Transport mechanisms governing serotonin clearance in vivo revealed by high-speed chronoamperometry

High-speed chronoamperometry was used to determine the kinetics of clearance of exogenously applied serotonin (5-HT) in the dorsal raphe nucleus (DRN), dentate gyrus, CA3 region of the hippocampus or corpus callosum of anesthetized rats. Maximal velocity (Vmax) for 5-HT clearance was greatest in the DRN > dentate gyrus > CA3 > corpus callosum. Apparent affinity (K(T)) of the serotonin transporter (5-HTT) was similar in DRN and CA3 but greater in dentate gyrus and corpus callosum. A 90% loss of norepinephrine transporters (NET) produced by 6-hydroxydopamine pretreatment, resulted in a two-fold reduction in Vmax and a 30% decrease in K(T) in the dentate gyrus, but no change in kinetic parameters in the CA3 region. Pretreatment with 5,7-dihydroxytryptamine that resulted in a 90% reduction in 5-HTT density, modestly reduced Vmax in dentate gyrus but not in CA3. The same treatment had no effect on K(T) in the dentate gyrus but increased K(T) two-fold in the CA3. Neurotoxin treatments had no effect on 5-HT clearance in the corpus callosum. In hippocampal regions of intact rats, local application of the selective serotonin reuptake inhibitor, fluvoxamine, inhibited 5-HT clearance most robustly when the extracellular concentration of 5-HT was less than the K(T) value. By contrast, the NET antagonist, desipramine, significantly inhibited 5-HT clearance when extracellular concentrations of 5-HT were greater than the K(T) value. These data indicate that hippocampal uptake of 5-HT may be mediated by two processes, one with high affinity but low capacity (i.e. the 5-HTT) and the other with low affinity but a high capacity (i.e. the NET). These data show for the first time in the whole animal that 5-HT clearance in brain is regionally distinct with regard to rate and affinity.     

Effects of chronic antidepressant treatments on serotonin transporter function, density, and mRNA level.

To investigate functional changes in the brain serotonin transporter (SERT) after chronic antidepressant treatment, several techniques were used to assess SERT activity, density, or its mRNA content. Rats were treated by osmotic minipump for 21 d with the selective serotonin reuptake inhibitors (SSRIs) paroxetine or sertraline, the selective norepinephrine reuptake inhibitor desipramine (DMI), or the monoamine oxidase inhibitor phenelzine. High-speed in vivo electrochemical recordings were used to assess the ability of the SSRI fluvoxamine to modulate the clearance of locally applied serotonin in the CA3 region of hippocampus in drug- or vehicle-treated rats. Fluvoxamine decreased the clearance of serotonin in rats treated with vehicle, DMI, or phenelzine but had no effect on the clearance of serotonin in SSRI-treated rats. SERT density in the CA3 region of the hippocampus of the same rats, assessed by quantitative autoradiography with tritiated cyanoimipramine ([(3)H]CN-IMI), was decreased by 80-90% in SSRI-treated rats but not in those treated with phenelzine or DMI. The serotonin content of the hippocampus was unaffected by paroxetine or sertraline treatment, ruling out neurotoxicity as a possible explanation for the SSRI-induced decrease in SERT binding and alteration in 5-HT clearance. Levels of mRNA for the SERT in the raphe nucleus were also unaltered by chronic paroxetine treatment. Based on these results, it appears that the SERT is downregulated by chronic administration of SSRIs but not other types of antidepressants; furthermore, the downregulation is not caused by decreases in SERT gene expression.     

The contribution of low-affinity transport mechanisms to serotonin clearance in synaptosomes

Although many studies assert that the serotonin (5-HT) transporter (SERT) is the predominant mechanism controlling extracellular 5-HT concentrations, accumulating evidence suggests that low affinity, high capacity transport mechanisms may contribute more to 5-HT clearance than previously thought. The goal of this study was to quantify the contributions of SERT relative to other mechanisms in clearing extracellular 5-HT concentrations ranging from 50 nM to 1 μM in synaptosomes prepared from wild-type and SERT knockout mice using rotating disk electrode voltammetry. SERT inhibitors combined with decynium-22 (D-22), a blocker of several low-affinity transporters, blocked all uptake of 5-HT into synaptosomes. We found that SERT is responsible for the majority of synaptosomal uptake only at relatively low 5-HT concentrations, but comprises a diminishing proportion of 5-HT clearance when extracellular 5-HT increases above 100 nM. The effect of D-22 was similar in wild-type and SERT knockout synaptosomes. Thus, there was no evidence of upregulation of low-affinity mechanisms in knockout mice across the concentrations of 5-HT tested. These are surprising results, in light of the prevailing view that SERT is the primary uptake mechanism for extracellular 5-HT at physiological concentrations. We conclude that non-SERT mediated 5-HT uptake is substantial even at modest 5-HT concentrations. These findings, in conjunction with other studies, have important implications for understanding serotonergic disorders and may explain the variable efficacy and stability of patients' responses to antidepressants, such as the selective serotonin reuptake inhibitors.     

The effects of antidepressant treatment on serotonergic and dopaminergic systems in Fawn-Hooded rats: a quantitative autoradiography study

Fawn-Hooded (FH) rats exhibit a phenotype including depressive behaviour and high alcohol preference, and as such tricyclic antidepressants and selective serotonin reuptake inhibitors (SSRIs) reduce alcohol consumption in this rat strain [Psychiatr. Genet. 12 (2002) 1-16]. However, the neurochemical effects of these antidepressants on monoamine systems in the brain, especially in mesolimbic areas have not been studied in FH rats. Therefore, the present study investigated neurochemical effects of subchronic treatment (10 days) with desipramine (DMI) and sertraline on several neurochemical markers of serotonin and dopamine systems. Binding to these markers including dopamine transporters (DATs), 5-HT transporters (SERTs), 5-HT(1A)- and 5-HT(2A)-receptors in rat brain sections was performed by quantitative autoradiography. The findings from the present study revealed that DMI and sertraline exhibited differential effects on SERTs and DATs in FH rat brain. For example, DMI caused a dramatic up-regulation of DATs whereas sertraline had no effect on DAT binding. In addition, both antidepressants showed some common and some differential effects on the binding to 5-HT(1A)- and 5-HT(2A)-receptors dependent upon region. These data demonstrate that DMI and sertraline differentially effect serotonergic and dopaminergic systems in mesolimbic regions in FH rats, suggesting that there may be different neurochemical mechanisms underlying their efficacy to reduce ethanol consumption in this animal model.     

Distinct effects of sleep deprivation on binding to norepinephrine and serotonin transporters in rat brain

There is evidence to suggest that the antidepressant activity of sleep deprivation may be due to an enhancement of serotonergic and/or noradrenergic neurotransmission in brain. In the present study we examined the possibility that such changes may occur at the level of the norepinephrine (NET) and serotonin (SERT) and transporters. Rats were deprived of sleep for 96 h using the modified multiple platform method and then sacrificed for autoradiographic assessments of NET and SERT binding throughout the brain. [3H]Nisoxetine binding to the NE transporter was generally decreased in 44 of 45 areas examined, with significant reductions occurring in the anterior cingulate cortex (-16%), endopiriform n. (-18%), anterior olfactory n. (-19%), glomerular layer of olfactory bulb (-18%), ventral pallidum (-14%), medial preoptic area (-16%), retrochiasmatic/arcuate hypothalamus (-18%), anteromedial thalamic n. (-15%), and rostral raphe (-17%). In contrast, SERT binding measured with [11C]DASB showed no clear directional trends in 61 brain areas examined, but was significantly reduced in subdivisions of the anterior olfactory nucleus (-22%) and substantia nigra (-18%). Thus, sleep deprivation induced widespread decreases in NET binding, and fewer and well-localized decreases in SERT binding. Significant down-regulation in one brain region, the anterior olfactory nucleus, was observed in the case of both transporters. These results suggest that mechanisms involved in the antidepressant action of sleep deprivation may involve generalized NET down-regulation as well as decreased SERT binding in specific areas. Insofar as these changes may be associated with increased levels of serotonin (5-HT) and norepinephrine (NE) in the synapse, they suggest that sleep deprivation may share some basic mechanisms of action with several current antidepressant medications.     

A comparison of the chronic treatment effects of venlafaxine and other antidepressants on serotonin and norepinephrine transporters.

BACKGROUND: Venlafaxine blocks both serotonin and norepinephrine transporters (SERT and NET), with higher affinity for SERT. Serotonergic effects occur with lower doses, whereas both serotonergic and noradrenergic effects occur with higher doses of venlafaxine. Chronic treatment of rats with selective serotonin reuptake inhibitors decreases SERT binding sites, whereas similar treatment with selective norepinephrine reuptake inhibitors decreases NET binding sites. We hypothesized that venlafaxine would affect monoamine transporters dose-dependently, with low doses causing selective reduction of SERT binding sites and higher doses reducing both SERT and NET binding sites. METHODS: Rats were treated for 21 days with a low (15 mg/kg/day) or high (70 mg/kg/day) dose of venlafaxine, vehicle, or other antidepressants. The SERT and NET density was determined by quantitative autoradiography. RESULTS: Neither dose of venlafaxine nor amitriptyline reduced binding to either the SERT or NET. In rats with noradrenergic terminals destroyed by 6-hydroxydopamine, venlafaxine still failed to reduce SERT binding. Also, rats treated simultaneously with sertraline plus desipramine exhibited reductions in both SERT and NET binding. CONCLUSIONS: Chronic venlafaxine treatment affected SERT and NET binding differently from paroxetine or desipramine. The inability of venlafaxine to reduce SERT or NET binding sites is not due to its dual uptake inhibiting properties.     

Muscarinic regulation of Ca2+ oscillation frequency in GH3 cells

The circadian timekeeping system exhibits many functional changes with aging, including a loss of sensitivity to time cues such as systemic injections of the serotonergic agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). In order to elucidate the neurochemical mechanisms responsible for this age-related loss of sensitivity of the circadian pacemaker to serotonin agonists, the present study used quantitative autoradiography to determine whether aging decreases serotonin receptor populations in male Syrian hamsters. Four neuroanatomical regions that regulate circadian timekeeping were studied (the suprachiasmatic nuclei [SCN], the lateral geniculate nuclei [LGN], and the median raphe nucleus [MRN] and dorsal raphe nucleus [DRN]). The specific binding of [3H]8-OH-DPAT to serotonin7 (5-HT7) and serotonin1A (5-HT1A) receptors was investigated by competitive inhibition with ritanserin and pindolol, respectively. The results showed that the SCN, IGL, MRN, and DRN of the male Syrian hamster exhibited specific binding of [3H]8-OH-DPAT to both the 5-HT7 and 5-HT1A receptors, and that the latter receptor subtype is more abundant in all of these regions. At 17-19 months of age, a 50% decrease in 5-HT7 receptors was found in the DRN but not in any other regions. No significant age-related changes in 5-HT1A receptors were observed in any regions examined. The finding that a marked decrease in 5-HT7 receptors occurs in the DRN at the age previously characterized by loss of sensitivity to 8-OH-DPAT suggests that this region and this receptor subtype play important roles in 8-OH-DPAT induction of circadian phase shifts in vivo and that they constitute an important locus of aging in the circadian timing system.     

Activation of 5-HT1A autoreceptors in the dorsal raphe nucleus reduces the behavioral consequences of social defeat

In animal models, serotonin (5-HT) activity contributes to stress-induced changes in behavior. Syrian hamsters (Mesocricetus auratus) exhibit a stress-induced change in behavior in which social defeat results in increased submissive and defensive behavior and a complete loss of normal territorial aggression directed toward a novel, non-aggressive opponent. We refer to this defeat-induced change in agonistic behavior as conditioned defeat. In this study we tested the hypothesis that 5-HT activity in the dorsal raphe nucleus (DRN) contributes to the acquisition and expression of conditioned defeat. We investigated whether injection of the selective 5-HT1A agonist flesinoxan (200ng, 400ng, or 800ng in 200nl saline) into the DRN would reduce the acquisition and expression of conditioned defeat. Additionally, we investigated whether injection of the selective 5-HT1A antagonist WAY 100635 (400ng in 200nl saline) into the DRN would enhance the acquisition and expression of conditioned defeat following a sub-optimal social defeat experience. We found that injection of flesinoxan into the DRN before exposure to a 15-min social defeat reduced the amount of submissive and defensive behavior shown at testing. We also found that injection of flesinoxan into the DRN before testing similarly reduced submissive and defensive behavior. In addition, we found that WAY 100635 enhanced conditioned defeat when injected either before social defeat or before testing. These data support the hypothesis that the activity of 5-HT cells in the DRN, as regulated by 5-HT1A autoreceptors, contributes to the formation and display of conditioned defeat. Further, our results suggest that 5-HT release in DRN projection regions augments defeat-induced changes in social behavior.     

Endogenous 5-HT, released by MDMA through serotonin transporter- and secretory vesicle-dependent mechanisms, reduces hippocampal excitatory synaptic transmission by preferential activation of 5-HT1B receptors located on CA1 pyramidal neurons

A multitude of different serotonin (5-HT) receptor types are expressed in the hippocampus, but the identity of receptors actually mediating the physiological response to endogenous 5-HT has not been determined. We combined pharmacologically induced release of 5-HT with patch-clamp recordings on disinhibited rat CA1 minislices to determine effects of endogenous 5-HT on the excitability of pyramidal neurons and synaptic transmission among them. We found that application of 5-HT releasers, 3,4-methylenedioxy-methamphetamine (MDMA) or p-methylthioamphetamine, at concentrations ranging from 2 to 50 microm, reduced the excitatory synaptic transmission between CA1 pyramidal neurons without altering their basal electrical properties. This effect of MDMA was blocked by the selective 5-HT1B antagonist GR 55562, was dependent on endogenous 5-HT content and was mediated by presynaptically located, pertussis-toxin sensitive mechanisms. We found no other MDMA effects in our preparation, which indicates that the release of endogenous 5-HT preferentially stimulates 5-HT1B receptors on CA1 pyramidal neurons. Therefore, 5-HT1B receptor activation may represent a predominant component of the physiological response to endogenous 5-HT in the CA1. The high sensitivity of the 5-HT1B receptor-mediated reduction of polysynaptic excitatory responses to the extracellular 5-HT level enabled us to study mechanisms of the 5-HT releasing action of MDMA. Block of the serotonin transporter (SERT) with citalopram slowed the time course and reduced overall 5-HT release by MDMA. Depletion of vesicular 5-HT, by inhibition of vesicular monoamine transporter type 2 with tetrabenazine prevented the release. Thus although the SERT reversal contributes, a direct vesicle-depleting action is essential for MDMA release of 5-HT.     

Antidepressants modulate the in vitro inhibitory effects of propofol and ketamine on norepinephrine and serotonin transporter function

Norepinephrine transporter (NET) and serotonin transporter (SERT) proteins regulate norepinephrine (NE) and serotonin via their reuptake function and are targets of antidepressants action. Several intravenous (IV) anesthetics have been shown to inhibit NET and SERT. The interactions between antidepressants and anesthetics on transporter function, however, are not well studied. We examined the effect of different IV anesthetics on NET and SERT function, with and without chronic antidepressant pretreatment, by measuring NE or 5-hydroxytryptamine (5-HT) uptake and determined NET and SERT protein expression via immunoblotting. Both ketamine and propofol inhibited NET dose-dependently (propofol 10(-4)M -22%+/-5.6%, and propofol 10(-3)M -35%+/-5.7%; ketamine 10(-4)M -23%+/-4.1% and ketamine 10(-3)M -73%+/-2.9%); and SERT (propofol 10(-4)M -11%+/-4.3% and propofol 10(-3)M -23%+/-3.8%; ketamine 10(-4)M -29%+/-5.2% and ketamine 10(-3)M -63%+/-6.4%). Etomidate and thiopental had no effect on either NET or SERT function. Desipramine and fluoxetine, specific inhibitors of NET and SERT, respectively, both enhanced the inhibitory effects of propofol but reduced the inhibitory effects of ketamine on NET and SERT functions. IV anesthetics treatment did not change transporter protein expression in the presence of its respective inhibitor. Our results demonstrate that both ketamine and propofol inhibited SERT and NET function, but the inhibition was differentially modulated by antidepressants. Therefore, in the clinical context, this would suggest that patients receiving antidepressant treatments might have altered response to IV anesthetics in an agent-specific manner.     

Release and uptake of catecholamines in the bed nucleus of the stria terminalis measured in the mouse brain slice

The release and clearance of electrically evoked catecholamine (CA) in the ventral portion of the bed nucleus of the stria terminalis (BSTV) in mouse brain slices was evaluated with fast-scan cyclic voltammetry at carbon-fiber microelectrodes (CFME). Uptake in this region was observed to be markedly slower than in the caudate putamen (CPu). Clearance rates were reduced in the BSTV in both norepinephrine transporter knockout (NET KO) and dopamine transporter knockout (DAT KO) mice when compared to results in wild-type (WT) mice. However, uptake was faster in the BSTV in both the DAT and NET KO mice than in the CPu of DAT KO mice. This indicates that both transporters play a role in CA clearance in the BSTV. The transporters restrict extracellular CA to the general area of the BSTV, as revealed by the diminished signal as the CFME is moved sequentially further and further from the site where CA release is evoked. However, in slices from the DAT KOs and NET KOs, CA release could be observed outside of the BSTV region during such experiments. These results show that the low rate uptake in the BSTV facilitates extrasynaptic diffusion of catecholamine, but that uptake still regulates and limits the range of the transmitter to the region. Slower clearance from the extracellular fluid allows the released CA to act as a volume transmitter and diffuse to distant sites within the region to exert its neurochemical action.     

Androgen actions on central serotonin neurotransmission: relevance for mood, mental state and memory.

Sex steroids exert potent effects on mood and mental state in the human. Our previous experimental findings in female rats suggest that these effects may be mediated, in part, by the action of estrogen on the 5-hydroxytryptamine2A receptor (5-HT(2A)R) and serotonin transporter (SERT) in brain. Here we review our recent findings on the effect of acute (approximately 32 h) testosterone manipulation on central 5-HT(2A)R and SERT in male rats. Castration decreased while testosterone or estrogen, but not 5alpha-dihydrotestosterone (5alpha-DHT), increased significantly the content of 5-HT(2A)R mRNA and SERT mRNA in the dorsal raphe nucleus (DR) and the density of 5-HT(2A)R and SERT binding sites in higher centers of the brain. The lack of effect of 5alpha-DHT, a potent androgen which cannot be converted to estrogen, suggests that the action of testosterone depends upon its conversion to estrogen by aromatase. This may also explain why estrogen, but not testosterone or 5alpha-DHT, increased the density of 5-HT(2A)R binding sites in the caudate-putamen, a brain region where aromatase is scarce. The estrogen induction of SERT mRNA is most prominent in the rostral DR and this together with the correlation between sensitivity of DR serotonin neurons to estrogen and neurotoxic amphetamine derivatives provides a potential topochemical handle with which to investigate testosterone/estrogen regulation of SERT gene expression. These findings are discussed in relation to the possible role of interactions between sex steroids and serotonin mechanisms in mood disorders, schizophrenia and Alzheimer's disease.     

Tetraethylammonium ions block the nicotinic cholinergic receptors of cochlear outer hair cells

An involvement of serotonin (5-HT) 1A receptors in the etiology of psychiatric disorders has been suggested. Hypo-responsiveness of the 5-HT1A receptor is linked to anxiety and constitutive deletion of the 5-HT1A receptor produces anxiety-like behaviors in the mouse. Evidence that 5-HT1A receptor inactivation increases the therapeutic effects of antidepressants has also been presented. The present studies used in vivo microdialysis and homologous recombination techniques to examine the contribution of 5-HT1A autoreceptors to these effects. Basal and fluoxetine-evoked extracellular concentrations of 5-HT were quantified in the striatum, a projection area of dorsal raphe neurons (DRN), of wild-type (WT) and 5-HT1A receptor knock out (KO) mice. The density of 5-HT transporters was also determined. Basal 5-HT concentrations did not differ in WT and KO mice. Fluoxetine (10 mg/kg) increased 5-HT concentrations in both genotypes. This increase was, however, 2-fold greater in KO mice. In contrast, no differences in K(+)-evoked 5-HT concentrations were seen. Similarly, neither basal nor stimulation-evoked DA differed across genotype. Autoradiography revealed no differences between genotype in the density of 5-HT transporters or post-synaptic 5-HT2A receptors, an index of 5-HT neuronal activity. These experiments demonstrate that, under basal and KCl stimulated conditions, adaptive mechanisms in the 5-HT system compensate for the lack of 5-HT1A autoreceptor regulation of DRN. Furthermore, they suggest that the absence of release-regulating 5-HT1A autoreceptors in the DRN can not account for the anxiety phenotype of KO mice. The enhanced response to fluoxetine in KO mice is consistent with pharmacological studies and suggests that adaptive mechanisms that occur in response to 5-HT1A receptor deletion are insufficient to oppose increases in 5-HT concentrations produced by acute inhibition of the 5-HT transporter.     

Acute and chronic effects of nicotine on serotonin uptake in prefrontal cortex and hippocampus of rats

We sought to investigate the effect of nicotine exposure (chronic and acute) on serotonin transporter (SERT) activity in two regions of the brain important for behavioral effects of nicotine. We first looked at the effects of chronic nicotine exposure (0.7 mg/kg nicotine, twice a day for 10 days) on [(3)H]5-HT uptake in prefrontal cortex (PFC) and hippocampus of rats. A significant increase in [(3)H]5-HT uptake was observed in synaptosomes prepared from both regions. To rule out the possibility that the increases were due to the last injection given, in a separate set of experiments a single injection of nicotine was administered the evening before sacrifice. No change in uptake occurred in either region, suggesting that the increases in uptake caused by nicotine was an effect of chronic exposure and not to an acute treatment. SERT binding studies, using prefrontocortical or hippocampal membrane preparations, revealed that chronic nicotine exposure significantly increased B(max) which correlated to an increase in SERT density. Lastly, we looked at the short-term effect of nicotine on [(3)H]5-HT uptake. Rats received a single nicotine injection 15-75 min before sacrifice. PFC synaptosomes displayed a time-dependent increase in uptake, whereas hippocampal synaptosomes showed an increase at only one time point.     

Effects of tryptophan depletion on the binding of [11C]-DASB to the serotonin transporter in baboons: response to acute serotonin deficiency.

BACKGROUND: The objective of this study was to evaluate the sensitivity of [(11)C]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)benzylamine (DASB) binding to the brain serotonin transporter (SERT) to changes in endogenous serotonin (5-hydroxytryptamine [5-HT]) levels. A ligand sensitive to endogenous competition (EC) would enable the measurement of fluctuations of intrasynaptic 5-HT. A ligand insensitive to EC can provide a measure of SERT unaffected by levels of 5-HT. Alternatively, serotonin depletion could accelerate internalization of SERT and reduce binding. METHODS: Eighteen (14 baseline and 9 tryptophan-depleted) positron emission tomography (PET) scans were carried out in two baboons (Papio anubis). A metabolite-corrected arterial input function was used to estimate the binding potential (BP = B(max)/K(D)). RESULTS: Depletion of plasma tryptophan by a mean of 65% from the baseline (p = .03) reduces [(11)C]-DASB BP in the six brain regions of interest (ROI). Lower DASB binding correlated with lower plasma tryptophan levels in the ROIs with higher SERT density. CONCLUSIONS: [(11)C]-DASB binding to SERT in vivo rapidly declines in response to acute reduction in serotonin availability, contrary to what is predicted by a simple competition model. This rapid reduction in SERT availability may be due to accelerated transporter internalization.     

Expression of norepinephrine and serotonin transporter mRNAs in the rat superior cervical ganglion

We investigated the gene expression of three monoamine transporters (norepinephrine transporter, NET; serotonin transporter, SERT; and dopamine transporter, DAT) in the rat superior cervical ganglion (SCG). Most of principal ganglion neurons abundantly expressed NET mRNA. In addition, about 30% of principal ganglion neurons also expressed SERT mRNA. However, DAT mRNA expression was not observed there. These results suggest that serotonin as well as norepinephrine works as a neurotransmitter in a subset of principal ganglion neurons.     

Serotonin-1A autoreceptor binding in the dorsal raphe nucleus of depressed suicides

Serotonergic dysfunction is present in mood disorders and suicide. Brainstem 5-HT1A somatodendritic autoreceptors regulate serotonin neuron firing but studies of autoreceptor binding in the dorsal raphe nucleus (DRN) in depressed suicides report conflicting results. We sought to determine: (1) the anatomical distribution of 5-HT1A receptor binding in the DRN in depressed suicides and psychiatrically normal controls; and (2) whether sex differences in 5-HT1A binding in the DRN contribute to differences between depressed suicides and controls. Previously collected quantitative receptor autoradiograms of [3H]8-hydroxy-2-(di-n-propyl)aminotetralin (3H-8-OH-DPAT) in postmortem tissue sections containing the DRN from drug-free suicide victims (n=10) and matched controls (n=10) were analyzed. Less total receptor binding (fmol/mg tissuexmm3) was observed in the entire DRN in depressed suicides compared with controls (p<0.05). Group differences along the rostrocaudal extent of the DRN were observed for cross-sectional 5-HT(1A) binding (fmol/mg tissue) and receptor binding (fmol/mgxmm3, p<0.05). Cross-sectional 5-HT1A DRN binding in depressed suicides compared with controls was higher rostrally and lower caudally. The differences between depressed suicides and controls were present in males and females, although females had more binding than males. Less autoreceptor binding in the DRN of depressed suicides may represent a homeostatic response to less serotonin release, increasing serotonin neuron firing. More autoreceptor binding in rostral DRN might contribute to deficient serotonin release in ventromedial prefrontal cortex by lower neuronal firing.     

Ectopic serotonin accumulation and efflux in rat mesencephalic slices after prior tissue 'radiolabelling'

Radiolabelling of brain tissue has long been used to facilitate detection of transmitter efflux, on the assumption that egress of tritiated monoamines reflects that of the endogenous transmitter. The present study tested the hypothesis that the application of exogenous serotonin (5-HT) to mesencephalic slices, in the manner used during a typical radiolabelling protocol, leads to efflux of 5-HT from physiologically inappropriate loci such as other non-serotonergic neurones. We used fast cyclic voltammetry (FCV) to determine the effect of tissue pre-incubation with 5-HT on electrically-stimulated 5-HT efflux and reuptake in rat mesencephalic slices. Seven subregions were studied, including the dorsal raphe nucleus (DRN), dorsomedial periaqueductal grey (PAGdm) and the oral part of the pontine reticular nucleus (PnO). In control slices (pre-incubated without 5-HT), stimulated 5-HT efflux was only detectable in DRN, PAGdm and occasionally in PnO. In slices incubated in 5-HT (100nM) for 30min, stimulated 5-HT efflux was detected in all seven subregions studied. In such slices, citalopram (75nM) increased efflux and reuptake t(1/2) in DRN to 201+/-21 and 487+/-117% of pre-drug values (P<0.05) but had no significant effect on either measure in PnO. The 5-HT1 autoreceptor agonist, 5-carboxamidotryptamine (5-CT, 100nM) decreased efflux in DRN by 54+/-6% (P<0.05), but was without effect (10+/-14%) in PnO. The present results show that pre-incubation in 5-HT allows stimulated 5-HT efflux from regions of the mesencephalon other than DRN and PAGdm. This stimulated 5-HT efflux is apparently not influenced by 5-HT transporters or 5-HT1 autoreceptors, suggesting that efflux is ectopic, an artefact of the pre-incubation process. In summary, incubation of rat mesencephalic tissue in 5-HT, in the manner of a typical radiolabelling protocol, results in stimulated 5-HT efflux from non-physiological sites. The results of such transmitter efflux studies should thus be interpreted with caution.     

Monoaminergic compensation in the neuropeptide Y deficient mouse brain

Neuropeptide Y (NPY) is an important central regulator of food consumption and energy expenditure via the hypothalamus. NPY containing neurons have a broad central distribution and are often colocalized with norepinephrine (NE). However, NPY deficient mice do not exhibit any substantial changes in food consumption, body weight or body composition when compared to wild type mice. Since NE and serotonin (5HT) are also important regulators of appetite and metabolism, we evaluated these systems in NPY deficient mice. Brain sections from NPY deficient and wild type mice were labeled with either (3)H-nisoxetine for the NE transporter (NET) or (3)H-citalopram for the 5HT transporter (SERT). Tyrosine hydroxylase expression was evaluated by radioimmunohistochemistry. Brain monoamines and metabolites were evaluated using HPLC. NPY deficient mice exhibited a substantial decrease in NET binding in most brain regions examined. NET binding was less than 50% of control binding in the cerebral cortex and subregions of the thalamus with the greatest decrease seen in the hypothalamus. In contrast, more modest and regionally variable changes were observed in the SERT binding with decreases in regions such as the accessory olfactory nucleus, glomerular layer of the olfactory bulb and the CA1 region of the hippocampus. Measurement of NE and 5HT content as well as the primary metabolites revealed increased NE turnover and decreased 5HT content in the hypothalamus. Therefore, developmental compensation by the NE and 5HT systems may contribute to the absence of a body weight phenotype in NPY deficient mice.     

Adaptive changes in serotonin neurons of the raphe nuclei in 5-HT(4) receptor knock-out mouse

Decreased serotonin (5-HT) transmission is thought to underlie several mental diseases, including depression and feeding disorders. However, whether deficits in genes encoding G protein-coupled receptors may down-regulate the activity of 5-HT neurons is unknown currently. Based on recent evidence that stress-induced anorexia may involve 5-HT(4)receptors (5-HT(4)R), we measured various aspects of 5-HT function in 5-HT(4)R knock-out (KO) mice. When compared to dorsal raphe nucleus (DRN) 5-HT neurons from wild-type mice, those from 5-HT(4)R KO mice exhibited reduced spontaneous electrical activity. This reduced activity was associated with diminished tissue levels of 5-HT and its main metabolite, 5-hydroxyindole acetic acid (5-HIAA). Cumulative, systemic doses of the 5-HT uptake blocker citalopram, that reduced 5-HT cell firing by 30% in wild-type animals, completely inhibited 5-HT neuron firing in the KO mice. This effect was reversed by administration of the 5-HT(1A) receptor (5-HT(1A)R) antagonist, WAY100635, in mice of both genotypes. Other changes in DRN of the KO mice included increases in the levels of 5-HT plasma membrane transporter sites and mRNA, as well as a decrease in the density of 5-HT(1A)R sites without any change in 5-HT(1A) mRNA content. With the exception of increased 5-HT turnover index in the hypothalamus and nucleus accumbens and a decreased density of 5-HT(1A)R sites in the dorsal hippocampus (CA1) and septum, no major changes were detected in 5-HT territories of projection, suggesting region-specific adaptive changes. The mechanisms whereby 5-HT(4)R mediate a tonic positive influence on the firing activity of DRN 5-HT neurons and 5-HT content remain to be determined.