Region-Specific Regulation of Presynaptic Dopamine Homeostasis by D2 Autoreceptors Shapes the In Vivo Impact of the Neuropsychiatric Disease-Associated DAT Variant Val559.

Disruptions of dopamine (DA) signaling contribute to a broad spectrum of neuropsychiatric disorders, including attention-deficit hyperactivity disorder (ADHD), addiction, bipolar disorder, and schizophrenia. Despite evidence that risk for these disorders derives from heritable variation in DA-linked genes, a better understanding is needed of the molecular and circuit context through which gene variation drives distinct disease traits. Previously, we identified the DA transporter (DAT) variant Val559 in subjects with ADHD and established that the mutation supports anomalous DAT-mediated DA efflux (ADE). Here, we demonstrate that region-specific contributions of D2 autoreceptors (D2AR) to presynaptic DA homeostasis dictate the consequences of Val559 expression in adolescent male mice. We show that activation of D2ARs in the WT dorsal striatum (DS), but not ventral striatum (VS), increases DAT phosphorylation and surface trafficking. In contrast, the activity of tyrosine hydroxylase (TH) is D2AR-dependent in both regions. In the DS but not VS of Val559 mice, tonic activation of D2ARs drives a positive feedback loop that promotes surface expression of efflux-prone DATs, raising extracellular DA levels and overwhelming DAT-mediated DA clearance capacity. Whereas D2ARs that regulate DAT are tonically activated in the Val559 DS, D2ARs that regulate TH become desensitized, allowing maintenance of cytosolic DA needed to sustain ADE. Together with prior findings, our results argue for distinct D2AR pools that regulate DA synthesis versus DA release and inactivation and offer a clear example of how the penetrance of gene variation can be limited to a subset of expression sites based on differences in intersecting regulatory networks.SIGNIFICANCE STATEMENT Altered dopamine (DA) signaling has been linked to multiple neuropsychiatric disorders. In an effort to understand and model disease-associated DAergic disturbances, we previously screened the DA transporter (DAT) in subjects with attention-deficit hyperactivity disorder (ADHD) and identified multiple, functionally impactful, coding variants. One of these variants, Val559, supports anomalous DA efflux (ADE) and in transgenic mice leads to changes in locomotor patterns, psychostimulant sensitivity, and impulsivity. Here, we show that the penetrance of Val559 ADE is dictated by region-specific differences in how presynaptic D2-type autoreceptors (D2ARs) constrain DA signaling, biasing phenotypic effects to dorsal striatal projections. The Val559 model illustrates how the impact of genetic variation underlying neuropsychiatric disorders can be shaped by the differential engagement of synaptic regulatory mechanisms.

Abrogated Freud-1/Cc2d1a Repression of 5-HT1A Autoreceptors Induces Fluoxetine-Resistant Anxiety/Depression-Like Behavior.

Freud-1/Cc2d1a represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the role of Freud-1 in vivo, we generated mice with adulthood conditional knock-out of Freud-1 in 5-HT neurons (cF1ko). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1A double knock-out (cF1/1A dko) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behavior was seen upon knock-out of Freud-1 on the 5-HT1A autoreceptor-negative background; rather, a reduction in depression-like behavior emerged. These studies implicate transcriptional dysregulation of 5-HT1A autoreceptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors, such as Freud-1, to restore transcriptional balance may augment response to antidepressant treatment.SIGNIFICANCE STATEMENT Altered regulation of the 5-HT1A autoreceptor has been implicated in human anxiety, major depression, suicide, and resistance to antidepressants. This study uniquely identifies a single transcription factor, Freud-1, as crucial for 5-HT1A autoreceptor expression in vivo Disruption of Freud-1 in serotonin neurons in mice links upregulation of 5-HT1A autoreceptors to anxiety/depression-like behavior and provides a new model of antidepressant resistance. Treatment strategies to reestablish transcriptional regulation of 5-HT1A autoreceptors could provide a more robust and sustained antidepressant response.

Epigenetic reprogramming of cortical neurons through alteration of dopaminergic circuits.

Alterations of the dopaminergic system are associated with the cognitive and functional dysfunctions that characterize complex neuropsychiatric disorders. We modeled a dysfunctional dopaminergic system using mice with targeted ablation of dopamine (DA) D2 autoreceptors in mesencephalic dopaminergic neurons. Loss of D2 autoreceptors abolishes D2-mediated control of DA synthesis and release. Here, we show that this mutation leads to a profound alteration of the genomic landscape of neurons receiving dopaminergic afferents at distal sites, specifically in the prefrontal cortex. Indeed, we observed a remarkable downregulation of gene expression in this area of ~2000 genes, which involves a widespread increase in the histone repressive mark H3K9me2/3. This reprogramming process is coupled to psychotic-like behaviors in the mutant mice. Importantly, chronic treatment with a DA agonist can revert the genomic phenotype. Thus, cortical neurons undergo a profound epigenetic reprogramming in response to dysfunctional D2 autoreceptor signaling leading to altered DA levels, a process that may underlie a number of neuropsychiatric disorders.

Somatodendritic 5-hydroxytryptamine1A (5-HT1A) autoreceptor function in major depression as assessed using the shift in electroencephalographic frequency spectrum with buspirone.

BACKGROUND: Positron emission tomography and post-mortem studies of the number of somatodendritic 5-hydroxytryptamine(1A) (5-HT(1A)) autoreceptors in raphé nuclei have found both increases and decreases in depression. However, recent genetic studies suggest they may be increased in number and/or function. The current study examined the effect of buspirone on the electroencephalographic (EEG) centroid frequency, a putative index of somatodendritic 5-HT(1A) receptor functional status, in a cohort of medication-free depressed patients and controls. METHOD: A total of 15 depressed patients (nine male) and intelligence quotient (IQ)-, gender- and age-matched healthy controls had resting EEG recorded from 29 scalp electrodes prior to and 30, 60 and 90 min after oral buspirone (30 mg) administration. The effect of buspirone on somatodendritic 5-HT(1A) receptors was assessed by calculating the EEG centroid frequency between 6 and 10.5 Hz. The effect of buspirone on postsynaptic 5-HT(1A) receptors was assessed by measuring plasma growth hormone, prolactin and cortisol concentrations. RESULTS: Analysis of variance revealed a significantly greater effect of buspirone on the EEG centroid frequency in patients compared with controls (F1,28 = 6.55, p = 0.016). There was no significant difference in the neuroendocrine responses between the two groups. CONCLUSIONS: These findings are consistent with an increase in the functional status of somatodendritic, but not postsynaptic, 5-HT1A autoreceptors, in medication-free depressed patients in line with hypotheses based on genetic data. This increase in functional status would be hypothesized to lead to an increase in serotonergic negative feedback, and hence decreased release of 5-HT at raphé projection sites, in depressed patients.

Increased serotonin-1A (5-HT1A) autoreceptor expression and reduced raphe serotonin levels in deformed epidermal autoregulatory factor-1 (Deaf-1) gene knock-out mice.

Altered regulation of the serotonin-1A (5-HT1A) receptor gene is implicated in major depression and mood disorders. The functional human 5-HT1A C(-1019)G promoter polymorphism (rs6295), which prevents the binding of Deaf-1/NUDR leading to dysregulation of the receptor, has been associated with major depression. In cell models Deaf-1 displays dual activity, repressing 5-HT1A autoreceptor expression in serotonergic raphe cells while enhancing postsynaptic 5-HT1A heteroreceptor expression in nonserotonergic neurons. A functional Deaf-1 binding site on the mouse 5-HT1A promoter was recognized by Deaf-1 in vitro and in vivo and mediated dual activity of Deaf-1 on 5-HT1A gene transcription. To address regulation by Deaf-1 in vivo, Deaf-1 knock-out mice bred to a C57BL/6 background were compared with wild-type siblings for changes in 5-HT1A RNA and protein by quantitative RT-PCR, in situ hybridization, and immunofluorescence. In the dorsal raphe, Deaf-1 knock-out mice displayed increased 5-HT1A mRNA, protein, and 5-HT1A-positive cell counts but reduced 5-HT levels, whereas other serotonergic markers, such as tryptophan hydroxylase (TPH)- or 5-HT-positive cells and TPH2 RNA levels, were unchanged. By contrast, 5-HT1A mRNA and 5-HT1A-positive cells were reduced in the frontal cortex of Deaf-1-null mice, with no significant change in hippocampal 5-HT1A RNA, protein, or cell counts. The region-specific alterations of brain 5-HT1A gene expression and reduced raphe 5-HT content in Deaf-1(-/-) mice indicate the importance of Deaf-1 in regulation of 5-HT1A gene expression and provide insight into the role of the 5-HT1A G(-1019) allele in reducing serotonergic neurotransmission by derepression of 5-HT1A autoreceptors.

[Development of serotonergic neurons of dorsal raphe nuclei in mice with knockout of monoamine oxidase A and 5-HT1A and 5-HT1B autoreceptor].

The morphological changes in the development of serotonergic neurons of the dorsal raphe nuclei in the medulla oblongata was studied by immunocytochemistry in mice with knockout of 1A and 1B serotonin autoreceptors as well as monoamine oxidase A. Serotonin autoreceptors regulate electric activity of serotonergic neurons as well as the synthesis and release of the neurotransmitter, while monoamine oxidase A catalyzes its degradation. These genetic modifications proved to have no effect on the number of serotonergic neurons in the medulla oblongata but induced morphofunctional changes. Decreased cell size and increased intracellular serotonin level were observed in the case of monoamine oxidase A deficiency, while excessive cell size and decreased intracellular serotonin level were observed in the case of autoreceptor deficiency. The data obtained confirm the hypothesis of autoregulation of serotonergic neurons in development.

Regulation of dopamine neurotransmission from serotonergic neurons by ectopic expression of the dopamine D2 autoreceptor blocks levodopa-induced dyskinesia.

Levodopa-induced dyskinesias (LID) are a prevalent side effect of chronic treatment with levodopa (L-DOPA) for the motor symptoms of Parkinson's disease (PD). It has long been hypothesized that serotonergic neurons of the dorsal raphe nucleus (DRN) are capable of L-DOPA uptake and dysregulated release of dopamine (DA), and that this "false neurotransmission" phenomenon is a main contributor to LID development. Indeed, many preclinical studies have demonstrated LID management with serotonin receptor agonist treatment, but unfortunately, promising preclinical data has not been translated in large-scale clinical trials. Importantly, while there is an abundance of convincing clinical and preclinical evidence supporting a role of maladaptive serotonergic neurotransmission in LID expression, there is no direct evidence that dysregulated DA release from serotonergic neurons impacts LID formation. In this study, we ectopically expressed the DA autoreceptor D2Rs (or GFP) in the DRN of 6-hydroxydopamine (6-OHDA) lesioned rats. No negative impact on the therapeutic efficacy of L-DOPA was seen with rAAV-D2Rs therapy. However, D2Rs treated animals, when subjected to a LID-inducing dose regimen of L-DOPA, remained completely resistant to LID, even at high doses. Moreover, the same subjects remained resistant to LID formation when treated with direct DA receptor agonists, suggesting D2Rs activity in the DRN blocked dyskinesogenic L-DOPA priming of striatal neurons. In vivo microdialysis confirmed that DA efflux in the striatum was reduced with rAAV-D2Rs treatment, providing explicit evidence that abnormal DA release from DRN neurons can affect LID. This is the first direct evidence of dopaminergic neurotransmission in DRN neurons and its modulation with rAAV-D2Rs gene therapy confirms the serotonin hypothesis in LID, demonstrating that regulation of serotonergic neurons achieved with a gene therapy approach offers a novel and potent antidyskinetic therapy.

Electrophysiological properties of catecholaminergic neurons in the norepinephrine-deficient mouse.

To determine how norepinephrine affects the basic physiological properties of catecholaminergic neurons, brain slices containing the substantia nigra pars compacta and locus coeruleus were studied with cell-attached and whole-cell recordings in control and dopamine beta-hydroxylase knockout (Dbh -/-) mice that lack norepinephrine. In the cell-attached configuration, the spontaneous firing rate and pattern of locus coeruleus neurons recorded from Dbh -/- mice were the same as the firing rate and pattern recorded from heterozygous littermates (Dbh +/-). During whole-cell recordings, synaptic stimulation produced an alpha-2 receptor-mediated outward current in the locus coeruleus of control mice that was absent in Dbh -/- mice. Normal alpha-2 mediated outward currents were restored in Dbh -/- slices after pre-incubation with norepinephrine. Locus coeruleus neurons also displayed similar changes in holding current in response to bath application of norepinephrine, UK 14304, and methionine-enkephalin. Dopamine neurons recorded in the substantia nigra pars compacta similarly showed no differences between slices harvested from Dbh -/- and control mice. These results indicate that endogenous norepinephrine is not necessary for the expression of catecholaminergic neuron firing properties or responses to direct agonists, but is necessary for auto-inhibition mediated by indirect alpha-2 receptor stimulation.

Desensitized D2 autoreceptors are resistant to trafficking.

Dendritic release of dopamine activates dopamine D2 autoreceptors, which are inhibitory G protein-coupled receptors (GPCRs), to decrease the excitability of dopamine neurons. This study used tagged D2 receptors to identify the localization and distribution of these receptors in living midbrain dopamine neurons. GFP-tagged D2 receptors were found to be unevenly clustered on the soma and dendrites of dopamine neurons within the substantia nigra pars compacta (SNc). Physiological signaling and desensitization of the tagged receptors were not different from wild type receptors. Unexpectedly, upon desensitization the tagged D2 receptors were not internalized. When tagged D2 receptors were expressed in locus coeruleus neurons, a desensitizing protocol induced significant internalization. Likewise, when tagged µ-opioid receptors were expressed in dopamine neurons they too were internalized. The distribution and lack of agonist-induced internalization of D2 receptors on dopamine neurons indicate a purposefully regulated localization of these receptors.

A Lack of Serotonin 1B Autoreceptors Results in Decreased Anxiety and Depression-Related Behaviors.

The effects of serotonin (5-HT) on anxiety and depression are mediated by a number of 5-HT receptors, including autoreceptors that act to inhibit 5-HT release. While the majority of anxiety and depression-related research has focused on the 5-HT1A receptor, the 5-HT1B receptor has a lesser known role in modulating emotional behavior. 5-HT1B receptors are inhibitory GPCRs located on the presynaptic terminal of both serotonin and non-serotonin neurons, where they act to inhibit neurotransmitter release. The autoreceptor population located on the axon terminals of 5-HT neurons is a difficult population to study due to their diffuse localization throughout the brain that overlaps with 5-HT1B heteroreceptors (receptors located on non-serotonergic neurons). In order to study the contribution of 5-HT1B autoreceptors to anxiety and depression-related behaviors, we developed a genetic mouse model that allows for selective ablation of 5-HT1B autoreceptors. Mice lacking 5-HT1B autoreceptors displayed the expected increases in extracellular serotonin levels in the ventral hippocampus following administration of a selective serotonin reuptake inhibitor. In behavioral studies, they displayed decreased anxiety-like behavior in the open field and antidepressant-like effects in the forced swim and sucrose preference tests. These results suggest that strategies aimed at blocking 5-HT1B autoreceptors may be useful for the treatment of anxiety and depression.

Dopamine D3 receptors expressed by all mesencephalic dopamine neurons.

A polyclonal antibody was generated using synthetic peptides designed in a specific sequence of the rat D(3) receptor (D(3)R). Using transfected cells expressing recombinant D(3)R, but not D(2) receptor, this antibody labeled 45-80 kDa species in Western blot analysis, immunoprecipitated a soluble fraction of [(125)I]iodosulpride binding, and generated immunofluorescence, mainly in the cytoplasmic perinuclear region of the cells. In rat brain, the distribution of immunoreactivity matched that of D(3)R binding, revealed using [(125)I]R(+)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino] tetralin ([(125)I]7-trans-OH-PIPAT), with dense signals in the islands of Calleja and mammillary bodies, and moderate to low signals in the shell of nucleus accumbens (AccSh), frontoparietal cortex, substantia nigra (SN), ventral tegmental area (VTA) and lobules 9 and 10 of the cerebellum. Very low or no signals could be detected in other rat brain regions, including dorsal striatum, or in D(3)R-deficient mouse brain. Labeling of perikarya of AccSh and SN/VTA appeared with a characteristic punctuate distribution, mostly at the plasma membrane where it was not associated with synaptic boutons, as revealed by synaptophysin immunoreactivity. In SN/VTA, D(3)R immunoreactivity was found on afferent terminals, arising from AccSh, in which destruction of intrinsic neurons by kainate infusions produced a loss of D(3)R binding in both AccSh and SN/VTA. D(3)R-immunoreactivity was also found in all tyrosine hydroxylase (TH)-positive neurons observed in SN, VTA and A8 retrorubral fields, where it could represent D(3) autoreceptors controlling dopamine neuron activities, in agreement with the elevated dopamine extracellular levels in projection areas of these neurons found in D(3)R-deficient mice.

Analysis of structure and expression of the Xenopus thyroid hormone receptor-beta gene to explain its autoinduction.

Transcription of both Xenopus thyroid hormone receptor (TR) genes, xTR alpha and -beta, is strongly up-regulated by their own ligand T3 during natural or T3-induced metamorphosis of tadpoles and in some Xenopus cell lines. To explain this autoinduction, we analyzed the sequence of 1.6 kilobases of xTR beta promoter for putative T3-responsive elements. Two direct repeat +4 AGGTCA hexamer motifs (DR+4), an imperfect distal (-793/-778) and a perfect proximal (-5/11) site, a DR+1 site, and some possible half-sites were located in the 1.6-kilobase promoter. Transfection of Xenopus XTC-2 cells (which express xTR alpha and -beta) and XL-2 cells (which predominantly express TR alpha) with chloramphenicol acetyltransferase reporter constructs of deletion mutants and promoter fragments showed that the distal and proximal DR+4 sites responded to T3, although other flanking sequences may also play a role. The thyroid hormone-responsive element half-site present as DR+1 in the up-stream sequence at -1260/-950, when cloned in front of a heterologous promoter, functions independently. T3 enhanced transcription from the two DR+4-containing fragments when present together by only 2- to 3-fold due to a high basal activity. Overexpression of unliganded xTR alpha and xTR beta in XTC-2 cells repressed basal activity, which was then enhanced 7- to 4-fold by T3, respectively; with XL-2 cells cotransfected with xTR beta, T3 inducibility increased to 16-fold. Electrophoretic mobility shift assays with recombinant Xenopus TR alpha, TR beta, retinoid-X receptor-alpha (RXR alpha) and RXR gamma proteins showed that TR-RXR heterodimers, but not TR or RXR monomers or homodimers, strongly bound the natural and synthetic distal and proximal DR+4 elements in a ligand-independent manner. TR/RXR heterodimers exhibited the highest binding affinity for a 28-mer oligonucleotide probe for the -5/11 proximal DR+4 site, with only slight binding to DR+1 (retinoid-X-responsive element-like) site. The xTR beta promoter binding to XTC-2 cell nuclear extract suggested the in vivo relevance of the findings with recombinant TR/RXR heterodimers. It is concluded that xTR alpha and -beta proteins are capable of regulating the expression of xTR beta gene, which can explain its autoinduction seen during T3-induced metamorphosis.

[Lack of association between the G681C polimorphism in the 5-HT1D(beta) autoreceptor gene and schizophrenia].

A major role of the serotonergic system has been hypothesized in the pathogenesis of schizophrenia, mostly based on the evidence of action of atypical antipsychotics. Disturbances of serotonergic pathways have been implicated in the etiology of schizophrenia. The aim of this study was to investigate the association between schizophrenia and the G861C polymorphism in the 5-HT1D(beta) autoreceptor gene. There was conducted a case-control analysis in a sample of 196 schizophrenic patients and 143 gender, age and ethnic matched controls. No statistically differences were found in allelic or genotypic distributions between cases and controls. Thus, the results do not support an association of the G861C polymorphism in the 5-HT1D(beta) autoreceptor gene with schizophrenia in the studied sample.

Is the 5-HT(1Dbeta) receptor gene implicated in the pathogenesis of obsessive-compulsive disorder?

OBJECTIVE: Obsessive-compulsive disorder (OCD) is a psychiatric condition for which strong evidence of a genetic component and serotonergic system involvement exists. Recent studies have shown that sumatriptan, a selective ligand of the serotonin (5-HT)(1Dbeta) autoreceptor, modifies OCD symptoms. The aim of this study was to investigate the presence of linkage disequilibrium between the 5-HT(1Dbeta) receptor gene, which has a variant caused by a silent G to C substitution at nucleotide 861 of the coding region, and OCD. METHOD: DNA was collected from 67 probands who met DSM-IV criteria for OCD and from their living parents or siblings. Transmission Disequilibrium Test/sib-Transmission Disequilibrium Test analyses were then conducted with the DNA data. RESULTS: Thirty-two families were informative for the analysis, which showed a preferential transmission of the G allele to the affected subjects. CONCLUSIONS: If the results are confirmed, there may be important implications for the 5-HT(1Dbeta) receptor gene in the pathogenesis and treatment of OCD.

Ovarian steroid regulation of serotonin-1A autoreceptor messenger RNA expression in the dorsal raphe of rhesus macaques.

It is widely hypothesized that ovarian steroids act on serotonin neurons to modulate mood and alter neuroendocrine function in women. However, information is needed on the molecular consequences of estrogen and progesterone action in serotonin neurons. This study examined the effect of estrogen, with and without progesterone, on the expression of messenger RNA for the serotonin-1A autoreceptor in monkeys using in situ hybridization and a 432-bp serotonin-1A probe generated with polymerase chain reaction. Monkeys were spayed/ovariectomized (control; n=4), estrogen treated (28 days, n=4) and estrogen+progesterone treated (14 days estrogen+14 days estrogen+progesterone, n=4). Perfusion-fixed midbrain sections containing the dorsal raphe (10 microm) were hybridized at 60 degrees C with 35S antisense complementary RNA. After a final wash in 0.1 x standard saline citrate at 70 degrees C, sections were apposed to betamax film for four days and then emulsion fixed. Adjacent sections were immunostained for serotonin to confirm the location of the dorsal raphe. Densitometric analysis of autoradiographs with gray level thresholding was performed at five levels of the dorsal raphe. The number of pixels exceeding background in defined areas was obtained (pixel number), as well as the mean optical density. In the estrogen- and the estrogen+progesterone-treated groups compared to the control group, there was a 38% and 43% decrease in serotonin-1A messenger RNA signal, respectively, represented by pixel number (P<0.05). Mean optical density for serotonin-1A was significantly decreased by estrogen treatment (21%; P<0.05) and then further decreased with the addition of progesterone treatment (45%; P<0.01). Also, the number of positive cells and the grains/cell were counted. There were significantly fewer serotonin-1A messenger RNA-positive cells in the serotonergic neurons of the dorsal raphe in estrogen- and estrogen+progesterone-treated groups (P<0.001) than controls. There were significantly lower single-cell levels of serotonin-1A messenger RNA in serotonergic neurons of the dorsal raphe only in the estrogen+progesterone-treated group (P<0.05). These results suggest that estrogen reduces serotonin-1A gene expression and that the addition of progesterone further reduces serotonin-1A gene expression in non-human primates. If the changes in gene expression are manifested by alterations in protein expression, then, together, these actions of estrogen and progesterone could increase serotonin neurotransmission, thereby elevating mood and/or altering neuroendocrine functions.

Antisense oligodeoxynucleotides against the muscarinic m2, but not m4, receptor supports its role as autoreceptors in the rat hippocampus.

Antisense oligodeoxynucleotides against muscarinic m2 and m4 receptors were used to investigate the role of these receptor subtypes as negative autoreceptors in the regulation of acetylcholine (ACh) release in the rat hippocampus. Following the continuous infusion of antisenses into the third ventricle (1 microgram microliter-1 h-1, 3 days), 3H-AF-DX 384/muscarinic M2-like binding was significantly decreased in the medial septum by the antisense against the m2 receptor whereas M2-like binding in the dorsal striatum was decreased by the antisense against the m4 receptor. In contrast, 3H-pirenzepine/muscarinic M1-like binding was unaffected by either antisense treatment in any of the brain areas investigated. When perfused into the hippocampus via a dialysis probe, the purported muscarinic M2 receptor antagonist AF-DX 384 (100 nM) increased hippocampal ACh release in freely moving rats. This effect of AF-DX 384 was significantly attenuated by the m2, but not the m4, receptor antisense treatment. Hippocampal choline acetyltransferase activity was not affected by either antisense treatments. Taken together, these results suggest that the molecularly defined muscarinic m2 receptor regulates hippocampal ACh release by acting as a negative autoreceptor. In contrast, the molecularly defined m4 receptor is unlikely to be directly involved in the negative regulation of ACh release in the rat hippocampus. Therefore, inhibiting muscarinic m2 receptor function may be an alternative approach to regulate the release of ACh in neurodegenerative diseases associated with impaired cholinergic functions.

Extracellular serotonin in the prefrontal cortex is limited through terminal 5-HT(1B) autoreceptors: a microdialysis study in knockout mice.

RATIONALE: Serotonin (5-HT) autoreceptors regulate extracellular 5-HT levels and have been suggested to limit the effects of acute treatment with selective serotonin reuptake inhibitors (SSRIs). OBJECTIVES: The role of terminal 5-HT(1B) autoreceptors was assessed by comparing the effects of a SSRI on extracellular 5-HT in wild-type and 5-HT(1B) receptor knockout (KO) mice and by using a 5-HT(1B) receptor antagonist. Since systemic SSRI administration also activates somatodendritic 5-HT(1A) autoreceptors, a SSRI was administered locally to study the role of terminal 5-HT(1B) autoreceptors. METHODS: In vivo microdialysis in wild-type and 5-HT(1B) receptor KO mice was used to study the effects of the 5-HT(1B) receptor agonist CP93129 (1 micro M), the SSRI fluvoxamine (0.3 micro M and 1.0 micro M) and the 5-HT(1B) receptor antagonist NAS-181 (1 micro M) on extracellular 5-HT in the medial prefrontal cortex. RESULTS: The 5-HT increase induced by local SSRI administration was augmented in 5-HT(1B) KO mice relative to wild-type mice and was augmented by simultaneous administration of a 5-HT(1B) receptor antagonist in the latter genotype. Basal 5-HT levels did not differ between the two genotypes. Activation of 5-HT(1B) receptors by CP93129 decreased extracellular 5-HT, whereas 5-HT levels in wild-type mice were not affected by the 5-HT(1B) receptor antagonist NAS-181. In 5-HT(1B) KO mice, NAS-181 did not affect extracellular 5-HT and did not further increase the effect of fluvoxamine, showing that NAS-181 is a selective 5-HT(1B) receptor antagonist. The greater increase in 5-HT levels following combined administration of a SSRI with NAS-181 in wild-type mice, relative to 5-HT(1B) KO mice, suggests possible adaptive changes in the KO mice. CONCLUSIONS: The present study shows that terminal 5-HT(1B) autoreceptors play a significant role in the regulation of 5-HT release in the prefrontal cortex.

The effect of imipramine on the amount of mRNA coding for rat dopamine D2 autoreceptors.

Several reports have investigated the possibility that chronic antidepressant treatment alters dopamine autoreceptors. Since radioligand binding studies do not differentiate between presynaptic and postsynaptic dopamine D2 receptors in the rat forebrain, we used the in situ hybridization technique to measure the amount of mRNA coding for dopamine D2 autoreceptors in the dopaminergic cell bodies. The amount of mRNA coding for dopamine D2 autoreceptors in the rat mesencephalon was analyzed following acute and repeated treatment with imipramine, the most widely used antidepressant drug. No significant changes in the amount of mRNA were observed in the substantia nigra of the rat, after acute or repeated treatment with imipramine. In the ventral tegmental area repeated treatment with imipramine (14 days, twice a day) increased the amount of dopamine D2 autoreceptor mRNA in the lateral part of this brain region (containing nucleus paranigralis and n. parabrachialis pigmentosus), without there being any significant changes in the more medial part (n. interfascicularis and n. linearis). The increase in the amount of dopamine D2 autoreceptor mRNA in the ventral tegmental area started to be significant 72 h after acute imipramine. Moreover, this increase was also observed after 14 drug-free days following the acute administration of the drug. The results indicate the different sensitivity of neurons synthesizing dopamine autoreceptors for imipramine. Another interesting finding is the observation that acute treatment with imipramine seems to be sufficient to trigger changes as a function of time regardless of whether imipramine is again administered, providing a possible explanation for the delayed therapeutic effect of the drug.

Improved efficacy of fluoxetine in increasing hippocampal 5-hydroxytryptamine outflow in 5-HT(1B) receptor knock-out mice.

To test for the contribution of the 5-HT(1B) receptor subtype in mediating the effects of fluoxetine, a selective serotonin reuptake inhibitor (SSRI), we used intracerebral in vivo microdialysis in awake, freely moving 5-HT(1B) receptor knock-out mice. We show that a single systemic administration of fluoxetine (1, 5 or 10 mg/kg, i.p.) increased extracellular serotonin levels [5-HT](ext) in the ventral hippocampus and frontal cortex of wild-type and mutant mice. However, in the ventral hippocampus, fluoxetine, at the three doses studied, induced a larger increase in [5-HT](ext) in knock-out than in wild-type mice. In the frontal cortex, the effect of fluoxetine did not differ between the two genotypes. The region-dependent response to fluoxetine described here in mutants confirms data we recently reported for another SSRI, paroxetine. These data suggest that 5-HT(1B) autoreceptors limit the effects of selective serotonin reuptake inhibitors on dialysate 5-HT levels at serotonergic nerve terminals located mainly in the ventral hippocampus. Alternative mechanisms, e.g., changes in 5-HT transporter and/or 5-HT(1A) receptor density in 5-HT(1B) receptor knock-out mice could also explain these findings.