Tryptophan Hydroxylase Immunoreactivity Research Articles

Enhanced responsiveness to hypoxic panicogenic challenge in female rats in late diestrus is suppressed by short-term, low-dose fluoxetine: Involvement of the dorsal raphe nucleus and the dorsal periaqueductal gray.

Acute hypoxia, which is panicogenic in humans, also evokes panic-like behavior in male rats. Panic disorder is more common in women and susceptibility increases during the premenstrual phase of the cycle. We here investigated for the first time the impact of hypoxia on the expression of panic-like escape behavior by female rats and its relationship with the estrous cycle. We also evaluated functional activation of the midbrain panic circuitry in response to this panicogenic stimulus and whether short-term, low-dose fluoxetine treatment inhibits the hyper-responsiveness of females in late diestrus. Male and female Sprague Dawley rats were exposed to 7% O2. Females in late diestrus were also tested after short-term treatment with fluoxetine (1.75 or 10 mg/kg, i.p.). Brains were harvested and processed for c-Fos and tryptophan hydroxylase immunoreactivity in the periaqueductal gray matter (PAG) and dorsal raphe nucleus (DR). Acute hypoxia evoked escape in both sexes. Overall, females were more responsive than males and this is clearer in late diestrus phase. In both sexes, hypoxia induced functional activation (c-Fos expression) in non-serotonergic cells in the lateral wings of the DR and dorsomedial PAG, which was greater in late diestrus than proestrus (lowest behavioral response to hypoxia). Increased responding in late diestrus (behavioral and cellular levels) was prevented by 1.75, but not 10 mg/kg fluoxetine. The response of female rats to acute hypoxia models panic behavior in women. Low-dose fluoxetine administered in the premenstrual phase deserves further attention for management of panic disorders in women.

Lethal avian influenza A (H5N1) virus replicates in pontomedullary chemosensitive neurons and depresses hypercapnic ventilatory response in mice.

The highly pathogenic H5N1 (HK483) viral infection causes a depressed hypercapnic ventilatory response (dHCVR, 20%↓) at 2 days postinfection (dpi) and death at 7 dpi in mice, but the relevant mechanisms are not fully understood. Glomus cells in the carotid body and catecholaminergic neurons in locus coeruleus (LC), neurokinin 1 receptor (NK1R)-expressing neurons in the retrotrapezoid nucleus (RTN), and serotonergic neurons in the raphe are chemosensitive and responsible for HCVR. We asked whether the dHCVR became worse over the infection period with viral replication in these cells/neurons. Mice intranasally inoculated with saline or the HK483 virus were exposed to hypercapnia for 5 min at 0, 2, 4, or 6 dpi, followed by immunohistochemistry to determine the expression of nucleoprotein of H5N1 influenza A (NP) alone and coupled with 1) tyrosine hydroxylase (TH) in the carotid body and LC, 2) NK1R in the RTN, and 3) tryptophan hydroxylase (TPH) in the raphe. HK483 viral infection blunted HCVR by ∼20, 50, and 65% at 2, 4, and 6 dpi. The NP was observed in the pontomedullary respiratory-related nuclei (but not in the carotid body) at 4 and 6 dpi, especially in 20% of RTN NK1R, 35% of LC TH, and ∼10% raphe TPH neurons. The infection significantly reduced the local NK1R or TPH immunoreactivity and population of neurons expressing NK1R or TPH. We conclude that the HK483 virus infects the pontomedullary respiratory nuclei, particularly chemosensitive neurons in the RTN, LC, and raphe, contributing to the severe depression of HCVR and respiratory failure at 6 dpi. NEW & NOTEWORTHY The H5N1 virus infection is lethal due to respiratory failure, but the relevant mechanisms remain unclear. In this study, we demonstrated a gradual diminution of hypercapnic ventilatory response to a degree, leading to respiratory failure over a 6-day infection. Death was associated with viral replication in the pontomedullary respiratory-related nuclei, especially the central chemosensitive neurons. These results not only provide insight into the mechanisms of the lethality of H5N1 viral infection but also offer clues in the development of corresponding treatments to minimize and prevent respiratory failure.

Distribution of Tryptophan Hydroxylase Immunoreactivity in the Spiral Ganglion Neurons of Mouse Cochlea.

Serotonin, also known as 5-hydroxytryptamine, is an important neurotransmitter and neuromodulator in the nervous system. Spiral ganglion neurons (SGNs) are the first station of the peripheral afferent auditory nervous system. We have previously demonstrated positive serotonin immunoreactivity in mouse SGNs. Whether serotonin in the SGNs is absorbed from the circumstances, or is synthesized by the neurons themselves is unknown. The purpose of this study was to find out the source of serotonin in the SGNs. Tryptophan hydroxylase is the rate-limiting enzyme in the process of serotonin biosynthesis. We detected tryptophan hydroxylase in the mouse SGNs using immunohistochemistry. The positive immunostaining of tryptophan hydroxylase was observed in the cytoplasm of SGNs. The cytoplasm of the SGNs was not get stained in the negative control group. Our results suggest that SGNs can synthesize serotonin themselves, and they may serve as an additional source of serotonin in the cochlea. The SGNs are serotonergic neurons. The receptors of serotonin on the membrane of SGNs interact with serotonin as auto-receptors. Serotonin in these neurons plays its physiological role in an autocrine/paracrine way.

Comparison of Peroxidase‐Anti‐Peroxidase and Avidin‐Biotin‐Peroxidase Complex (ABC) Immunohistochemical Staining Procedures for Tryptophan Hydroxylase in Rat Brain

The neurotransmitter serotonin (5‐hydroxytryptamine; 5‐HT) plays a key role in sleep, feeding behavior, motor activity and, importantly, mood and the mechanism of action of antidepressant drugs. Immunohistochemistry for tryptophan hydroxylase (TpOH), the synthetic enzyme for 5‐HT, can be used to identify 5‐HT neurons in the central nervous system. As part of a project examining 5‐HT in brain function in the rat, we compared 2 indirect methods of visualizing primary antibodies against TpOH, a peroxidase‐anti‐peroxidase (PAP) method and the more sensitive avidin‐biotin‐peroxidase (ABC) procedure.A cryostat microtome was used to cut 40 micron sections from brains from rats transcardially perfused with 0.9% saline and 4% p‐formaldehyde in 0.1 M phosphate buffer (PB). Brains were post‐fixed 24 h in p‐formaldehyde and cryoprotected in 20% sucrose in 0.1 M PB. Sections were washed in phosphate‐buffered saline (PBS) before and between all steps. Tissues were treated with 0.4% H2O2 in methanol for 20 min to quench endogenous peroxidase activity and 1% sodium borohydride in 0.1 M PB for 30 min to enhance permeability of the tissue and improve staining quality. Sections were then treated with 1% normal donkey (PAP method) or horse (ABC method) serum in PBS with 0.2% Triton X‐100 detergent for 1 h and then 0.8% bovine serum albumin in PBS for 40 min. Tissues were incubated for 48 h in mouse monoclonal anti‐TpOH antibody (Sigma, 1:1000) in PBS with 0.3% Triton X‐100 and serum. Some sections were incubated without the primary antibody (omit negative controls). For the PAP protocol, tissues were treated with donkey anti‐mouse antibody (Jackson; 1:100) and then mouse PAP (Jackson; 1:400), both in PBS with 0.3% Triton X‐100 for 1 h. For the ABC protocol, tissues were treated with biotinylated horse anti‐mouse antibody and then avidin‐biotin‐peroxidase complex (Vector Elite kit), both in PBS with 0.3% Triton X‐100 and serum for 1 h. All sections were reacted with 0.0005% diaminobenzidine with 0.0001% H2O2 for 45 – 120 sec and mounted on gelatin‐coated slides that were coverslipped with DPX. Stained and negative control sections from both methods were examined using standard light microscopy.Staining using the PAP method was faint, homogenous and only slightly darker than negative controls. Individual neurons could not be identified. Increasing concentrations (1:500, 1:250, 1:100) of anti‐TpOH antibody had no effect on specific labeling but slightly increased background staining. This was surprising because the exact same reagents and PAP procedure has produced outstanding staining of tyrosine hydroxylase in brainstem catecholamine neurons (e.g., Muma et al., Exp. Neurology, 168:135–143, 2001). In contrast, labeling of TpOH using the ABC method was excellent. TpOH immunoreactivity was highly visible in the somata and primary dendrites of neurons in the dorsal and median raphe nuclei. These results add to and amplify the enhanced staining quality of the ABC procedure over the PAP method in formaldehyde‐fixed rat brain sections.Support or Funding InformationSupported by the School of Biological Sciences and the Department of Psychology, Illinois State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating Tryptophan Hydroxylase Immunoreactivity in the Dorsal Raphe of Rats Exposed to Early Adversity

Prior studies link anxiety to alterations in serotonergic neurotransmission. Tao and Menard (unpublished data) found that female rats exposed to intermittent physical stress during early adolescence display greater levels of anxiety-like behaviours when tested as adults in social interaction tests. Interestingly, these behavioural changes were associated with changes in serotonin (5-HT) fiber density in the medial prefrontal cortex. Whether these changes in 5-HT fibre density are accompanied by changes in the number of 5-HT-producing cells in the dorsal raphe is not known. To examine this research question, tryptophan-hydroxylase (TPH) immunoreactivity (-IR) was used to label 5-HT cells in the dorsal raphe. Half the animals from the original adolescent stress study were exposed to IPS, and the remaining animals were handled only, serving as the control (CON) group. In adulthood, half of each of those groups were tested in the SI test. The remaining animals were not behaviourally tested, serving as home-cage controls (HCC). A two-factor analysis of variance will be performed with Treatment (IPS vs CON) and Testing (SI vs HCC) as the two factors. It is expected that, regardless of Testing, IPS rats will display higher levels of TPH-IR in the dorsal raphe than no-stress controls. The number of TPH-IR-positive cells will also be correlated with archival behavioural data. It is expected that that higher levels of TPH-IR will be associated with higher levels of anxiety-like behaviour. This study provides insights into the role that early adversity plays in shaping the structure and function of the brain’s serotonergenic system.

Anti-Apoptotic Protein Bcl-xL Expression in the Midbrain Raphe Region Is Sensitive to Stress and Glucocorticoids.

Anti-apoptotic proteins are suggested to be important for the normal health of neurons and synapses as well as for resilience to stress. In order to determine whether stressful events may influence the expression of anti-apoptotic protein Bcl-xL in the midbrain and specifically in the midbrain serotonergic (5-HT) neurons involved in neurobehavioral responses to adverse stimuli, adult male rats were subjected to short-term or chronic forced swim stress. A short-term stress rapidly increased the midbrain bcl-xl mRNA levels and significantly elevated Bcl-xL immunoreactivity in the midbrain 5-HT cells. Stress-induced increase in glucocorticoid secretion was implicated in the observed effect. The levels of bcl-xl mRNA were decreased after stress when glucocorticoid elevation was inhibited by metyrapone (MET, 150 mg/kg), and this decrease was attenuated by glucocorticoid replacement with dexamethasone (DEX; 0.2 mg/kg). Both short-term stress and acute DEX administration, in parallel with Bcl-xL, caused a significant increase in tph2 mRNA levels and slightly enhanced tryptophan hydroxylase immunoreactivity in the midbrain. The increasing effect on the bcl-xl expression was specific to the short-term stress. Forced swim repeated daily for 2 weeks led to a decrease in bcl-xl mRNA in the midbrain without any effects on the Bcl-xL protein expression in the 5-HT neurons. In chronically stressed animals, an increase in tph2 gene expression was not associated with any changes in tryptophan hydroxylase protein levels. Our findings are the first to demonstrate that both short-term stress and acute glucocorticoid exposures induce Bcl-xL protein expression in the midbrain 5-HT neurons concomitantly with the activation of the 5-HT synthesis pathway in these neurons.

5-HT1A-receptor agonist modified amygdala activity and amygdala-associated social behavior in a valproate-induced rat autism model

Accumulating evidence suggests that dysfunction of the amygdala is related to abnormal fear processing, anxiety, and social behaviors noted in autistic spectrum disorders (ASDs). In addition, studies have shown that disrupted brain serotonin homeostasis is linked to ASD. With a valproate (VPA)-induced rat ASD model, we investigated the possible role of amygdala serotonin homeostasis in autistic phenotypes and further explored the underlying mechanism. We first discovered that the distribution of tryptophan hydroxylase immunoreactivity in the caudal raphe system was modulated on postnatal day (PD) 28 of the VPA-exposed offspring. Then, we found a significantly higher serotonin transporter availability in the amygdala of the VPA-exposed offspring on PD 56 by using single photon emission computed tomography and computed tomography co-registration following injection of (123)I-labeled 2-((2-(dimethylamino)methyl)phenyl)thio)-5-iodophenylamine((123)I[ADAM]). Furthermore, treatment with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, increased social interaction and improved fear memory extinction in the VPA-exposed offspring. 8-OH-DPAT treatment also reversed the characteristics of miniature excitatory post-synaptic currents as well as paired pulse facilitation observed in lateral amygdala slices. These results provided further evidence to support the role of the amygdala in characteristic behavioral changes in the rat ASD model. The serotonergic projections that modulate the amygdala function might play a certain role in the development and treatment of behavioral symptoms exhibited in individuals with ASD.

Effect of fluoxetine on the expression of tryptophan hydroxylase and 14-3-3 protein in the dorsal raphe nucleus and hippocampus of rat

The serotonergic system is one of the major systems targeted in the pharmacological treatment of mood disorders including depression. Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been reported to induce the expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of serotonin. The 14-3-3 protein family not only activates neuronal enzymes, including TPH, but also plays a role in a wide variety of cell signaling. The aim of the present study was to determine whether fluoxetine regulates both the interaction of TPH and 14-3-3 proteins as well as the increase of those proteins in the dorsal raphe nucleus and the hippocampus. Sprague-Dawley rats were administered fluoxetine or vehicle for 5 and 14 days and sacrificed at 5 and 14 days after initial treatment. The intensity of immunoreactivity for TPH and 14-3-3 proteins in the dorsal raphe nucleus of the midbrain and in the hippocampus was measured, and the colocalization of both proteins was observed with double-labeling immunofluorescence. At 5 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein increased in both the dorsal raphe nucleus and the hippocampus, while that of TPH did not change in either region. In addition, at 14 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein significantly increased in both the dorsal raphe nucleus and hippocampus, while that of TPH showed few changes in either region. Colocalization of TPH and 14-3-3 proteins was observed in the cell bodies of dorsal raphe nucleus, whereas it was not observed in the hippocampus. These results suggest that the time-dependent regulation of 14-3-3 protein may be one of the various factors associated with delayed pharmacological effects of SSRIs.

The comparative effects of environmental enrichment with exercise and serotonin transporter blockade on serotonergic neurons in the dorsal raphe nucleus

We have previously reported that inhibition of the serotonin transporter (SERT) by selective serotonin reuptake inhibitor (SSRI) fluoxetine significantly reduces the number of tryptophan hydroxylase (TPH)-positive cells in the dorsal raphe nucleus (DRN). We have been interested in exploring whether this SSRI-induced change in TPH might be modified by housing in an enriched environment. Like SSRI antidepressants, environmental enrichment (EE) and physical exercise have been found to have efficacy in the prevention and alleviation of depression. We postulated that EE with exercise and SERT inhibition would similarly affect TPH regulation and that EE with exercise might modify the effect of fluoxetine on TPH. Three week old male Sprague-Dawley rats were housed in either a standard cage (SE) or an enriched environment (EE). SE animals were singly housed with no access to enrichment objects. EE animals were group housed and were provided with various enrichment objects (e.g. running wheel) that were changed and rearranged regularly. Nine weeks after the experiment began, the rats were randomly assigned to one of four treatment groups: (1) SE control; (2) SE fluoxetine; (3) EE control; or (4) EE fluoxetine. Fluoxetine (5 mg/kg/day) was placed in the drinking water. Sections of DRN were processed for TPH immunohistochemistry. The number of TPH-positive cells was determined by blinded, manual counting. Results were analyzed by analysis of variance (ANOVA) followed by post-hoc Tukey tests. Significance was set at P < 0.05. For animals housed in a standard environment, fluoxetine induced a significant 29% reduction in the number of TPH-immunoreactive cells in the DRN. A similar reduction in TPH immunoreactivity was observed in animals that were housed in an enriched environment but not exposed to fluoxetine (39%). The number of TPH-positive cells in the DRN for animals housed in an enriched environment and exposed to fluoxetine was not significantly different than animals housed in an enriched environment and not exposed to fluoxetine. The reduction of TPH immunoreactivity in the DRN by EE with exercise suggests that a modified housing environment and voluntary exercise affects regulation of TPH, possibly via a mechanism similar to that of SERT inhibitors. This downregulation of serotonin biosynthesis by fluoxetine and EE with exercise may ultimately play a role in the therapeutic action of both interventions.

Effect of Selective Serotonin Reuptake Inhibitors via 5-HT1A Receptors on L-DOPA-Induced Rotational Behavior in a Hemiparkinsonian Rat Model

l-Dihydroxyphenylalanine (l-DOPA) is considered the gold standard for the treatment of Parkinson’s disease (PD). However, long-term administration of l-DOPA can induce abnormal side effects. On the other hand, selective serotonin reuptake inhibitors (SSRIs) including fluoxetine have gained tremendous popularity in the treatment of depression in PD. SSRIs are thought to influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic networks, which are complex and not yet fully understand. In this study, intranigral injection of 6-hydroxydopamine (6-OHDA) in rats caused a significant loss of tyrosine hydroxylase immunoreactivity in the striatum and substantia nigra. However, tryptophan hydroxylase immunoreactivity of the striatum and raphe nucleus was unaffected by 6-OHDA. Immunohistochemical analysis reveal that the serotonergic system was unaffected by the injection of 6-OHDA. We demonstrated also that pre-treatment with fluoxetine significantly suppressed l-DOPA-induced rotational behavior. Additionally, fluoxetine suppressed l-DOPA-induced ERK1/2 and histone H3 phosphorylation. These effects of fluoxetine were abolished by pre-treatment with WAY 100135, a 5-HT1A antagonist. These results suggest that fluoxetine may influence motor function in PD via pharmacological modification of interactions between serotonergic and dopaminergic neuronal networks.

Activity-Dependent Expression of Lmx1b Regulates Specification of Serotonergic Neurons Modulating Swimming Behavior

Genetic programs, environmental factors, and electrical activity interact to drive the maturation of the brain. Although the cascade of transcription factors that leads to specification of the serotonergic phenotype has been well characterized, its interactions with electrical activity are not known. Here we show that spontaneous calcium spike activity in the hindbrain of developing Xenopus laevis larvae modulates the specification of serotonergic neurons via regulation of expression of the Lmx1b transcription factor. Activity acts downstream of Nkx2.2 but upstream of Lmx1b, leading to regulation of the serotonergic phenotype. Using global manipulation of activity and targeted alteration of Lmx1b expression, we also demonstrate that changes in the number of serotonergic neurons change larval swimming behavior. The results link activity-dependent regulation of a transcription factor to transmitter specification and altered behavior.

Postnatal changes in tryptophan hydroxylase and serotonin transporter immunoreactivity in multiple brainstem nuclei of the rat: Implications for a sensitive period

Previously, we found that the brainstem neuronal network in normal rats undergoes abrupt neurochemical, metabolic, and physiological changes around postnatal days (P) 12-13, a critical period when the animal's response to hypoxia is also the weakest. This has special implications for sudden infant death syndrome (SIDS), insofar as seemingly normal infants succumb to SIDS when exposed to respiratory stressors (e.g., hypoxia) during a narrow postnatal window. Because an abnormal serotonergic system has recently been implicated in SIDS, we conducted a large-scale investigation of the 5-HT-synthesizing enzyme tryptophan hydroxylase (TPH) and serotonin transporter (SERT) with semiquantitative immunohistochemistry in multiple brainstem nuclei of normal rats aged P2-21. We found that 1) TPH and SERT immunoreactivity in neurons of raphé magnus, obscurus, and pallidus and SERT in the neuropil of the pre-Bötzinger complex, nucleus ambiguus, and retrotrapezoid nucleus were high at P2-11 but decreased markedly at P12 and plateaued thereafter until P21; 2) SERT labeling in neurons of the lateral paragigantocellular nucleus (LPGi) and parapyramidal region (pPy) was high at P2-9 but fell significantly at P10, followed by a gradual decline until P21; 3) TPH labeling in neurons of the ventrolateral medullary surface was stable except for a significant fall at P12; and 4) TPH and SERT immunoreactivity in a number of other nuclei was relatively stable from P2 to P21. Thus, multiple brainstem nuclei exhibited a significant decline in TPH and SERT immunoreactivity during the critical period, suggesting that such normal development can contribute to a narrow window of vulnerability in postnatal animals.

Immunohistochemical evidence for the presence of tryptophan hydroxylase in the brains of insects as revealed by sheep anti-tryptophan hydroxylase polyclonal antibody

Immediately following the discovery of tryptophan hydroxylase in Drosophila, we demonstrated the presence of tryptophan hydroxylase in the brain of the beetle Harmonia axyridis (Coleoptera: Coccinellidae). However, whether tryptophan hydroxylase is present in the brains of other insects is still a matter of discussion. In the current study, sheep anti-tryptophan hydroxylase polyclonal antibody has been applied to test for tryptophan hydroxylase immunoreactivity in a broader taxonomic range of insect brains, including holometabolous and hemimetabolous insects: one species each of Coleoptera, Hymenoptera, Diptera, and Blattaria, and two species of Lepidoptera. All species show consistent tryptophan hydroxylase immunoreactivity with distribution patterns matching that of serotonin. The immuno-positive results of such an antibody in brains from diverse orders of insects suggest that specific tryptophan hydroxylase responsible for central serotonin synthesis is probably present in the brains of all insects.

Single dose of MDMA causes extensive decrement of serotoninergic fibre density without blockage of the fast axonal transport in Dark Agouti rat brain and spinal cord

Prolonged neurotoxicity of the recreational drug, MDMA (3,4-methylenedioxymethamphetamine) on serotoninergic axon terminals has been suggested. The effect of a single (15 mg/kg) dose of intraperitoneally administered MDMA on serotoninergic fibre density, defined by tryptophan hydroxylase (TpH) and serotonin transporter (5-HTT) immunoreactivity, has been evaluated in the spinal cord and brain areas in Dark Agouti rats, 7 and 180 days after MDMA applications. Immunostaining for amyloid precursor protein (APP) has been performed to examine possible defects of the fast axonal transport, and 5-HTT mRNA expressions were quantified in neurones of medullary raphe nuclei. Seven days after MDMA treatment, a substantial decrease in the density of TpH-immunoreactive fibres was detectable in the frontal cortex, the caudate-putamen, the CA1 region of the hippocampus, and marked decreases were found in the spinal cord. These changes in TpH density showed a high correlation with 5-HTT densities. In contrast, APP-immunoreactive axonal bulbs were not detected in any of the brain regions studied. Seven days after MDMA administrations, significantly elevated 5-HTT mRNA expressions were found in the raphe pallidus and obscurus. Our results suggest that a single dose of MDMA elicits widespread depletion of TpH and 5-HTT immunoreactivity in serotoninergic axons without morphological sign of the blockage of the fast anterograde axonal transport. Our results do not support the notion of MDMA-induced axotomy of serotoninergic neurones. The up-regulation of 5-HTT mRNA expressions 1 week after MDMA injections might indicate the potential recovery of the serotonin system.

Serotonin Concentration, Synthesis, Cell Origin, and Targets in the Rat Caput Epididymis During Sexual Maturation and Variations Associated With Adult Mating Status: Morphological and Biochemical Studies

The caput epididymis of some mammals contains large quantities of serotonin whose origin, targets, and physiological variations have been poorly studied. We combined morphological and biochemical techniques to begin approaching these aspects of serotonin in the rat caput epididymis. Serotonin immunostaining was detected in mast, epithelial, and neuroendocrine cells. Epithelial cells displayed immunoreactivity to 5HT(1A), 5HT(2A,) and 5HT(3) serotonin receptors. Endothelial and mast cells labeled positive for 5HT(1B) serotonin receptors and spermatozoa displayed 5HT(2A) and 5HT(3) serotonin receptor immunoreactivity. Epithelial, endothelial, and mast cells stained positive for serotonin transporters. Only epithelial cells showed tryptophan hydroxylase immunoreactivity; this enzyme catalyzes the limiting step in the serotonin synthetic pathway. In addition, Western blot analyses of caput homogenates documented the presence of 2 protein bands ( approximately 51 kd and approximately 48 kd) that were immunoreactive for tryptophan hydroxylase. Chromatographic analyses documented the presence of tryptophan hydroxylase in the caput, and showed that both its activity and serotonin availability increased with sexual maturation and decreased following p-chlorophenylalanine treatment, an inhibitor of tryptophan hydroxylase activity. Interestingly, serotonin concentration and tryptophan hydroxylase activity tended to be higher in breeding males than in those with no mating experience. We think that these results support the existence of a local serotoninergic system in the rat caput epididymis that might regulate some aspects of male reproductive function.

Increased tryptophan hydroxylase immunoreactivity in the dorsal raphe nucleus of alcohol-dependent, depressed suicide subjects is restricted to the dorsal subnucleus

Considerable evidence suggests that alcoholics with co-occurring depressive disorder are at greater risk for developing psychosocial problems particularly suicidal behavior. Moreover, dysfunction in serotonin (5-HT) neurotransmission has been implicated in depression, suicide and alcoholism. In the present study, we measured the levels of tryptophan hydroxylase (TPH), the main synthetic enzyme of 5-HT synthesis, in specific nuclei of the dorsal raphe (DR) in depressed suicide victims with alcohol dependence and matched psychiatrically normal controls. TPH immunoreactivity (IR) was quantified in frozen tissue sections containing the DR from 8 suicide victims with a diagnosis of major depression and alcohol dependence, and 8 psychiatrically normal control subjects by using immunoautoradiographic methods. We found that the levels of TPH-IR were significantly increased by 46% in the dorsal subnucleus of the DR in depressed suicide victims with alcohol dependence when compared with controls. In contrast, TPH-IR levels did not significantly differ in the other DR subnuclei between depressed, alcoholic suicide subjects, and controls. Our results indicate that abnormalities in 5-HT biosynthesis in the brain of depressed alcoholic suicide subjects are restricted within distinct regions of the DR.

Electrophysiological Heterogeneity of Spinally Projecting Serotonergic and Nonserotonergic Neurons in the Rostral Ventromedial Medulla

This study examined the passive membrane and action potential properties of serotonergic and nonserotonergic neurons in the rostral ventromedial medulla (RVM) of the rat using whole cell patch-clamp recording techniques in the slice. Serotonergic neurons were identified by immunoreactivity for tryptophan hydroxylase (TrpH). Spinally projecting neurons were retrogradely labeled with 1'-dioactadecyl-3,3,3',3'-tetramethylindocarbodyanine perchlorate (DiI). Three types of neurons were identified within both spinally projecting serotonergic and nonserotonergic populations. Type 1 neurons exhibited irregular or sporadic spontaneous activity interspersed with periods of quiescence. Type 2 neurons were not spontaneously active and were additionally discriminated by a more negative resting membrane potential and a larger-amplitude action potential. Type 3 neurons fired repetitively without pause. Serotonergic neurons had a higher membrane resistance and greater action potential half-width than their nonserotonergic counterparts and rarely exhibited a fast afterhyperpolarization. Serotonergic type 3 neurons also fired more slowly and regularly than nonserotonergic type 3 neurons. Comparison of electrophysiological and immunohistochemical characteristics suggested that the smallest type 3 serotonergic neurons had an increased risk of immunohistochemical "misclassification" due to failure to detect TrpH, possibly due to more complete dialysis of intracellular contents during lengthy recordings. This risk was minimal for type 1 or 2 serotonergic neurons. The three different types of spinally projecting serotonergic neurons also differed markedly in their responsiveness to the mu opioid receptor agonist D-Ala2, NMePhe4, Gly5-ol]enkephalin. These results provide important new electrophysiological and pharmacological evidence for a significant heterogeneity among spinally projecting serotonergic RVM neurons. They may also provide a basis for resolving the controversy concerning the role of serotonergic RVM neurons in opioid analgesia.

Chronic stress, as well as acute stress, reduces BDNF mRNA expression in the rat hippocampus but less robustly

Daily restraint for 3 weeks was shown to atrophy dendrites of hippocampal pyramidal neurons in rats. Brain-derived neurotrophic factor (BDNF), which maintains neuronal survival and morphology, has been shown to decrease in response to acute stress. Plasma glucocorticoid (GC) and serotonergic projections from the raphe nuclei play major roles in reducing BDNF synthesis in the hippocampus. We investigated BDNF mRNA levels there, together with plasma GC levels, GC receptors in the hippocampus/hypothalamus and 5-HT synthesizing enzyme, tryptophan hydroxylase in the raphe nuclei, in animals chronically stressed for 1–3 weeks, using in situ hybridization and immunohistochemistry. In these animals, BDNF mRNA levels were significantly decreased in the hippocampus after 6 h of restraint, but the ability of restraint to reduce BDNF synthesis seemed less robust than that seen in acute stress models. HPA axis response to stress in these animals assessed by plasma GC levels was delayed and sustained, and the GC receptor in the paraventricular hypothalamic nucleus was increased at 1 week. Tryptophan hydroxylase immunoreactivity was increased in the median raphe nucleus at 2–3 weeks. Repetitive stress-induced reduction of BDNF may partly contribute to the neuronal atrophy/death and reduction of hippocampal volume observed both in animals and humans suffering chronic stress and/or depression.

More tryptophan hydroxylase in the brainstem dorsal raphe nucleus in depressed suicides

Deficient serotonin neurotransmission in suicide is indicated by reduced brainstem serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), fewer 5-HT 1A autoreceptors and reduced cortical serotonin transporter binding in suicide victims. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of 5-HT, and alterations in TPH could explain some of these findings. We sought to determine the amount of TPH immunoreactivity (TPH-IR) in the dorsal (DRN) and median (MRN) raphe nuclei in suicides and controls. Brainstems of suicide victims and controls ( n = 11 pairs) were collected at autopsy, matched for age, sex and postmortem interval, frozen and sectioned (20 μm). Immunoautoradiography, using an antibody to label TPH, was performed, slides exposed to film and autoradiograms quantified by a computer-based image analysis system. We examined sections every 1000 μm throughout the whole length of the nucleus, performing statistical analysis only on those subjects for whom the raphe was complete ( n = 8 pairs). TPH-IR (μCi/g) was higher in suicides than controls (S: 300.8 ± 70.8 vs. C: 259.6 ± 40.7, t = 2.57, df = 7, P = 0.04) in the dorsal raphe nucleus (DRN), and not different between suicides and controls (S: 251.3 ± 44.2 vs. C: 235.9 ± 27.4, t = 1.49, df = 7, P = 0.18) in the MRN. DRN TPH-IR was higher in male suicide victims (MS) compared to male controls (MC; MS: 318.4 ± 54.4 vs. MC: 271.9 ± 22.5, t = 2.66, df = 6, P = 0.03). The analysis of TPH-IR area and density at each DRN rostrocaudal levels showed higher area and density in suicides compared to controls in the rostral DRN and lower area and density in the caudal DRN. TPH-IR, an index of the amount of TPH enzyme, in the DRN is higher in depressed suicides. More TPH may be an upregulatory homeostatic response to impaired serotonin release or less autoreceptor activation. Alternatively, the serotonin impairment in suicide may be due to hypofunctional serotonin-synthesizing enzyme.

ϵ‐Sarcoglycan immunoreactivity and mRNA expression in mouse brain

Myoclonus dystonia (M-D) is a hereditary movement disorder caused by a maternally imprinted gene that is often associated with psychiatric symptoms. Most cases of M-D are believed to result from mutations of the epsilon-sarcoglycan protein. The neuroanatomical distribution of epsilon-sarcoglycan-like immunoreactivity in mouse was investigated by using an antiserum against the epsilon-sarcoglycan protein. The expression of epsilon-sarcoglycan mRNA was studied by a sensitive fluorescence in situ hybridization (FISH) method. Immunohistochemistry and FISH revealed a wide distribution of epsilon-sarcoglycan protein and mRNA throughout the mouse brain. High expression levels of epsilon-sarcoglycan mRNA and immunoreactivity were found in the mitral cell layer of the olfactory bulb, the Purkinje cell layer in cerebellum, and the monoaminergic neurons in the mouse midbrain. Immunohistochemistry revealed a similar distribution of epsilon-sarcoglycan protein. Double-labeling FISH showed colocalization of tyrosine hydroxylase and epsilon-sarcoglycan mRNAs within all the midbrain dopaminergic (DAergic) cell groups. By combining FISH with fluorescence immunohistochemistry, coexpression of epsilon-sarcoglycan mRNA and tryptophan hydroxylase immunoreactivity was found in the serotonergic (5-HTergic) neurons within the dorsal raphe nucleus. The distribution of epsilon-sarcoglycan in the mouse brain suggests that the symptom complex of M-D may be related to the effects of decreased epsilon-sarcoglycan activity on the development or function of monoaminergic neurons.