first blocked by infusing citalopram into the hippocampus, systemic administration of citalopram or paroxetine resulted in a 50-70% decrease in hippocampal 5-HT overflow. This presumably reflected the inhibition of 5-HT release subsequent to reuptake blockade in the raphe nuclei and, in turn, activation of somatodendritic autoreceptors. In support, pretreatment with (+/-)-pindolol or (+)-WAY100135 ((+)-N-tert-butyl-3-(4-(2- methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide dihydrochloride), to block 5-HT1A autoreceptors, abolished the decrease in 5-HT produced by systemic injection of the uptake blockers.

Regulation of serotonin release by GABA and excitatory amino acids

Regulation of serotonin release by γ-aminobutyric acid (GABA) and glutamate was examined by microdialysis in unanaesthetized rats. The GABAA receptor agonist muscimol, or the glutamate receptor agonists kainate, γ-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate or N-methyl-D-aspartate were infused into the dorsal raphe nucleus (DRN) while extracellular serotonin was measured in the DRN and nucleus accumbens. Muscimol produced decreases, and the glutamate receptor agonists produced increases in serotonin. To determine if these receptors have a tonic influence on serotonergic neurons, glutamate or GABAA receptor antagonists were infused into the DRN. Kynurenate, a nonselective glutamate receptor blocker, produced a small, 30% decrease in serotonin. A similar decrease was obtained with combined infusion of AP-5 and DNQX into the DRN. The GABAA receptor blocker bicuculline produced an approximately three-fold increase in DRN serotonin. In conclusion, glutamate neurotransmitters have a weak tonic excitatory influence on serotonergic neurons in the rat DRN. However, the predominate influence is mediated by GABAA receptors.

Differences in hypothalamic serotonin between estrous phases and gender: an in vivo microdialysis study

The aim of the present study was to assess whether there are gender differences in (1) levels of extracellular serotonin (5-HT) in the forebrain, and (2) the effect on 5-HT of a reuptake inhibitor, paroxetine, or a releasing drug, fenfluramine. In vivo microdialysis was used to measure 5-HT in the hypothalamus of male and regularly cycling female rats. Hypothalamic 5-HT was significantly lower in estrous females (0.83 +/- 0.05 pg/sample, n=33) than in male rats (1.04 +/- 0.06 pg, n=38). Levels in diestrous females (0.98 +/- 0.09 pg, n=38) were not significantly different from males. Paroxetine (1 mg/kg) increased hypothalamic 5-HT in males, and diestrous and estrous females to approximately 2 pg/sample. However, the increase in hypothalamic 5-HT produced by a maximally effective dose of paroxetine (10 mg/kg) was significantly greater in male rats and during diestrous than during estrous. d,l-Fenfluramine (10 mg/kg) evoked an increase in extracellular 5-HT to approximately 15 pg/sample in all groups. A higher dose of d,l-fenfluramine (20 mg/kg) produced a significantly greater increase in hypothalamic 5-HT in males than in females during estrous or diestrous. These results are consistent with other evidence that during estrous, when rats are responding to peak levels of estrogen and progesterone, 5-HT release is decreased.

Increased extracellular serotonin in rat brain after systemic or intraraphe administration of morphine

Abstract: The effect of morphine on serotonin (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) in the CNS of unanesthetized rats was investigated by microdialysis. Morphine was administered either subcutaneously, by local perfusion into the diencephalon, or by intraraphe microinjection. Systemic administration of morphine resulted in a significant increase in both extracellular 5‐HT and 5‐HIAA in the diencephalon. The effect of morphine on 5‐HT was dose dependent during local perfusion of the diencephalon with inhibitors of uptake or monoamine oxidase. Systemic morphine also produced significant increases in extracellular 5‐HT in the striatum and hippocampus during uptake inhibition. The site of opioid effects on 5‐HT was tested by locally perfusing morphine into the diencephalon. This had no effect on 5‐HT or 5‐HIAA. In contrast, intraraphe injection of morphine caused a dose‐dependent increase in extracellular 5‐HT and 5‐HIAA in the diencephalon. These results suggest that systemic morphine induces an increase in 5‐HT release in widespread areas of the forebrain. This appears to be due to an effect on 5‐HT cell bodies and not on 5‐HT nerve endings in projection sites.

Effect of chronic administration of the selective serotonin (5-HT) uptake inhibitor citalopram on extracellular 5-HT and apparent autoreceptor sensitivity in rat forebrain in vivo

Rats were administered the selective serotonin (5-HT) uptake blocker citalopram or saline for 14 days to determine if prolonged treatment would lead to changes in extracellular 5-HT or autoreceptor sensitivity. One day after drug withdrawal, dialysis probes were implanted in the frontal cortex and dorsal hippocampus. Dialysis experiments were carried out using chloral hydrate anesthetized rats. The experimental protocol comprised the administration of three consecutive drug challenges: (1) After stable baseline levels were obtained, citalopram was infused through the dialysis probes to locally block uptake in the forebrain. (2) Subsequently, a 5-HT1B receptor agonist (RU24969 or CP93,129) was infused through the probe to test for changes in terminal autoreceptor sensitivity. (3) Last, citalopram was administered systemically to test the effect of indirect activation of somatodendritic autoreceptors. Under these conditions, with uptake already blocked locally in the forebrain, systemic citalopram produces a decrease in extracellular 5-HT, an effect that can be inhibited by pretreatment with antagonists of 5-HT1A receptors. The results indicate that during local infusion of citalopram extracellular 5-HT was significantly higher in the dorsal hippocampus of the chronic citalopram as compared to saline treatment group. This difference persisted throughout the full time course of the experiment. However, the decreases in 5-HT levels produced by local infusion of a 5-HT1B receptor agonist or after systemic citalopram administration were not significantly different between the chronic citalopram and saline treated groups. There were no significant differences between chronic citalopram and saline treated animals in frontal cortex. These results suggest that prolonged inhibition of 5-HT uptake may produce a selective change in the regulation of release from median raphe 5-HT neurons, but this change could not be clearly linked to a change in nerve terminal or somatodendritic autoreceptor sensitivity.