Serotonergic Axon Terminals Research Articles

Serotonin improves behavioral contrast sensitivity of freely moving rats.

Serotonin (5-HT) is a neuromodulator secreted from serotonergic neurons located in the pons and upper brain stem in a behavioral context-dependent manner. The serotonergic axon terminals innervate almost the whole brain, causing modulatory actions on various brain functions including vision. Our previous study demonstrated the visual responses of neurons in the primary visual cortex (V1) of anesthetized monkeys were modulated by the activation of 5-HT receptors depending on the response magnitude, in which 5-HT2A receptor-selective agonists enhanced weak visual responses but not strong responses. This observation suggests that the activation of serotonergic receptors modulates neuronal visual information processing to improve the behavioral detectability of a stimulus. However, it remains unknown if 5-HT improves visual detectability at the behavioral level. To investigate this point, visual detectability was measured as contrast sensitivity (CS) in freely moving rats using a two-alternative forced-choice visual detection task (2AFC-VDT) combined with the staircase method. The grating contrast was decreased or increased step by step after a correct choice (hit) or incorrect choice (miss), respectively. CS was evaluated as an inverse value of the visual contrast threshold. The effect of the intraperitoneal administration of fluoxetine (FLX, 5 mg/kg), a selective serotonin reuptake inhibitor, on CS was tested. The CS of rats was significantly higher in FLX than control conditions, and the drug effect showed specificity for the spatial frequency (SF) of a grating stimulus, in which CS improvement was observed at optimal SF but not non-optimal high SF. Thus, we conclude that endogenously-secreted serotonin in the brain improves visual detectability, which may be mediated by vision-related neurons acquiring SF information of the visual stimulus.

Detection of a High-Turnover Serotonin Circuit in the Mouse Brain Using Mass Spectrometry Imaging.

SummaryMonoamine neurotransmitters are released by specialized neurons regulating behavioral, motor, and cognitive functions. Although the localization of monoaminergic neurons in the brain is well known, the distribution and kinetics of monoamines remain unclear. Here, we generated a murine brain atlas of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) levels using mass spectrometry imaging (MSI). We found several nuclei rich in both 5-HT and a catecholamine (DA or NE) and identified the paraventricular nucleus of the thalamus (PVT), where 5-HT and NE are co-localized. The analysis of 5-HT fluctuations in response to acute tryptophan depletion and infusion of isotope-labeled tryptophan in vivo revealed a close kinetic association between the raphe nuclei, PVT, and amygdala but not the other nuclei. Our findings imply the existence of a highly dynamic 5-HT-mediated raphe to PVT pathway that likely plays a role in the brain monoamine system.

Localization of organic cation transporter 2 (OCT2) in monoaminergic and cholinergic axon terminals of the mouse brain

Organic cation transporters (OCTs) are low-affinity, high-capacity carriers that mediate sodium-independent transport for biogenic cations, including catecholamine, serotonin, histamine, and choline/acetylcholine. Among them, OCT2 is expressed in neurons of the central nervous system. Although previous studies show OCT2 expression in several populations of cholinergic and monoaminergic neurons, the regional distribution of OCT2 in the brain remains largely unknown. Here we performed immunohistochemical analyses to reveal the distribution of OCT2 throughout the mouse forebrain. OCT2 immunoreactivity was widely distributed, with substantial regional specificity in cortical and subcortical structures including the hippocampus, striatum, and some subdivisions of the amygdala and extended amygdala. Interestingly, OCT2 appeared as punctate, bouton-like labeling in cholinergic, dopaminergic, and serotonergic axon terminals that were co-labeled with presynaptic neurochemical markers. We also co-labeled OCT2 and an anterograde tract-tracer injected into the locus coeruleus, demonstrating that OCT2 was localized to presumptive noradrenergic terminals in the forebrain. Together, our results demonstrated that the polyspecific cation transporter OCT2 is distributed in cholinergic and monoaminergic terminals in various forebrain regions, suggesting that OCT2 could play a role in regulating presynaptic reuptake and recycling of choline and monoamines.

Serotonergic regulation of excitability of principal cells of the dorsal cochlear nucleus.

The dorsal cochlear nucleus (DCN) is one of the first stations within the central auditory pathway where the basic computations underlying sound localization are initiated and heightened activity in the DCN may underlie central tinnitus. The neurotransmitter serotonin (5-hydroxytryptamine; 5-HT), is associated with many distinct behavioral or cognitive states, and serotonergic fibers are concentrated in the DCN. However, it remains unclear what is the function of this dense input. Using a combination of in vitro electrophysiology and optogenetics in mouse brain slices, we found that 5-HT directly enhances the excitability of fusiform principal cells via activation of two distinct 5-HT receptor subfamilies, 5-HT2A/2CR (5-HT2A/2C receptor) and 5-HT7R (5-HT7 receptor). This excitatory effect results from an augmentation of hyperpolarization-activated cyclic nucleotide-gated channels (Ih or HCN channels). The serotonergic regulation of excitability is G-protein-dependent and involves cAMP and Src kinase signaling pathways. Moreover, optogenetic activation of serotonergic axon terminals increased excitability of fusiform cells. Our findings reveal that 5-HT exerts a potent influence on fusiform cells by altering their intrinsic properties, which may enhance the sensitivity of the DCN to sensory input.

Acute treatment with fluvoxamine elevates rat brain serotonin synthesis in some terminal regions: An autoradiographic study

A considerable body of evidence indicates the involvement of the neurotransmitter serotonin (5-HT) in the pathogenesis and treatment of depression. The acute effect of fluvoxamine, on 5-HT synthesis rates was investigated in rat brain regions, using α-(14)C-methyl-L-tryptophan as a tracer. Fluvoxamine (25 mg/kg) and saline (control) were injected intraperitoneally, one hour before the injection of the tracer (30 μCi). There was no significant effect of fluvoxamine on plasma free tryptophan. After Benjamini-Hochberg False Discovery Rate correction, a significant decrease in the 5-HT synthesis rate in the fluvoxamine treated rats, was found in the raphe magnus (-32%), but not in the median (-14%) and dorsal (-3%) raphe nuclei. In the regions with serotonergic axon terminals, significant increases in synthesis rates were observed in the dorsal (+41%) and ventral (+43%) hippocampus, visual (+38%), auditory (+65%) and parietal (+37%) cortex, and the substantia nigra pars compacta (+56%). There were no significant changes in the 5-HT synthesis rates in the median (+11%) and lateral (+24%) part of the caudate-putamen, nucleus accumbens (+5%), VTA (+16%) or frontal cortex (+ 6%). The data show that the acute administration of fluvoxamine affects 5-HT synthesis rates in a regionally specific pattern, with a general elevation of the synthesis in the terminal regions and a reduction in some cell body structures. The reasons for the regional specific effect of fluvoxamine on 5-HT synthesis are unclear, but may be mediated by the presynaptic serotonergic autoreceptors.

Inhibition of NGF-induced neurite outgrowth of rat pheochromocytoma cells (PC12) following administration of dioxyamphetamine

Amphetamine analogs are known to induce not only neurotoxicity at serotonergic axon terminals but also neocortical neuronal degeneration. However, a much less studied aspect involves the impact of amphetamine exposure on neuronal development. The present study investigated whether pretreatment of PC12 cells with dioxyamphetamine (DA) alters differentiation of PC12 cells by NGF and, if so, which components of the Ras/Raf/MEK/ERK pathway known to be involved in the differentiation response to NGF are particularly affected. Though exposure of PC12 cells to DA 1 h prior to NGF treatment resulted in apopotosis, several PC12 cells survived. However, neurite outgrowth of these NGF-responsive cells was repressed. Immunoblots of whole cell extracts revealed a strong induction rather than inhibition of ERK phosphorylation up to 48 h after DA/NGF treatment. Our results indicate that NGF-mediated neurite outgrowth was inhibited by pretreatment with DA, and this blockage of NGF-induced neuritogenesis was not due to an inhibition of ERK phosphorylation.

Serotonin‐immunoreactive axon terminals innervate pyramidal cells and interneurons in the rat basolateral amygdala

The basolateral nuclear complex of the amygdala (BLC) receives a dense serotonergic innervation that appears to play a critical role in the regulation of mood and anxiety. However, little is known about how serotonergic inputs interface with different neuronal subpopulations in this region. To address this question, dual-labeling immunohistochemical techniques were used at the light and electron microscopic levels to examine inputs from serotonin-immunoreactive (5-HT+) terminals to different neuronal subpopulations in the rat BLC. Pyramidal cells were labeled by using antibodies to calcium/calmodulin-dependent protein kinase II, whereas different interneuronal subpopulations were labeled by using antibodies to a variety of interneuronal markers including parvalbumin (PV), vasoactive intestinal peptide (VIP), calretinin, calbindin, cholecystokinin, and somatostatin. The BLC exhibited a dense innervation by thin 5-HT+ axons. Electron microscopic examination of the anterior basolateral nucleus (BLa) revealed that 5-HT+ axon terminals contained clusters of small synaptic vesicles and a smaller number of larger dense-core vesicles. Serial section reconstruction of 5-HT+ terminals demonstrated that 76% of these terminals formed synaptic junctions. The great majority of these synapses were symmetrical. The main targets of 5-HT+ terminals were spines and distal dendrites of pyramidal cells. However, in light microscopic preparations it was common to observe apparent contacts between 5-HT+ terminals and all subpopulations of BLC interneurons. Electron microscopic analysis of the BLa in sections dual-labeled for 5-HT/PV and 5-HT/VIP revealed that many of these contacts were synapses. These findings suggest that serotonergic axon terminals differentially innervate several neuronal subpopulations in the BLC.

Presynaptic modulation of 5-HT release in the rat septal region

5-HT released from serotonergic axon terminals in the septal nuclei modulates the activity of septal output neurons (e.g. septohippocampal cholinergic neurons) bearing somatodendritic 5-HT receptors. Therefore, we studied the mechanisms involved in the presynaptic modulation of 5-HT release in the lateral (LS) and medial septum (MS), and the diagonal band of Broca (DB). HPLC analysis showed that tissue concentrations of noradrenaline, dopamine and 5-HT were highest in DB (DB>MS>LS). Slices prepared from LS, MS and DB regions were preincubated with [3H]5-HT, superfused in the presence of 6-nitro-2-(1-piperazinyl)-quinoline (6-nitroquipazine) and electrically stimulated up to three times (first electrical stimulation period (S1), S2, S3; 360 pulses, 3 Hz, 2 ms, 26–28 mA). In all septal regions the Ca2+-dependent and tetrodotoxin-sensitive electrically-evoked overflow of [3H] was inhibited by the 5-HT1B agonist CP-93,129 and the α2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline tartrate (UK-14,304). Also the μ- and κ-opioid receptor agonists (d-Ala2, N-Me-Phe4, glycinol5)-enkephalin (DAMGO) and [trans-(1S,2S(-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]-benzenacetamide hydro-chloride] (U-50,488H), respectively, acted inhibitory (although less potently), whereas the δ-opioid receptor agonist (d-Pen2, d-Pen5)-enkephalin (DPDPE), the dopamine D2 receptor agonist quinpirole and the adenosine A1 receptor agonist N6-cyclopentyladenosine were all ineffective; the GABAB receptor agonist baclofen had weak effects. All inhibitory effects of the agonists were antagonized by the corresponding antagonists (3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide dihydrochloride (GR-55,562), idazoxan, naloxone, nor-binaltorphimine), which also significantly enhanced the evoked release of 5-HT at S1. It is concluded that 5-HT release in septal nuclei of the rat is modulated by presynaptic 5-HT1B autoreceptors, as well as by α2-, μ- and κ-opioid heteroreceptors. All of these receptors seem to be under a tonic inhibitory influence of the corresponding endogenous agonists and show qualitatively comparable modulatory properties along the dorso-ventral distribution of the 5-HT terminals.

Neurotoxic methamphetamine regimen severely impairs recognition memory in rats.

Methamphetamine (mAMPH), when administered repeatedly to rodents or primates, is neurotoxic to some cortical neurons and to forebrain dopaminergic and serotonergic axon terminals. The aim of the present study was to investigate the effects of a neurotoxic regimen of mAMPH on two hippocampus-dependent memory tasks: object recognition, a nonspatial memory task, and the Morris water maze, a spatial memory task. Male rats were treated with mAMPH (4 x 4.0 mg/kg, s.c.) or saline and trained in the object recognition task 1 week and 3 weeks later. During training, animals explored two identical copies of the same object. In retention test sessions one of the objects was replaced by a novel object. mAMPH-treated rats showed no recognition memory during the short-term memory (STM) test, given 90 min after the training session, and showed marked impairments in the long-term memory (LTM) test, given 24 h after training. Even 3 weeks after drug injections, the mAMPH-treated animals were unable to discriminate between the novel and familiar objects during both STM and LTM tests. Despite the severe deficits observed in the recognition memory, no effects of prior mAMPH treatment were seen in the water maze task. Damage to monoamine terminals was confirmed by significant 30-40% losses of [(125)I]RTI-55 binding to striatal dopamine transporter and hippocampal serotonin transporter sites at both 1 and 3 weeks after mAMPH treatments. Thus, administration of mAMPH restricted to a single day can produce a profound, persistent, and selective deficit in a nonspatial hippocampus-dependent memory.

Methamphetamine enhances the cleavage of the cytoskeletal protein tau in the rat brain

The view that methamphetamine is neurotoxic to dopaminergic and serotonergic axon terminals has been based largely on biochemical and histological studies. In the present study, methamphetamine-induced structural damage to axons was quantified using a sensitive sandwich enzyme-linked immunosorbent assay developed for the detection of the cleaved form of the cytoskeletal protein tau. The administration of a monoamine-depleting regimen of methamphetamine (4×10 mg/kg, i.p. every 2 hours for a total of four injections) produced a time-dependent increase in the concentration of cleaved tau in the striatum. Maximal concentrations of cleaved tau were detected 3 days following methamphetamine administration. Cleaved tau concentrations also were significantly elevated in the dorsal hippocampus and, to a lesser extent, in the prefrontal cortex of methamphetamine-treated rats. Maintenance of rats in a cold (4 °C) environment not only prevented the methamphetamine-induced depletion of striatal dopamine and serotonin but also prevented the methamphetamine-induced increase in striatal cleaved tau concentrations. The novel findings from this study are supportive of the view that methamphetamine produces acute structural damage to neurons that may lead to the long-term neurotoxic effects of repeated, high-dose administration of the drug and that cleaved tau reliably quantifies the time-dependent neurotoxic effects of methamphetamine.

Differential subcellular location of mitochondria in rat serotonergic neurons depends on the presence and the absence of monoamine oxidase type B

Monoamine oxidase type A and type B are major neurotransmitter-degrading enzymes in the CNS. The type A is present on mitochondrial outer membranes in the whole extent of noradrenergic and dopaminergic neurons, including their axon terminals. The type B is present in serotonergic neurons, but its subcellular localization has not been elucidated. In the present study, we used both a double-labeling immunofluorescence method and electron microscopic immunohistochemistry to examine the subcellular localization of monoamine oxidase type B in serotonergic neurons projecting from the dorsal raphe nucleus to the suprachiasmatic nucleus in the rat brain. In the dorsal raphe nucleus, serotonin-positive neuronal cell bodies were clustered, and virtually all of these cell bodies were also positive for monoamine oxidase type B. By contrast, serotonin-negative neuronal cell bodies were mostly free of this enzyme. Within the neuronal cell bodies and dendrites that were positive for monoamine oxidase type B, most mitochondria contained this enzyme on their outer membranes, but a substantial proportion of mitochondria lacked this enzyme. In the suprachiasmatic nucleus, serotonin-positive varicosities were concentrated, but none of these varicosities exhibited monoamine oxidase type B. In this nucleus, mitochondria were found in almost all serotonin-positive axon terminals, but monoamine oxidase type B was not observed in any axon terminal that contained mitochondria. Our results show that there are two kinds of mitochondria in serotonergic neuronal cell bodies and dendrites: one containing monoamine oxidase type B on their outer membranes, and the other lacking this enzyme. In addition, mitochondria in serotonergic axon terminals do not possess monoamine oxidase type B. It is suggested in serotonergic neurons that only mitochondria lacking monoamine oxidase type B are transported by axonal flow up to axon terminals. It is also probable that mitochondria containing monoamine oxidase type B are transported along the axons, but that this enzyme undergoes a change, for example, conformational change, decomposition or removal from the membranes.

Putative oxidative metabolites of 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-β-carboline of potential relevance to the addictive and neurodegenerative consequences of ethanol abuse

Ethanol is metabolized in the brain by catalase/H 2O 2 to yield acetaldehyde and by an ethanol-inducible form of cytochrome P450 (P450 IIE1) in a reaction that yields oxygen radicals. Within the cytoplasm of serotonergic axon terminals these metabolic pathways together provide conditions for the endogenous synthesis of 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-β-carboline ( 1), by reaction of acetaldehyde with unbound 5-hydroxytryptamine (5-HT), and for the oxygen radical-mediated oxidation of this alkaloid. The major initial product of the hydroxyl radical (HO·)-mediated oxidation of 1 in the presence of free glutathione (GSH), a constituents of nerve terminals and axons, is 8- S-glutathionyl-1-methyl-1,2,3,4,-tetrahydro-β-carboline-5,6-dione ( 6). When administered into the brains of mice, 6 is a potent toxin (LD 50 = 2.9μg) and evokes episodes of hyperactivity and tremor. Compound 6 binds at the GABA B receptor and evokes elevated release and turnover of several neurotransmitters. Furthermore, the GABA B receptor antagonist phaclofen attenuates the behavioral response caused by intracerebral administration of 6. These observations suggest that 6 might be an inverse agonist at the GABA B receptor site. Accordingly, it is speculated that ethanol drinking might potentiate formation of 6 that contributes to elevated release of several neurotransmitters including dopamine (DA) and endogenous opioids in regions of the brain innervated by serotonergic axon terminals. Subsequent interactions of DA and opioids with their receptors might be related to the initial development of dependence on ethanol. Redox cycling of 6 (and of several putative secondary metabolites) in the presence of intraneuronal antioxidants and molecular oxygen to produce elevated fluxes of cytotoxic reduced oxygen species might contribute to the degeneration of serotonergic pathways. Low levels of 5-HT in certain brain regions of the rat predisposes these animals to drink or augments drinking. Accordingly, 6, formed as a result of ethanol metabolism in the cytoplasm of certain serotonergic axon terminals, might contribute to the initial development of dependence on ethanol, by mediating DA and opioid release, and long-term preference and addiction to the fluid as a result of the progressive degeneration of these neurons.

Evidence for 5-HT autoreceptor-mediated, nerve impulse-independent, control of 5-HT synthesis in the rat brain.

To gain further insight into the operation of 5-HT autoreceptor-mediated feedback control of 5-HT biosynthesis in serotonergic nerve terminal areas, the effect of the 5-HT1B and the 5-HT1A receptor agonists, TFMPP and 8-OH-DPAT, respectively, were investigated in the rat central nervous system (CNS) using in vivo and in vitro neurochemical approaches. TFMPP suppressed 5-HT synthesis (5-HTP accumulation after decarboxylase inhibition) both in vivo and in vitro. In vivo, the 5-HT synthesis-suppressing effect of the drug (3.0 mg/kg, s.c.) proved resistant to either acute hemitransection or reserpine (5 mg/kg, i.p.; 90 min before) pretreatment. In vitro, in cortical, hippocampal and striatal slice preparations, TFMPP (0.1-10 microM) decreased 5-HT synthesis under basal and stimulated (30 mM K+) conditions, an effect which was unaltered by prior in vivo reserpine-induced 5-HT depletion but was attenuated in the presence of 5-HT1B receptor antagonists such as methiothepin, cyanopindolol or propranolol. The 8-OH-DPAT (0.1 mg/kg, s.c.)-induced decrease of 5-HT synthesis in vivo was abolished by hemitransection but resistant to acute reserpine pretreatment; 8-OH-DPAT (10 microM) did not decrease 5-HT synthesis in vitro. In conclusion, the present study confirms the importance of 5-HT autoreceptors in the feedback control of nerve terminal 5-HT biosynthesis. Specifically, our data indicate: (1) that the reduction of rat brain 5-HT synthesis after TFMPP is mediated by 5-HT1B autoreceptors located on the serotonergic axon terminals, and (2) that the effect is directly mediated and occurs independently of 5-HT neuronal firing and intact monoamine stores.

Microglial response to degeneration of serotonergic axon terminals.

The neurotoxic drug p-chloramphetamine (PCA) causes widespread degeneration of fine, unmyelinated serotonergic (5-HT) axons in the forebrain. PCA toxicity is selective for 5-HT axon terminals; preterminal axons and cell bodies are spared. Degeneration is followed by slowly progressive axonal sprouting and partial reinnervation. PCA is injected subcutaneously; this route of administration avoids mechanical disruption of the blood brain barrier. The present study analyzed the response of microglia and astrocytes in rat brain to selective ablation of 5-HT axons by PCA. Several microglial markers were analyzed with immunocytochemical methods. An increase in the number of microglial processes and in immunoreactive staining was observed with antibodies directed against CR-3, MHC-I, CD4, and rat LCA. The microglial response was maximal 3 weeks after PCA treatment, became less evident 6 weeks after treatment, and by 9 weeks no difference was observed between treated and control rats. No change was detected in MHC-II or the macrophage marker ED1, nor in expression of GFAP by astrocytes. Thus, degeneration of 5-HT axon terminals affects only a subset of the microglial markers examined; in comparison, retrograde reaction to facial nerve transection causes a robust increase in all of these markers and in GFAP. The microglial response to PCA-induced axon loss is slow in onset and small in magnitude. These findings indicate that CNS microglia are activated by degeneration of fine, unmyelinated 5-HT axon terminals; furthermore, sensitive microglial markers can detect a subtle axonal lesion that provokes no detectable increase in GFAP expression by astrocytes.

The organization of serotonergic projections to cerebral cortex in primates: Regional distribution of axon terminals

Serotonergic axons are widely distributed in the primate forebrain and represent the most abundant ascending projection from the reticular formation. Immunocytochemical methods have been utilized to examine the density, laminar distribution and morphology of serotonergic axons in both primary projection (motor, somatosensory) and association areas (dorsolateral prefrontal, area 5) as well as in the hippocampus and in cingulate cortex of rhesus and cynomolgus macaques. Serotonergic axons are present in all areas of cortex examined, and all cortical layers receive serotonergic afferents. However, the intracortical distribution of serotonergic axon terminals is not uniform; rather, different regions of cortex exhibit dissimilarities in both the density and laminar distribution of serotonergic axons. Thus, there are local patterns of serotonin innervation that are characteristic of each cortical area. Highly diverse patterns of serotonin innervation are found in heterotypical areas of cortex; more subtle variations are present among homotypical areas. Two morphologic types of serotonergic axon terminals, fine and beaded, are present in all cortical areas, and they typically exhibit different laminar distributions: in most areas of neocortex, beaded axons predominate in layer I while fine axons predominate in layers II—VI. However, exceptions to this pattern were observed in primary visual cortex and in the hippocampal formation. The distinctive local patterns of serotonin innervation observed in this study indicate that raphe-cortical projections are likely to have differential influences on particular cytoarchitectonic areas of cerebral cortex in the primate. Moreover, the discrete laminar distribution of serotonin axons suggests that serotonergic projections selectively innervate particular neuronal elements in cerebral cortex. The present findings suggest that the two classes of serotonergic axons, fine and beaded, which have different patterns of termination, affect different sets of cortical neurons. In addition, these two serotonergic projections may be associated with different sets of serotonergic receptors and thus produce selective effects on cortical function.

Effects of repeated trazodone administrations on serotonergic neurotransmission: Biochemical studies

1. 1. Repeated administrations of trazodone as well as imipramine or mianserin(10 mg/kg i.p. twice daily for 3 weeks) attenuated the norepinephrine (NE) stimulation of adenylate cyclase studied in brain minces. Therefore trazodone shares with “tricyclic” (imipramine) and “atypic” (mianserin) antidepressants the capability to modulate the beta-adrenergic function. 2. 2. Daily treatments with imipramine or trazodone enhanced the Vmax of neural uptake of serotonin (5HT) in minces prepared from rat frontal cortex; in contrast mianserin failed to modify the [ 3H]-5HT uptake. 3. 3. Repeated administrations of imipramine but not of trazodone or mianserin reduced the maximum number of [ 3H]-imipramine recognition sites which are located on serotonergic axon terminals. 4. 4. Differently, only repeated administration of trazodone decreased Bmax values of [ 3H]-mianserin binding sites which are located on membranes innervated by serotonergic neurons. Moreover trazodone did not change the number or affinity of 5HT 2 receptors either after single or repeated administrations; in contrast even a single administration with mianserin or repeated administrations with imipramine down-regulated [ 3H]-ketanserin specific binding in membranes prepared from the frontal cortex. 5. 5. Our observations therefore suggest that trazodone, imipramine or mianserin exerts similar effects on the adenylate cyclase system, by acting on a interneuronal loop which links serotonergic and noradrenergic transmission function. However, its exact mechanism of action, in part resembling both tricyclic and atypic antidepressants, requires further exhamination.

Distinct morphologic classes of serotonergic axons in primates exhibit differential vulnerability to the psychotropic drug 3,4-methylenedioxymethamphetamine

Immunohistochemical methods were used to analyse the distribution and morphology of serotonergic axons in normal macaque monkeys and in monkeys given (±)3,4-methylenedioxymethamphetamine. In untreated monkeys, we observed two morphologic classes of serotonergic axon terminals, which differ in regional and laminar distribution. These two axon types, fine and beaded, correspond to the serotonergic axon types which have been described in the rat. In 3,4-methylenedioxymethamphetamine-treated monkeys, there is a profound loss of serotonergic axon terminals, yet some are consistently spared. The surviving axon terminals are nearly all of the beaded type; in contrast, fine serotonergic axons are markedly reduced in density. There are regional differences in the magnitude of denervation, which reflect differences in the distribution of these two types of serotonergic axons in controls. The present study demonstrates that 3,4-methylenedioxymethamphetamine has differential neurotoxic effects on fine and beaded serotonergic axons. These results indicate that in the primate there are two distinct classes of serotonergic axon terminals, which differ in morphology, distribution, and vulnerability to psychotropic drugs. We hypothesize that in the primate, as demonstrated in the rat, these two classes of serotonergic axon terminals may arise from different raphe nuclei. In both rodent and primate, the dorsal and median raphe nuclei give rise to parallel ascending serotonergic projections, which are likely to have different pharmacologie properties and functions.

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.

Serotonin-storing secretory granules from thyroid parafollicular cells

The subcellular storage of 5-HT was studied in sheep thyroid parafollicular cells. These cells are neural crest derivatives and were investigated as a serotonergic model system. Light and electron microscopic immunocytochemistry was used to examine the distributions of 5-HT, 45 and 56 kDa forms of 5-HT binding protein (SBP), and calcitonin. A single type of parafollicular cell was found that contained calcitonin, 5-HT, and 45 kDa SBP but not 56 kDa SBP. The secretory granules of parafollicular cells all displayed calcitonin immunoreactivity, and many were also immunoreactive for 5-HT and 45 kDa SBP. Granules were isolated, first by size and then by density, on successive metrizamide gradients. These provided a granular fraction that was enriched in calcitonin, endogenous 5-HT, and 45 kDa SBP. Immunoblots confirmed the presence of 45 kDa SBP in the isolated granules and in suspensions of parafollicular cells that were purified by an affinity chromatographic technique. Parafollicular cell granules did not appear to contain substantial stores of ATP. Granules isolated on Percoll gradients were morphologically homogeneous and took up 3H-5-HT. The specificity of this uptake was confirmed by quantitative electron microscopic radioautography. The granular uptake of 3H-5-HT was inhibited by reserpine (10 microM). It is concluded that parafollicular cell granules are different from other amine-storing vesicles that do contain ATP; nevertheless, since parafollicular cell granules store 5-HT and have the same 45 kDa SBP as is found in serotonergic axon terminals, parafollicular cell granules may be analogous to the synaptic vesicles of serotonergic neurons.

Evidence for a Selective Localization of Voltage‐Sensitive Ca2+ Channels in Nerve Cell Bodies of Corpus Striatum

Specific binding sites for [3H]nitrendipine, an organic Ca2+ channel antagonist, were abolished in crude synaptosomal membranes of kainic acid-lesioned caudate nuclei. In contrast, specific lesions of dopaminergic or serotonergic axon terminals in caudate nuclei failed to alter the density or the affinity of [3H]nitrendipine binding sites. In addition, the basal and veratridine-stimulated 45Ca2+ accumulations were greatly impaired in slices prepared from kainic acid-lesioned caudate nuclei. The veratridine-elicited accumulation of 45Ca2+ in control slices was attenuated by addition of tetrodotoxin in the incubation medium. The present data provide evidence that most of the [3H]nitrendipine binding sites and the voltage-dependent Ca2+ channels are located in intrinsic neurons or interneurons in caudate nucleus. In contrast, destruction of dopaminergic or serotonergic nerve terminals emanating from other brain areas and innervating the caudate nucleus failed to change the apparent Bmax value for [3H]nitrendipine binding.