Differentiation of monoamine accumulating neuron systems in cultured chick retina: an immunohistochemical and fluorescence histochemical study

Monoamine-containing neurons (dopamine and serotonin) have been reported to be localized among the cells in the inner nuclear layer (INL) of the chick retina. To examine the development of these neurons and whether they have an ability to synthesize serotonin (5-HT) or dopamine from exogenous precursors, we administered monoamines and their precursors in vitro as well as in situ and examined monoamine neuronal differentiation histochemically. In the chick retina, serotonin-containing neurons were found among amacrine cells. Two other types of serotonin-accumulating neurons were observed in the INL. One also had the ability to take up tryptophan and 5-hydroxytryptophan (5-HTP) and convert them to serotonin. In the monolayer culture from 61/2-day-old chick embryonic retinas, serotonin-containing neurons were observed on the 4th day of culture, increased in number until the 8th day, but had almost disappeared by the 15th day. After incubation of 8 day-cultures with 5-hydroxytryptophan, the number of serotonin-immunoreactive cells was at least twice that seen in untreated cultures. On the 20th day of culture, the same percentage of cells had the capability to take up serotonin, but the number of those which took up 5-hydroxytryptophan and decarboxylated it to serotonin had decreased markedly. Dopaminergic amacrine cells have also been found in the INL and shown to take up exogenously administered L-DOPA. In cultures, no dopamine-containing cells were observed with the present fluorescence histochemistry without prior incubation with DOPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the electrophysiological and pharmacological properties of serotonin-containing neurons in the nucleus raphe dorsalis, raphe medianus and raphe pallidus recorded from mouse brain slices in vitro: role of autoreceptors

The potential role of autoreceptors in regulating the activity of serotonin-containing nucleus raphe dorsalis (RD), raphe medianus (RM) and raphe pallidus (RPA) neurons was examined by recording the activity of these neurons under a variety of conditions both in vivo and in vitro. Raphe neurons recorded in vivo displayed the characteristic slow, rhythmic discharge pattern previously described for rat and cat raphe cells. The activity of these neurons was suppressed in a dose-dependent manner by tryptophan, LSD and chlorimipramine administered intravenously. There were no significant changes in the spontaneous discharge rate of raphe neurons over time when recorded in vitro, even though tissue serotonin and its metabolite, 5-hydroxyindoleacetic acid, decreased dramatically. RPA neurons fired significantly faster than either RD or RM neurons both in vivo and in vitro. Prior depletion of brain serotonin by p-chlorophenylalanine administration resulted in no significant change in raphe unit activity recorded in vitro. Elevation of brain serotonin by monoamine oxidase inhibition produced a total inhibition of raphe unit activity in vitro. Similarly, increasing the concentration of serotonin in the tissue slice by adding serotonin directly to the incubation medium resulted in a profound, though transitory, depression of unit activity. This depressant effect of serotonin was rapidly reversible upon drug wash-out. Serotonin receptor blockers, methiothepin, cypoheptadine, and methysergide, produced no significant change in unit activity. The serotonin reuptake blocker, fluoxetine, produced a total inhibition of raphe unit activity in all three nuclei in vitro. These data suggest that excess serotonin suppresses the activity of raphe neurons, apparently by an action on autoreceptors, but that a deficiency, or normal concentration, of serotonin does not influence the spontaneous activity of these cells. The data also show that RD and RM are much more sensitive to the depressant effects of serotonin than the caudal RPA neurons. More generally, these studies provide a data base for examining the electrophysiological and pharmacological characteristics of serotonergic neurons in the three major serotonin-containing nuclei in mouse brain. The mouse has proven to be a much easier species than the rat to use in these types of studies, based on the finding that mouse brain slices are more viable in vitro than are rat brain slices.     

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.     

The organization of indoleamine neurons in the brain of the rhesus monkey (Macaca mulatta)

The organization of indoleamine-containing neurons throughout the CNS of Macaca mulatta was examined with Falck-Hillarp histofluorescnce and radioenzymatic biochemical techniques. Indoleamine-containing cell bodies, corresponding to the serotonin-containing groups B1 to B9, were observed within the brain stem. The pontine cell populations, however, were far more numerous than in the rat and represented caudal subpopulations of cells within the nucleus raphe dorsalis (B7) and nucleus centralis superior (B8). Additionally, the pontine indoleamine-containing cells in M. mulatta extended laterally through the tegmentum such that they were often adjacent to catecholamine-containing neurons of the locus coeruleus complex. Ascending indolaemine-containing fiber bundles, similar to those defined in nonprimate mammals, were also observed within the pons and mesencephalon. Regional differences in the distribution of serotonin within hypothalamic nuclei and other forebrain regions of M. mulatta were revealed through a highly sensitive radioenzymatic assay in conjunction with microdissection. Significant concentrations of serotonin were evident in most areas, although the levels of this neurotransmitter were much lower in the median eminence region and nucleus suprachiasmaticus than in the rat. These differences may reflect variations in the control of neuroendocrine events in the two species.     

Variation of tryptophan-5-hydroxylase concentration in the rat raphe dorsalis nucleus after p-chlorophenylalanine administration. II. Anatomical distribution of the tryptophan-5-hydroxylase protein and regional variation of its turnover rate

Distribution of tryptophan-5-hydroxylase (TpOH)-containing cells and TpOH protein tissue concentrations were evaluated in the nucleus raphe dorsalis (NRD) of rat brain by immunocytochemistry and direct transfer onto nitrocellulose filters of unfixed adjacent brain sections. This work has demonstrated that: (1) the direct transfer onto nitrocellulose filters could be easily used for the quantitative analysis of TpOH protein distribution; (2) the origin of the TpOH in this brain nucleus was preferentially cellular; (3) classical subdivisions, qualitatively defined from morphometric and topographic observations could be precisely described in terms of cellular density, tissue and cellular concentrations and turnover of TpOH protein. Such differences could imply a physiological control of TpOH gene expression in the serotoninergic neurons.     

Dreams and hallucinations: A common neurochemical mechanism mediating their phenomenological similarities

Dreams and drug-induced hallucinations have several phenomenological similarities, especially with respect to their visual and emotive components. This similarity is hypothesized to be due to a neurochemical mechanism which is common to both states: the inactivation of the brain serotonin system. This is supported by electrophysiological data indicating that the activity of serotonin-containing neurons is depressed during both dreaming (in REM and non-REM sleep) and in response to hallucinogenic drugs. Further support for the hypothesis derives from neuropharmacological data demonstrating that decreases in synaptic serotonin are associated with increased hallucinatory-like behavior or hallucinatory experience during waking, and increased duration of REM periods during sleep. Reciprocally, increases in synaptic serotonin are associated with decreased hallucinatory-like behavior or hallucinatory experience, and with decreased REM sleep time and dream reports. Neuroanatomical evidence that serotonin is heavily concentrated in brain areas which mediate visual perception and emotive experience is consonant with the strong visual and emotive components of dreams and hallucinations. When these data are considered in conjunction with the exclusively inhibitory synaptic action of serotonin in the forebrain, an explicit hypothesis can be formulated: A cessation, or decrease, in the discharge rate of serotonin-containing neurons, either spontaneously during REM and non-REM sleep, or in response to drugs such as LSD, precipitates, through disinhibition, a dramatic increase in activity of their target neurons in brain areas mediating visual sensation and emotional experience. These latter neural events are a primary physiological substrate for the emergence of strong sensory and emotive processes during dreams and drug-induced hallucinations.     

Calcium regulates the activity of serotonin-containing dorsal raphe neurons in vitro

Small elevations of calcium ions (15%) significantly depressed the activity of serotonin-containing dorsal raphe neurons by 35% in mouse brain slices in vitro, while large increases in calcium ion concentration (300%) dramatically decreased the incidence of spontaneously active raphe neurons. Neurochemical studies indicated that these effects were not attributable to increased release and metabolism of serotonin. These findings may have implications for the treatment of mood disorders, for which disturbances in both calcium and serotonin metabolism have been demonstrated.     

Variation of tryptophan-5-hydroxylase concentration in the rat raphe dorsalis nucleus afterp-chlorophenylalanine administration. II. Anatomical distribution of the tryptophan-5-hydroxylase protein and regional variation of its turnover rate

Distribution of tryptophan-5-hydroxylase (TpOH)-containing cells and TpOH protein tissue concentrations were evaluated in the nucleus raphe dorsalis (NRD) of rat brain by immunocytochemistry and direct transfer onto nitrocellulose filters of unfixed adjacent brain sections. This work has demonstrated that: (1) the direct transfer onto nitrocellulose filters could be easily used for the quantitative analysis of TpOH protein distribution; (2) the origin of the TpOH in this brain nucleus was preferentially cellular; (3) classical subdivisions, qualtitatively defined from morphometric and topographic observations could be precisely described in terms of cellular density, tissue and cellular concentrations and turnover of TpOH protein. Such differences could imply a physiological control of TpOH gene expression in the serotoninergic neurons.     

Hyperinnervation of the striatum by dorsal raphe afferents after dopamine-depleting brain lesions in neonatal rats

Dopamine-depleting brain lesions produced in neonatal rats by intraventricular injection of 6-hydroxydopamine (6-OHDA) are known to increase striatal levels of serotonin by adulthood. We now report that such lesions lead to an increased innervation of the striatum by serotonin-containing neurons from the dorsal raphe nucleus. This hyperinnervation was revealed as a 300% increase in the number of retrogradely labeled neurons in the dorsal raphe after horseradish peroxidase was injected into the adult striatum. The degree of hyperinnervation was reduced sharply when serotonin-depleting brain lesions were given to adult animals that had received 6-OHDA as neonates.     

Neurochemical evidence that 5-hydroxytryptaminergic neurons tonically inhibit noradrenergic neurons terminating in the hypothalamus

The medial zona incerta (MZI) and dorsomedial nucleus of the hypothalamus (DMN), which contain cell bodies and terminals of incertohypothalamic dopaminergic (DA) neurons, are densely innervated by both noradrenergic (NE) and 5-hydroxytryptaminergic (5-HT) neurons. In view of emerging anatomical and pharmacological evidence suggesting possible interactions between 5-HT and catecholaminergic neurons, the effects of experimental procedures that inhibit or disrupt 5-HT neurons on the activities of catecholaminergic neurons terminating in these regions were examined in the present study. Catecholaminergic neuronal activity was estimated by measuring catecholamine synthesis (accumulation of 3,4-dihydroxyphenylalanine [DOPA] after administration of a decar☐ylase inhibitor) and metabolism (concentrations of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and the norepinephrine metabolite 3-methoxy-4-hydroxyphenyleneglycol (MHPG) in the MZI and DMN of both male and female rats. Inhibition of 5-HT neurons following administration of the 5-HT_1A autoreceptor agonist8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) increasedthe accumulation of DOPA in the DMN and the concentrations of DOPAC in the MZI and DMN, indicating an activation of catecholaminergic neurons in these regions. Concentrations of MHPG were increased in the MZI and DMN by 8-OH-DPAT or 5,7-dihydroxytryptamine-induced lesions of 5-HT neurons, revealing that NE neurons terminating in these regions were activated following procedures that decrease 5-HT neuronal function. Following destruction of NE neurons projecting to the MZI and DMN, 8-OH-DPAT no longer increased DOPAC concentrations in these brain regions. Taken together, these results reveal that 5-HT neurons tonically inhibit the activity of NE neurons terminating in the MZI and DMN, but do not influence the activity of incertohypothalamic DA neurons.     

Phylogeny of enteric serotonergic neurons

Serotonergic neurons have previously been identified in the enteric nervous systems of humans, subhuman primates, rodents, and rabbits. The distribution of enteric serotonergic neurons in lower vertebrates was examined in order to determine if these neurons are restricted to mammals and, if they are found more generally amongst vertebrates, when they first appear in vertebrate phylogeny. Since mammalian enteric serotonergic neurons take up ³H-serotonin by a highly specific mechanism, the radioautographic demonstration of axonal uptake of ³H-serotonin was used as the primary tool in looking for these neurons. As controls, conditions known to interfere with ³H-serotonin uptake by mammalian enteric neurons were also examined. These controls included incubation with 10 μ fluoxetine, a specific antagonist, incubation in Na⁺-free medium, and incubation in the presence of a 100-fold excess of nonradioactive serotonin. Radioautographic labeling had to be absent or greatly reduced under all three control conditions for labeling by ³H-serotonin to be considered specific. Labeled enteric axons were found in cyclostomes (hagfish), teleosts (goldfish), and amphibia (bullfrog) but not in tunicates (sea squirt; sea vase) or echinoderms (sea cucumber). In addition, the serotonin concentration was measured in the intestine of two vertebrates, hagfish and goldfish, that do not have serotonin-containing enterochromaffin cells. Serotonin was found in both; in hagfish, the amine concentration was highest in preparations of muscularis externa containing the myenteric plexus. It is concluded that enteric serotonergic neurons arose early in vertebrate evolution, possibly in an ancestral chordate resembling amphioxus, although probably not in more primitive prevertebrates, and that they are a general feature of the vertebrate bowel.     

Origin of serotonin innervation of the arcuate and ventromedial hypothalamic region

Combined fluorescence serotonin immunohistochemistry and retrograde transport labelling with Fast blue and Fluoro-gold were used to identify serotonin-immunoreactive neurons in the midbrain and pons which project to the region of the arcuate and ventrome-dial hypothalamic nuclei. Approximately 90% of doubly labelled neurons were located in the 3 major mesencephalic serotonin-containing cell groups: dorsal raphe (38%), median raphe (21%) and medial lemniscus group (29%). Within these groups, there were numerous non-retrogradely labelled serotonin-immunoreactive neurons as well as numerous non-serotonin-immunoreactive retrogradely labelled neurons. No doubly labelled neurons were observed caudal to raphe pontis although non-serotonin-immunoreactive neurons were retrogradely labelled in the more caudal raphe nuclei.     

Serotonin‐immunoreactive sensory neurons in the antenna of the cockroach Periplaneta americana

The antennae of insects contain a vast array of sensory neurons that process olfactory, gustatory, mechanosensory, hygrosensory, and thermosensory information. Except those with multimodal functions, most sensory neurons use acetylcholine as a neurotransmitter. Using immunohistochemistry combined with retrograde staining of antennal sensory neurons in the cockroach Periplaneta americana, we found serotonin‐immunoreactive sensory neurons in the antenna. These were selectively distributed in chaetic and scolopidial sensilla and in the scape, the pedicel, and first 15 segments of the flagellum. In a chaetic sensillum, A single serotonin‐immunoreactive sensory neuron cohabited with up to four serotonin‐negative sensory neurons. Based on their morphological features, serotonin‐immunopositive and ‐negative sensory neurons might process mechanosensory and contact chemosensory modalities, respectively. Scolopidial sensilla constitute the chordotonal and Johnston's organs within the pedicel and process antennal vibrations. Immunoelectron microscopy clearly revealed that serotonin‐immunoreactivities selectively localize to a specific type of mechanosensory neuron, called type 1 sensory neuron. In a chordotonal scolopidial sensillum, a serotonin‐immunoreactive type 1 neuron always paired with a serotonin‐negative type 1 neuron. Conversely, serotonin‐immunopositive and ‐negative type 1 neurons were randomly distributed in Johnston's organ. In the deutocerebrum, serotonin‐immunoreactive sensory neuron axons formed three different sensory tracts and those from distinct types of sensilla terminated in distinct brain regions. Our findings indicate that a biogenic amine, serotonin, may act as a neurotransmitter in peripheral mechanosensory neurons. J. Comp. Neurol. 522:414–434, 2014. © 2013 Wiley Periodicals, Inc.     

Effects of gonadal steroids on the in vivo binding of [125I]alpha-bungarotoxin to the suprachiasmatic nucleus

Serotonergic neurons throughout the brain were destroyed by early postnatal treatment of rats with an intracisternal injection of 5,7-dihydroxytryptamine (5,7-DHT), as demonstrated with biochemical measurements of serotonin and immunocytochemical localization of serotonin-containing neurons. Using these methods, it was shown that approximately 75-98% of serotonergic neurons underwent cell death in rats which were treated on day 3. In contrast, intracisternal administration of 5,7-DHT in adult rats led to the loss of distal serotonergic terminals without apparent loss of the cell bodies. Desipramine pretreatment prevented significant effects of 5,7-DHT on noradrenergic neurons.     

Immunocytochemical localization of GTP cyclohydrolase I in the brain,adrenal gland,and liver of mice

Summary GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH₄), the cofactor of phenylalanine, tyrosine, and tryptophan hydroxylases, the enzymes that synthesize tyrosine, catecholamines (dopamine, noradrenaline, and adrenaline), and serotonin, respectively. We produced for the first time polyclonal antibody with highly sensitive immunoreactivity against an oligopeptide of rat enzyme, GFPERELPRPGA, by immunization of rabbits with the peptide conjugated to hemocyanin by glutaraldehyde. The specificity of the antibody was confirmed by Western blot analysis. Using this antibody specific for GCH, we observed strong GCH immunostaining in the liver cells, in the dopamine-, noradrenaline-, adrenaline-, or serotonin-containing cells of the brain, and in the adrenal gland of mice. Immunocytochemical studies revealed GCH to be localized in monoamine-containing perikarya in the periglomerular cells of the olfactory bulb, zona incerta, arcuate nucleus, ventral tegmental area, substantia nigra pars compacta, locus ceruleus, nucleus tractus solitarius, area postrema, and ventrolateral area of the medulla oblongata. GCH immunostaining was particularly strong in serotoninergic nuclei, such as dorsal and median raphe nuclei, nucleus raphe pallidus, and nucleus raphe magnus. By immunoelectron micoscopy, GCH-labeled cytoplasm and microtubules in the processes were observed ultrastructurally, but no staining was found in the mitochondria, and Golgi apparatus. Immunostaining was observed neither in the group D neurons that contain only aromatic amino acid decarboxylase without tyrosine hydroxylase, nor in glial cells and endothelial cells. These results indicate the abundant presence of GCH in catecholaminergic and serotoninergic neurons as well as in the adrenal medulla and liver, where BH₄ is synthesized as the cofactor of tyrosine, tryptophan, and phenylalanine hydroxylases.     

Variation of tryptophan-5-hydroxylase concentration in the rat raphe dorsalis nucleus after p-chlorophenylalanine administration. I. A model to study the turnover of the enzymatic protein

An immunoblot procedure was developed to quantify the amount of tryptophan hydroxylase (TpOH), the rate limiting enzyme in the synthesis of serotonin, in the rat raphe dorsalis nucleus (NRD). Using this method we have studied the time course variations in TpOH protein level after a single p-chlorophenylalanine (PCPA) i.p. injection (300 mg/kg). PCPA provoked a rapid and large decrease of TpOH in the NRD, without affecting neuron-specific enolase in the NRD or TpOH in the locus coeruleus. The decrease in TpOH was maximum (-60% of the control value) 2 days after the drug administration and followed a monoexponential law which allowed us to estimate the half-life of this enzymatic protein as 1.43 days and to postulate that, during these 2 days, TpOH synthesis was inhibited. The neosynthesis of TpOH molecules from 2 to 7 days was estimated to be 57.8 U TpOH/NRD/day which was comparable to the initial steady state of synthesis (48.44 U TpOH/NRD/day). In vivo administration of 6-fluorotryptophan or in vitro incubation of raphe homogenates with either halogenated derivative had no effect on TpOH protein levels. PCPA should be an interesting tool to study the turnover rate of TpOH protein.     

Atlas of serotonin-containing neurons in the optic lobes and brain of the crayfish, Cherax destructor

An atlas of neurons in the brain of the crayfish Cherax destructor that are immunoreactive to antibodies raised against serotonin has been compiled from whole mount preparations. Neuronal networks of serotonin-containing cells are identified in the optic lobes and protocerebrum, in the deutocerebrum, and in the tritocerebrum. The consistency of the whole-mount technique allows 50 out of a total of about 100 immunoreactive cells to be individually identified according to their neuronal architecture or the location of their cell somata or axons. Apart from six neurons with axons in the oesophageal connectives, all the immunoreactive cells are intrinsic to the optic lobes and brain.     

Early postnatal administration of 5,7-DHT: Effects on serotonergic neurons and terminals

Serotonergic neurons throughout the brain were destroyed by early postnatal treatment of rats with an intracisternal injection of 5,7-dihydroxytryptamine (5,7-DHT), as demonstrated with biochemical measurements of serotonin and immunocytochemical localization of serotonin-containing neurons. Using these methods, it was shown that approximately 75–98% of serotonergic nuerons underwent cell death in rats which were treated on day 3. In contrast, intracisternal administration of 5,7-DHT in adult rats led to the loss of distal serotonergic terminals without apparent loss the cell bodies. Desipramine prevented significant effects of 5,7-DHT on noradrenergic neurons.