Postnatal developmental changes of tryptophan hydroxylase activity in serotonergic cell bodies and terminals of rat brain

Tryptophan hydroxylase (TPH) is the rate limiting enzyme in serotonin biosynthesis, so its development is very important to the functional maturation of the serotonergic neurons. In the present study, we examined changes of TPH activity in serotonergic cell bodies and terminals of rat brain during postnatal development. TPH activity reached its peak in the cell body regions at 24 days after birth, while the enzyme activity in a terminal region rose to its plateau at day 30. TPH activity in adult rat is the highest in nucleus raphe dorsalis, then nucleus raphe centralis and hypothalamus in a decreasing order.     

Origin of the serotonergic innervation to the rat dorsolateral hypothalamus: retrograde transport of cholera toxin and upregulation of tryptophan hydroxylase mRNA expression following selective nerve terminals lesion.

The regulation of serotonin synthesis was investigated in the serotonergic neurons, which provide afferents to the dorsolateral hypothalamus (DLH). The origin of the DLH projection neurons within the raphe nucleus was identified by retrograde transport of Cholera toxin (CTb) and their serotonergic nature confirmed by tryptophan hydroxylase (TPH) immunocytochemistry. Disruption of serotonin synthesis steady-state was induced unilaterally by a selective and local destruction of serotonergic nerve terminals with 5,7-dihydroxytryptamine (5,7-DHT), stereotaxically injected in the right DLH. The results show that most of the serotonergic dorsal raphe neurons projecting to the DLH have an ipsilateral localization within the lateral aspects of the nucleus. In rats with unilateral DLH lesion, a population of serotonergic cells within the raphe nucleus exhibited a clear increase in TPH mRNA. These cells were about five times more numerous in the ipsilateral as compared to the contralateral dorsal raphe nucleus and they had, for the most part, a lateral localization within the raphe nucleus. Sham-operated rats did not exhibit any upregulation of TPH mRNA. Together, the present results provide the first demonstration that a discreet and selective destruction of serotonergic terminals induces a circumscribed and striking increase in TPH mRNA expression in a subset of brainstem serotonergic neurons projecting to and/or passing through the DLH. On the basis of these results and previous in vivo measurements of TPH activity (e.g., 5-HT synthesis), we suggest that this upregulation in TPH mRNA expression results from the loss of pre-synaptic and/or post-synaptic regulation of serotonin synthesis. These new findings raise important issues related to the repercussions of a local disruption in serotonergic neurotransmission on brain areas remote from the site of injury.     

Evidence for dual serotonergic projections to neocortex: axons from the dorsal and median raphe nuclei are differentially vulnerable to the neurotoxin p-chloroamphetamine (PCA)

Previous studies have shown that there are morphologically dissimilar serotonergic (5-HT) axon types in rat cerebral cortex which are differentially sensitive to the neurotoxic effects of certain psychotropic drugs: methylenedioxyamphetamines (MDA and MDMA) and p-chloroamphetamine (PCA) cause degeneration of fine 5-HT axon terminals in cortex, while sparing beaded axons. Moreover, a recent anterograde transport study suggests that fine and beaded 5-HT axons arise from the dorsal raphe (DR) and median raphe (MR) nuclei, respectively. These data led us to propose that the DR projection to neocortex is selectively vulnerable to the neurotoxic effects of PCA, while the MR projection is resistant; this hypothesis was tested in the present study by comparing retrograde axonal transport of the fluorescent tracer Fluoro-Gold in PCA-treated and control rats. Using this method, only axons that survive PCA treatment can take up and transport the injected label back to the cell bodies of origin, thus allowing us to determine which raphe-cortical projections remain intact after PCA. The results show that PCA administration produces a loss of fine 5-HT axon terminals in neocortex and a concomitant reduction in the number of retrogradely labeled neurons in the DR (77% decrease), when compared to controls. In contrast, beaded 5-HT axon terminals are spared and the number of labeled neurons in the MR remains unchanged after PCA. These results demonstrate that DR and MR projections to cortex are differentially vulnerable to PCA: fine axon terminals arise from neurons in the DR and are highly sensitive to the neurotoxic effects, whereas beaded axons from the MR are resistant. We therefore propose that there are two anatomically and functionally separate 5-HT projections to cortex having different (1) nuclei of origin, (2) axon morphology, (3) regional distributions, and (4) pharmacological properties. Since the mood-altering substances MDA, MDMA, and PCA act specifically upon 5-HT axon terminals from the dorsal raphe nucleus, DR neurons may be preferentially involved in the control of affective state.     

Selective effects of apomorphine on dorsal raphe neurons: A cytofluorimetric study

Using a quantitative cytofluorimetric method to detect changes in the intracellular levels of serotonin (5-HT) in individual neurons in rat brain, we have found that the dopaminergic agonist apomorphine increases 5-HT content of dorsal raphe cell bodies without affecting cells in the median raphe nucleus. Liquid chromatographic studies revealed that apomorphine also elevated the concentrations of both 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in striatum, the projection site for dorsal raphe neurons. Conversely, the dopaminergic antagonist haloperidol, at a dose of 0.8 mg/kg, decreased 5-HT levels in dorsal raphe cells. A lower dose of haloperidol (0.4 mg/kg), which had no significant effect alone, completely blocked the effect of apomorphine in the dorsal raphe. These results support the hypothesis that the effects of apomorphine on serotonergic neurons are secondary to dopamine receptor stimulation.     

3H]-serotonin retrograde labelling in serotonergic fibers

Autoradiography, following retrograde axonal transport with [3H]-serotonin (10(-4) M) was used to identify, selectively, the serotonergic neurons of medullary raphe that innervate the spinal cord. In combination with this technique, immunocytochemical detection of endogenous serotonin showed some raphe magnus cell bodies immunostained by 5-HT antibodies and [3H]5-HT radiolabelled. Serotonergic fibers of lateral and dorsolateral funiculus of ligated spinal cord were also characterized following retrograde axonal transport with 10(-4) M [3H]-serotonin. At the ultrastructural level, mainly myelinated, although unmyelinated labelled fibers were also identified as swollen axons. Clusters of large membranous organelles, predominantly mitochondria, some dense lamellar bodies, multivesicular bodies and lysosomes accumulated on the distal segment of the ligation. Autoradiographic reaction was always observed to be very intense and silver grains overlapped mainly mitochondria (41%) and clusters of membranous organelles (37%). However, smooth endoplasmic reticulum seemed to be devoid of silver grains. From our results, serotonergic myelinated spinal fibers were labelled after uptake-retrograde axonal transport with [3H]5-HT. Mitochondria and membrane organelles could convey tritiated compounds derived from [3H]-serotonin uptake towards the serotonergic cell bodies.     

Retrograde axonal transport following injection of [3H]serotonin in the olfactory bulb. I. Biochemical study

A retrograde axonal transport from the serotonergic nerve terminals in the olfactory bulb (OB) to their parent cell bodies in the midbrain raphe nuclei has been demonstrated after stereotaxic injection of [2H]5-HT into the OB of rats pretreated with a monoamine oxidase (MAO) inhibitor: at various time intervals thereafter (4-92 h) there was a preferential accumulation of radioactivity mainly in the raphe dorsalis nucleus (RDN). Maximal accumulation occurred at 24 h. Of this radioactivity, 30-50% was recovered as 5-HT. The accumulation was estimated to take place at two rates: a fast one (48 mm/day) and a slower one (16 mm/day). Under the same experimental conditions there was no clear evidence for a retrograde accumulation of [3H]norepinephrine in the RDN. A passive diffusion mechanism could be excluded since the diffuson of tracer towards the cerebrospinal fluid was prevented by prior mechanical obstruction of the olfactory diverticle of the lateral ventricle. Furthermore, colchicine strongly reduced (by 80%) the radioactive accumulatin in the RDN. Destruction of serotonergic nerve terminals by 5,6-dihydroxytryptamine or inhibiton of 5-HT uptake by fluoxetine decreased this retrograde accumulation whereas destruction of catecholaminergic nerve terminals by 6-hydroxydopamine was without effect. Pretreatment with reserpine decreased the amount of radioactivity transported to the RDN by 40%. In the absence of MAO inhibition pretreatment, animals still presentd 35% of the tracer transported to the RDN. Intrabulbar injection of MAO inhibitor did not affect the accumulation rates when compared with animals which received the inhibitor by the intraperitoneal route. In conclusion, the retrograde axonal transport following [3H]5-HT injection in the serotonergic RDN-OB system occurs via an active process which depends on a colchicine-sensitive mechanism and is partially linked to a reserp ine-sensitive structure. During its transport, the amine seems to be relatively protected from metabolic inactivation.     

Methylenedioxyamphetamine (MDA) and methylenedioxymethamphetamine (MDMA) cause selective ablation of serotonergic axon terminals in forebrain: immunocytochemical evidence for neurotoxicity

The psychotropic amphetamine derivatives 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) have been used for recreational and therapeutic purposes in man. In rats, these drugs cause large reductions in brain levels of serotonin (5-HT). This study employs immunocytochemistry to characterize the neurotoxic effects of these compounds upon monoaminergic neurons in the rat brain. Two weeks after systemic administration of MDA or MDMA (20 mg/kg, s.c., twice daily for 4 d), there is profound loss of serotonergic (5-HT) axons throughout the forebrain; catecholamine axons are completely spared. Regional differences in drug toxicity are exemplified by partial sparing of 5-HT axons in hippocampus, lateral hypothalamus, basal forebrain, and in some areas of neocortex. The terminals of 5-HT axons are selectively ablated, while axons of passage and raphe cell bodies are spared. Thickened preterminal fibers exhibit increased staining due to damming-up of neurotransmitter and other axonal constituents. Fine 5-HT axon terminals are extremely vulnerable to these drugs, whereas terminal-like axons with large varicosities survive, raising the possibility that some 5-HT axons may be resistant to the neurotoxic effects. At short survivals, visualization of greatly swollen, fragmented 5-HT axons provides anatomic evidence for degeneration of 5-HT projections. The results establish that MDA and MDMA produce structural damage to 5-HT axon terminals followed by lasting denervation of the forebrain. Both drugs have similar effects, but MDA produces a greater reduction of 5-HT axons than does MDMA at the same dosage. The selective degeneration of 5-HT axons indicates that these drugs may serve as experimental tools to analyze the organization and function of 5-HT projections. Caution should be exercised until further studies determine whether these compounds may be hazardous in man.     

The Expression of Brain-Derived Neurotrophic Factor and Tryptophan Hydroxylase in the Dorsal Raphe Nucleus during Repeated Stress

The mechanisms of development of stress-induced depression symptoms may include interaction between the elements of the serotonergic (5-HT) system and brain-derived neurotrophic factor (BDNF) at the level of the cell bodies of 5-HT neurons. In order to evaluate this situation, we used immunofluorescent staining of BDNF protein and the enzyme of serotonin synthesis tryptophan hydroxylase (TPH) in the dorsal raphe nucleus (DRN) of adult male rats that were subjected daily to a 15-min forced swim stress for 1, 2, 7, or 14 days. The expression of the BDNF protein was substantially decreased in the DRN after 7- or 14-day stress, whereas the expression of the TPH protein significantly increased after the 14-day stress and did not differ from the control after the shorter duration of repeated stress. The absence of correlation between some indices indicates their independence from each other at the level of the cell bodies of 5-HT neurons.     

Distribution and synaptic relations of NOS neurons in the dorsal raphe nucleus: A comparison to 5-HT neurons

Anti-nitric oxide synthase antibody was used to study the distribution, cytowchhecture, and synaptic relations of nitric oxide synthase-like immunoreactive neurons in the whole rostral-caudel length of the dorsal raphe nucleus of the rat and compared them with serotonergic neurons. Results showed that the distribution of the nitric oxide synthase in the dorsal raphe nucleus was similar to that of the serotonergic neurons at the rostral part of the dorsal raphe nucleus, including the mediodorsal and the medioventral cell groups, and changed at the middle and caudal parts of the dorsal raphe nucleus. The cytoarchitecture of the nitric oxide synthase-like immunoreactive neurons in the medioventrai cell group of the dorsal raphe nucleus was similar to that of the serotonergic neurons. Similar to the serotonergic neurons there, nitric oxide synthase-like immunoroactive neurons also received synapses from axon terminals that contained round, or flattened vesicles, or both kinds. Different to the serotonergic neurons, the few nitric oxide synthase-like immunoroactive axon terminals that were in this area formed synapses.     

TPH2 in the ventral tegmental area of the male rat brain

This study surveyed the distribution of tryptophan hydroxylase 2 (TPH2) mRNA, protein, and enzymatic activity throughout the male Sprague-Dawley rat brain.TPH2 is the genetic isoform of TPH that catalyzes the rate-limiting step in serotonin biosynthesis within the central nervous system. Although cell bodies of serotonergic neurons are located mainly in the raphe, serotonin-containing axons innervate many regions of the brain. In the present study, we assessed the levels of mRNA, protein expression, and enzyme activity of TPH2 in the rat raphe, ventral tegmental area (VTA), substantia nigra, hippocampus, cerebellum, dorsal striatum, nucleus accumbens, amygdala, and medial prefrontal cortex to more fully understand the distribution of this enzyme throughout the central nervous system. The pineal gland was used as a control tissue that expresses TPH1 (the peripheral enzyme), but not TPH2. As expected, the raphe showed the highest brain TPH2 activity and protein expression. In the contrast to other reports, however, the VTA followed the raphe as the region with the second-highest amount of TPH2 activity, mRNA and protein expression. There were significantly lower TPH activities and levels of TPH2 protein in the other regions. In addition, TPH2 immunocytochemistry demonstrated the presence of TPH-positive cell bodies within the VTA. The results of this study indicate that TPH2 and serotonergic signaling may play an important role in the mesolimbic/mesocortical reward pathway.     

Retrograde axonal transport following injection of [3H]-serotonin into the olfactory bulb. II. Radioautographic study

Radioautography was used to study the intraneuronal distribution of [3H]-serotonin (5-HT) and/or its derivatives selectively taken up by the olfactory bulb (OB) serotonergic terminals and subsequently transported to their parent cell bodies in the midbrain raphe nuclei. This was done 24 h after injection of [3H]5-HT into the main OB of rats either pretreated or not with monoamine oxidase (MAO) inhibitor. A prior mechanical obstruction of the rostral ventricular cavities prevented diffusion of the tracer towards cerebrospinal fluid. Heavily labelled nerve cell bodies were found mainly in the ipsilateral raphe dorsalis nucleus (RDN) and to a lesser extent in the raphe centralis nucleus. The radioautographic reaction often extended to dendritic processes while sparing the nucleus. A diffuse reaction was also observed but limited to the raphe area. The supraependymal 5-HT fibers were found to be free of labelling. Neither local destruction of catecholaminergic terminals with 6-OHDA, nor absence of MAO inhibition, impaired this radioautographic pattern, while destruction of serotonergic terminals with 5,6-dihydroxytryptamine in OB resulted in the disappearance of labelled axonal varicosities and neurons in the OB and the RDN respectively. At the electron microscopy level, labelled cell bodies in the RDN were medium-sized (12-15 micrometers). Silver grains were localized mainly on mitochondria and, to a lesser extent, on lysosomes and endoplasmic reticulum but spared the nucleus and the nucleolus. Silver grains were also found near the nuclear membrane and outside the neuronal membrane. The observation of heavy metal impregnated thick sections confirmed the preferential localization of silver grains on mitochondria with or without inhibition of MAO. These results could account for the subcellular compartments involved in the retrograde axonal transport of [3H]5-HT and its subsequent degradation and/or dendritic release.     

Degenerative changes in epinephrine tonic vasomotor neurons in Alzheimer's disease

The C-1 region in the rostral ventral lateral medulla contains mainly epinephrine (Epi) neurons. These neurons are the tonic vasomotor center of the brain. We previously demonstrated changes in the enzymatic activity of phenylethanolamine N-methyltransferase (PNMT) in axon terminals and cell bodies of Epi neurons from the medulla of Alzheimer's disease (AD) brains. In this study, we investigated the perikarya of C-1 neurons for the morphometric, immunohistochemical and histochemical changes that are seen in severely affected regions of Alzheimer brain. The mean areas and size distributionsof C-1 neurons from 6 AD and 6 neurologically normal patients were compared using the Wilcoxon rank sum test and Kolmogorov-Smirnov z tests respectively. Additional brain sections from the C-1 region of AD and control individuals were stained with cresyl violet or immunostained with antibodies to the lysosomal hydrolase cathepsin D, Tau-2, Alz-50 and β-amyloid protein. The average area of C-1 neurons in AD brains was decreased 18.3%(P < 0.001) compared to the areas of the same cell population in age-matched control brains. A shift toward smaller sized C-1 neurons was seen in the AD cases. Nissl stain demonstrated a central chromatolytic appearance in 3.7% of AD neurons sampled. No β-amyloid deposits were detected histologically or immunocytochemically in the C-1 region of AD brains. Both Tau-2 and Alz-50 immunoreactivity was observed in occasional (1%) C-1 neurons from AD brains but not in controls. A small proportion (30%) of the C-1 neurons showing atrophy displayed increased cathepsin D immunoreactivity. Accumulation of hydrolase-laden lysosomes was marked only in atrophic neurons in contrast to at-risk neurons of the prefrontal cortex and hippocampus where lysosomal alterations precede degenerative changes. Mechanisms underlying degeneration of these neurons are discussed.     

Routing of transmitter and other changes in fast axonal transport after transection of one branch of the bifurcate axon of an identified neuron

The regulation of the quantities and types of organelles that leave the neuronal cell body destined for use in the axon and its terminals is not well understood. We had previously found that transport of transmitter undergoes a precise down regulation when most of one branch of the bifurcate axon of an identified serotonergic neuron was removed. We have now investigated further the nature of the regulatory event and the reason for its initiation by eliminating portions of the axonal tree of this neuron. We find that the down regulation is more likely to be due to the loss of synapses than of axon because transport of [3H]serotonin decreases as much when an axonal branch is transected distally as after a proximal transection. Transport of [3H]fucosyl glycoprotein, which normally is associated with the serotonergic vesicle in this axon, decreases to the same extent as transport of [3H]serotonin following proximal transection. The glycoprotein down regulation occurs much more rapidly, possibly due to an inhibition of vesicle synthesis. A secondary rise in transport of [3H] fucosyl glycoprotein 3 days to 2 weeks after axotomy suggests that the radiolabeled glycoprotein has undergone a redistribution into organelles not normally labeled and transported in intact neurons in large amounts, since [3H]serotonin transport remains stably diminished during this period. We also describe here a case of routing of rapidly transported material. When one axonal branch is cut far from the point of bifurcation (approximately 10 mm), [3H]serotonin is directed away from the ranch lacking its synaptic terminals and into the remaining intact branch even though the transected branch is physically capable of transporting its normal amount of [3H]serotonin.(ABSTRACT TRUNCATED AT 250 WORDS)     

鱼藤酮处理大鼠脑内VMAT2及TPH、5-HT的表达改变

目的观察鱼藤酮处理大鼠脑内单胺囊泡转运体2(VMAT2)。色氨酸羟化酶(TPH)及5—羟色胺(5—HT)的表达变化;探讨其对5—HT能神经元的影响及可能机制。方法选用健康、成年雄性Wistar大鼠,背部皮下注射鱼藤酮3d或28d制作大鼠动物模型;利用免疫细胞化学分析VMAT2在大鼠黑质、中缝背核和尾壳核以及TPH、5—HT在中缝背核的表达变化;以透射电镜观察轴突超微结构的改变,探讨鱼藤酮毒性作用的可能机制。结果鱼藤酮3d组:(1)鱼藤酮组大鼠黑质、中缝背核VMAT2的表达增加,免疫反应强度吸光度值显著高于对照组;尾壳核VMAT2的表达减弱,免疫反应强度吸光度值明显低于对照组(P0.01);(2)鱼藤酮组大鼠中缝背核TPH、5—HT的表达减少,免疫反应强度吸光度值显著低于对照组(P0.01);(3)透射电镜观察显示鱼藤酮处理大鼠中缝背核轴突变性、其内微管解聚。鱼藤酮28d组:(1)鱼藤酮组大鼠黑质、中缝背核及尾壳核VMAT2的表达均减弱,免疫反应强度吸光度值显著低于对照组(P0.01或P0.05);(2)与对照组相比,鱼藤酮组大鼠中缝背核TPH、5—HT的表达降低,免疫反应强度吸光度值显著低于对照组(P0.01)。结论鱼藤酮降低5—HT能神经元TPH、5—HT的表达,其毒性作用可能与轴突微管解聚导致突触囊泡VMAT2轴浆顺行转运障碍有关。     

Axonal transport in serotonin neurons of the midbrain raphe.

The projections of serotonin-containing neurons of the midbrain raphe nuclei (nucleus raphe dorsalis, nucleus centralis superior) are studied by analysis of axonal transport of labeled amino acids. These results are correlated with regional alterations of serotonin content following midbrain raphe lesions which produce significant serotonin depletion in nearly all regions of the central nervous system. Twenty-four hours following injection of 100 muCi [3H]proline, raphe neurons have taken up labeled material and transported it, presumably as protein, to telencephalon, diencephalon, brain stem, the cerebellum and the spinal cord. This transport appears to take place predominantly in serotonin neurons. After injection of 100 muCi [3H]5-HTP into nucleus raphe dorsalis or nucleus centralis superior, the pattern of regional distribution of transported material is very similar to that obtained with tritiated proline. Selective lesions of serotonin terminals with 5.6-DHT result in greatly diminished axonal transport of proteins to all telencephalic, diencephalic and mesencephalic areas as well as to cerebellum, pons-medulla and spinal cord. Unilateral destruction of the medial forebrain bundle results in significant reduction in axonal transport of labeled material to ipsilateral telencehalon and thalamus. These results provide further support for the view that serotonin neurons of the midbrain raphe nuclei project widely throughout the neuraxis to telencephalon, diencephalon, brain stem, cerebellum and spinal cord.     

Ultrastructural analysis of the innervation of TRH-immunoreactive neuronal elements located in the periventricular subdivision of the paraventricular nucleus of the rat hypothalamus

A combination of electron microscopic immunocytochemistry and autoradiography was employed to examine the synaptic organization of thyrotropin-releasing hormone (TRH) neurons in the periventricular subdivision of the paraventricular nucleus of the rat hypothalamus. TRH neurons were identified by immunocytochemistry. Selective uptake of tritiated serotonin (5-HT) was used to identify serotoninergic elements. TRH-immunoreactive axon terminals were found to be in synaptic contact with TRH-immunoreactive dendrites and with unlabeled dendritic branchlets. There were direct appositions between radiolabeled 5-HT terminals and TRH-immunoreactive dendrites, but differential synaptic contacts between 5-HT axonal elements and TRH neurons were not seen. TRH-immunopositive cell bodies and dendrites received a very intense innervation by unlabeled axon terminals or axonal varicosities showing morphologically defined synaptic junctions. These were mostly of the asymmetrical variety and different types could be distinguished. The findings substantiate the view that TRH neurons of the periventricular subvision of the paraventricular nucleus may be influenced by TRH axons, serotoninergic fibers and a large number of unidentified nerve terminals.     

Melanin-concentrating hormone does not modulate serotonin release in primary cultures of fetal raphe nucleus neurons

Melanin-concentrating hormone (MCH) is a neuropeptide present in neurons located in the hypothalamus that densely innervate serotonergic cells in the dorsal raphe nucleus (DRN). MCH administration into the DRN induces a depressive-like effect through a serotonergic mechanism. To further understand the interaction between MCH and serotonin, we used primary cultured serotonergic neurons to evaluate the effect of MCH on serotonergic release and metabolism by HPLC-ED measurement of serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) levels. We confirmed the presence of serotonergic neurons in the E14 rat rhombencephalon by immunohistochemistry and showed for the first time evidence of MCHergic fibers reaching the area. Cultures obtained from rhombencephalic tissue presented 2.2 ± 0.7% of serotonergic and 48.9 ± 5.4% of GABAergic neurons. Despite the low concentration of serotonergic neurons, we were able to measure basal cellular and extracellular levels of 5-HT and 5-HIAA without the addition of any serotonergic-enhancer drug. As expected, 5-HT release was calcium-dependent and induced by depolarization. 5-HT extracellular levels were significantly increased by incubation with serotonin reuptake inhibitors (citalopram and nortriptyline) and a monoamine-oxidase inhibitor (clorgyline), and were not significantly modified by a 5-HT1A autoreceptor agonist (8-OHDPAT). Even though serotonergic cells responded as expected to these pharmacological treatments, MCH did not induce significant modifications of 5-HT and 5-HIAA extracellular levels in the cultures. Despite this unexpected result, we consider that assessment of 5-HT and 5-HIAA levels in primary serotonergic cultures may be an adequate approach to study the effect of other drugs and modulators on serotonin release, uptake and turnover.     

Retrograde axonal transport following injection of [H]serotonin in the olfactory bulb. I. Biochemical study

A retrograde axonal transport from the serotonergic nerve terminals in the olfactory bulb (OB) to their parent cell bodies in the midbrain raphe nuclei has been demonstrated after stereotaxic injection of [³H]5-HT into the OB of rats pretreated with a monoamine oxidase (MAO) inhibitor: at various time intervals thereafter (4–92 h) there was a preferential accumulation of radioactivity mainly in the raphe dorsalis nucleus (RDN). Maximal accumulation occurred at 24 h. Of this radioactivity, 30–50% was recovered as 5-HT. The accumulation was estimated to take place at two rates: a fast one (48 mm/day) and a slower one (16 mm/day). Under the same experimental conditions there was no clear evidence for a retrograde accumulation of [³H]norepinephrine in the RDN.A passive diffusion mechanism could be excluded since the diffusion of tracer towards the cerebrospinal fluid was prevented by prior mechanical obstruction of the olfactory diverticle of the lateral ventricle. Furthermore, colchicine strongly reduced (by 80%) the radioactive accumulation in the RDN.Destruction of serotonergic nerve terminals by 5,6-dihydroxytryptamine or inhibition of 5-HT uptake by fluoxetine decreased this retrograde accumulation whereas destruction of catecholaminergic nerve terminals by 6-hydroxydopamine was without effect. Pretreatment with reserpine decreased the amount of radioactivity transported to the RDN by 40%.In the absence of MAO inhibition pretreatment, animals still presented 35% of the tracer transported to the RDN. Intrabulbar injection of MAO inhibitor did not affect the accumulation rates when compared with animals which received the inhibitor by the intraperitoneal route.     

Nucleus-specific alteration of raphe neurons in human neurodegenerative disorders

Neurodegenerative diseases share symptoms suggested to be related to the serotonergic system. To evaluate the involvement of serotonergic raphe nuclei, we compared the percentage of neurons synthesizing serotonin in the nucleus centralis superior (NCS), raphe obscurus and pallidus (NROP) in Alzheimer's disease (AD), progressive supranuclear palsy (PSP), Parkinson's disease (PD), multiple system atrophy (MSA), and control brains. We used immunohistochemistry for tryptophan hydroxylase (TpOH), phosphorylated tau, and alpha-synuclein. We observed a significant decrease in the NCS in the NROP in AD, but a significant increase in PSP and MSA. Cytoskeletal pathology was present in the NCS and NROP to a variable degree. We conclude that there is disease- and nucleus-specific alteration of serotonin synthesis in the raphe.