The habenulo-raphe serotonergic circuit encodes an aversive expectation value essential for adaptive active avoidance of danger.

Anticipation of danger at first elicits panic in animals, but later it helps them to avoid the real threat adaptively. In zebrafish, as fish experience more and more danger, neurons in the ventral habenula (vHb) showed tonic increase in the activity to the presented cue and activated serotonergic neurons in the median raphe (MR). This neuronal activity could represent the expectation of a dangerous outcome and be used for comparison with a real outcome when the fish is learning how to escape from a dangerous to a safer environment. Indeed, inhibiting synaptic transmission from vHb to MR impaired adaptive avoidance learning, while panic behavior induced by classical fear conditioning remained intact. Furthermore, artificially triggering this negative outcome expectation signal by optogenetic stimulation of vHb neurons evoked place avoidance behavior. Thus, vHb-MR circuit is essential for representing the level of expected danger and behavioral programming to adaptively avoid potential hazard.

[The effect of serotonin precursor 5-hydroxytryptophan and neurotoxic analogue 5,7-dihydroxytryptamine on defensive conditioning in a snail].

This study is devoted to investigation of the influence of precursor of serotonin 5-hydroxytryptophane (5-HTP) and neurotoxic analogue 5,7-dihydroxytryptamine (5,7-DHT) on defensive conditioning and electrical characteristics of command neurones of defensive behaviour after learning. Snails injected with 5-HTP learned faster as compared to control group injected with physiological solution. After the 5,7-DHT injection, snails failed to form the conditioned reflex. Injection of 5-HTP after the preliminary injection of neurotoxin 5,7-DHT restored the capability of snails for learning. Injections of 5-HTP prevented the effect of 5,7-DHT at the behavioural level, but not at the level of electrical characteristics of the command neurones.

Serotonin--more than a neurotransmitter: transglutaminase-mediated serotonylation of C6 glioma cells and fibronectin.

In the central nervous system serotonin plays important roles as a neurotransmitter as well as during neuronal development and in synaptogenesis. Outside the central nervous system, serotonin is covalently transamidated to procoagulant proteins involved in blood clotting. This process is mediated by transglutaminases and named "serotonylation". Serotonylated proteins then tightly bind to specific serotonin binding sites on fibrinogen and thrombospondin to form stable extracellular multivalent complexes needed for thrombus formation. Here, we have investigated whether transglutaminases can also covalently incorporate extracellular serotonin to neural proteins and whether this might affect extracellular protein expression. Our data reveal that recombinant transglutaminase specifically transamidates [(3)H]-serotonin to cell-surface proteins from C6 glioma cells and the extracellular matrix protein fibronectin. Serotonylation of [(3)H]-serotonin was inhibited by the transglutaminase inhibitor cystamine and unlabelled serotonin. Transglutaminase-mediated transamidation of unlabelled serotonin to C6 cells induced an aggregation of extracellular protein matrices adjacent to and between single cells. Transglutaminase also transamidated the autofluorescent serotonin analogue 5,7-dihydroxytryptamine and monodansylcadaverine (MDC) into living C6 glioma cells. Electrophoretic separation of MDC-labelled C6 cells identified several distinct fluorescent proteins one of which was fibronectin.

Serotonin1B heteroreceptor activation induces an antidepressant-like effect in mice with an alteration of the serotonergic system.

OBJECTIVE: We sought to demonstrate whether the specific activation of serotonin1B (5-HT1B) heteroreceptors by systemic or local administration of the selective 5-HT1B receptor agonist anpirtoline could mediate antidepressant-like effects in mice. METHODS: We confirmed the selectivity of action of anpirtoline in the forced swim test (FST) in 5-HT1B knockout mice. We then evaluated the behavioural effects of anpirtoline on 5-HT-lesioned (5,7-dihydroxytryptamine creatinine [5,7-DHT]) and 5-HT-depleted (p-CPA) mice. We estimated the depletion level and selectivity of action of 5,7-DHT and p-CPA by measuring the neurotransmitter levels and [3H]-citalopram binding. We investigated the antidepressant-like effect of anpirtoline when locally perfused in an area of the brain where the response is mainly attributable to presynaptic (cortex and hippocampus) or postsynaptic receptors (substantia nigra and caudate putamen). Furthermore, we evaluated the effect of the 5-HT1B receptor antagonist GR127935 on the activity of various antidepressants in the FST. RESULTS: Anpirtoline was devoid of effects in 5-HT1B receptor knockout mice. It induced a greater effect in p-CPA and 5,7-DHT pretreated mice compared with control subjects, suggesting that the antidepressant-like activity of anpirtoline mainly depends on 5-HT1B heteroreceptor stimulation (autoreceptors being destroyed by 5,7-DHT). This observation was confirmed by the results showing the antidepressant-like effect of anpirtoline when locally perfused in areas of the brain that contain postsynaptic receptors. The blockade of 5-HT1B receptors antagonizes the effect of selective serotonin reuptake inhibitors (SSRIs). CONCLUSION: Our results demonstrate that the antidepressant-like effect of SSRIs in the FST requires the activation of 5-HT1B heteroreceptors.

Hydroxylated serotonin and dopamine as substrates and inhibitors for human cytosolic SULT1A3.

Sulfation as catalyzed by the cytosolic sulfotransferases (SULTs) is known to play an important role in the regulation and homeostasis of monoamine neurotransmitters. The current study was designed to examine the occurrence of the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine by human cytosolic SULTs and to investigate the inhibitory effects of these hydroxylated derivatives on the sulfation of their unhydroxylated counterparts, serotonin and dopamine. A systematic study using 11 known human cytosolic SULTs revealed SULT1A3 as the responsible enzyme for the sulfation of 7-hydroxyserotonin and 6-hydroxydopamine. The pH-dependence and kinetic constants of SULT1A3 with 7-hydroxyserotonin or 6-hydroxydopamine as substrate were determined. The inhibitory effects of 7-hydroxyserotonin and 6-hydroxydopamine on the sulfation of serotonin and dopamine were evaluated. Kinetic analyses indicated that the mechanism underlying the inhibition by these hydroxylated monoamine derivatives is of a competitive-type. Metabolic labeling experiments showed the generation and release of [(35) S]sulfated 7-hydroxyserotonin and [(35) S]sulfated 6-hydroxydopamine when SK-N-MC human neuroblastoma cells were labeled with [(35) S]sulfate in the presence of 7-hydroxyserotonin or 6-hydroxydopamine. Upon transfection of the cells with siRNAs targeted at SULT1A3, diminishment of the SULT1A3 protein and concomitantly the sulfating activity toward these hydroxylated monoamines was observed. Taken together, these results indicated clearly the involvement of sulfation in the metabolism of 7-hydroxyserotonin and 6-hydroxydopamine. By serving as substrates for SULT1A3, these hydroxylated monoamines may interfere with the homeostasis of endogenous serotonin and dopamine.

Staphylococcal enterotoxin induces emesis through increasing serotonin release in intestine and it is downregulated by cannabinoid receptor 1.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are the most recognizable bacterial superantigenic toxins causing food poisoning in humans throughout the world. However, it remains unclear how SEs induce emesis and its emetic signal pathway. We investigated a mechanism of SEA-induced emesis using a small emetic animal model, house musk shrew. SEA-induced emesis in the animals was inhibited by a 5-hydroxytryptamine (5-HT) synthesis inhibitor and a 5-HT(3) receptor antagonist. SEA could increase 5-HT release in the small intestine. Pre-treatment with 5,7-dihydroxytryptamine (5,7-DHT) markedly inhibited SEA-induced emesis. SEA-induced emesis was also abolished by surgical vagotomy. Furthermore, cannabinoid (CB) receptor agonists inhibited SEA-induced emesis, and the action was reversed by a CB1 antagonist. Both 5-HT release and CB1 receptor expression were found in the mucosal and myenteric plexus of the intestine. Moreover, a CB1 receptor agonist significantly decreased the 5-HT release in the intestine. These results demonstrate that SEA induces 5-HT release in intestine, rather than in brain, and that the 5-HT(3) receptors on vagal afferent neurons are essential for SEA-stimulated emesis. In addition, SEA-induced emesis is downregulated by the CB system through decreasing 5-HT release in intestine.

5,7-Dihydroxytryptamine--a selective marker of dopaminergic or serotonergic neurons?

The aim of the present study was to investigate whether 5,7-dihydroxytryptamine (5,7-DHT), an autofluorescent serotonin derivative, can be used as a specific marker for serotonergic or dopaminergic neurons in rat mesencephalic cultures. To this end, primary cultures were prepared from the ventral brain stem of 14-day-old Wistar rat foetuses and kept in culture for 10 days (DIV10). At DIV10, the cultures were characterized immunocytochemically with antibodies raised against tyrosine hydroxylase (TH; a marker for catecholaminergic/ dopaminergic neurons) and serotonin (5-HT). 5,7-DHT labelling of the neurons was investigated after incubation with 25 microM of the serotonin derivative (plus 0.005% ascorbic acid) for 60 min at 37 degrees C, followed by incubation with primary antibodies against TH or serotonin and a fluorescence (Cy3)-labelled secondary antibody. Using confocal laser scanning microscopy, this double immunofluorescence approach demonstrated that all cells which had accumulated 5,7-DHT additionally displayed anti-5-HT immunoreactivity, whereas no evidence was found for 5,7-DHT labelling of TH immunoreactive cells. Preincubation with the selective serotonin reuptake inhibitor fluvoxamine maleate (10 microM) prevented the loading of the 5-HT-positive cells with 5,7-DHT. In conclusion, the present data indicate that 5,7-DHT specifically labels serotonergic cells in rat midbrain cultures. Thus, 5,7-DHT can be used for the identification of living serotonergic neurons even in the presence of dopaminergic neurons.

Modulations of spinal serotonin activity affect the development of morphine tolerance.

To test whether modulations of spinal serotonin (5-HT) levels would affect the development of morphine tolerance, we treated rats with either intrathecal 5-HT or 5,7-dihydroxytryptamine (5,7-DHT; a 5-HT neurotoxin) in addition to systemic infusion with morphine (2 mg x kg(-1) x h(-1)). Continuous infusion of 5-HT (10 microg x 6 microL(-1) x h(-1)) into the lumbar subarachnoid space of rats for 9 h accelerated the development of morphine tolerance. The area under the curve for the tail-flick latency test was 454.1 +/- 35.1 in the Sham Control group vs 327.6 +/- 41.0 in the 5-HT-Infused group. mu-opioid receptor binding in the lumbar spinal cord showed a decrease in the Bmax (maximal binding -46.5%), but not the binding affinity (Kd), in 5-HT-infused rats. However, intrathecal injection of 5,7-DHT (50 microg), which resulted in a 48% reduction in 5-HT and 51% reduction in 5-hydroxyindoleacetic acid concentrations, led to an attenuation of morphine tolerance (the area under the curve was 613.0 +/- 24.7 in the 5,7-DHT-Lesioned group). The binding study indicated that the affinity of lumbar micro-opioid receptors decreased 196% in 5-HT-depleted rats, whereas there was no effect on apparent binding. The infusion of 5-HT (10 microg x 6 microL(-1) x h(-1)) was not analgesic and the 5,7-DHT-induced lesion did not affect acute morphine-induced analgesia. We conclude that activity of spinal 5-HT-containing neurons plays a crucial role during the development of morphine tolerance.

Determination of NADH in frozen rat brain sections by laser-induced fluorescence.

Methods to assess metabolism are important analytical tools in neuroscience. The fluorophore nicotinamide adenine dinucleotide (NADH) is a parameter of cellular metabolism. NADH fluorescence was measured using a laser-based fluorescence detector with spectral and temporal filters. Distribution and intensity of NADH fluorescence were investigated in frozen brain sections. In sections containing hippocampus the intensity of NADH fluorescence was correlated to brain structures. In order to investigate the consequences of neurotoxic lesions, 5,7-dihydroxytryptamine was injected into the dorsal raphe nucleus 4 to 240 days prior to the measurement. NADH fluorescence decreased in the affected region by 50%, indicating that no recovery in metabolic activity had occurred.

Brain-derived neurotrophic factor and trkB are essential for cAMP-mediated induction of the serotonergic neuronal phenotype.

Serotonergic neurons in the central nervous system are crucial in the control of autonomic functions and behavior. Mechanisms by which development and maintenance of the serotonergic transmitter phenotype is regulated include activation of protein kinase A (PKA). Using cultures established from the E14 rat raphe we show here that forskolin (10 microM) increases numbers of neurons expressing tryptophan hydroxylase (TpOH), the key enzyme of serotonin synthesis, and uptake of the false serotonergic transmitter 5, 7-dihydroxytryptamine (5,7-DHT). As shown by short-term treatments the effect is due to phenotype induction rather than survival. To begin to understand downstream or parallel signaling pathways required for the PKA-mediated induction of serotonergic markers, we have studied the putative implication of brain-derived neurotrophic factor (BDNF) and its receptor trkB. Treatment of raphe neurons with forskolin induced BDNF mRNA assayed by competitive RT-PCR. Moreover, trkB-IgG receptor bodies fully prevented the forskolin-induced numerical increase in TpOH- and 5,7-DHT-positive cells suggesting an implication of a TrkB-activated pathway. TrkC-IgG had no effect. K252b, a specific inhibitor of trk kinase activity likewise abolished the induction of serotonergic markers by forskolin. In turn, the inductive effect of BDNF on serotonergic markers was blocked by KT5720, a specific inhibitor of PKA. Taken together, these data suggest that co-activation of cAMP- and trkB-dependent signaling pathways plays a crucial role in the regulation of the serotonergic neuronal phenotype.

Effect of central 5-hydroxytryptamine depletion on inter-temporal choice: a quantitative analysis.

RATIONALE: It has been proposed that the ascending 5-hydroxytryptaminergic (5-HTergic) pathways are involved in "impulse control". Previous experiments have shown that rats whose 5-HTergic pathways have been destroyed are more liable than intact rats to select a smaller, immediate reinforcer rather than a larger, delayed reinforcer (impulsive choice). However, it remains unclear whether this effect of central 5-HT depletion reflects a change in the rate of time discounting (i.e. a change in the rate at which reinforcers become devalued as a function of delay) or a change in sensitivity to reinforcer size. OBJECTIVE: We examined the effect of central 5-HT depletion on time discounting using a quantitative model of inter-temporal choice (multiplicative hyperbolic model), which enables effects on time discounting to be differentiated from effects on sensitivity to reinforcer size. METHODS: Rats received injections of 5,7-dihydroxytryptamine into the dorsal and median raphe nuclei or sham lesions. They were trained to press two levers for food-pellet reinforcers in a discrete-trials adjusting-delay schedule. In free-choice trials, selection of lever A resulted in a brief fixed delay (dA) followed by delivery of one pellet; selection of lever B resulted in a longer variable delay (dB) followed by delivery of two pellets; dB was adjusted in accordance with the subject's choices. The value of dA was varied (0.5-8.0 s) in successive phases of the experiment, and the indifference value of dB was determined in each case. RESULTS: In both groups, the indifference value of dB was linearly related to the value of dA, in accordance with the multiplicative hyperbolic model. The lesioned group showed shorter indifference delays than the sham-lesioned group, this being reflected in a parallel displacement of the linear indifference function. In both experiments, the levels of 5-HT and 5-hydroxyindole-acetic acid were reduced in the brains of the lesioned rats, but the levels of noradrenaline and dopamine were not altered. CONCLUSIONS: According to the multiplicative hyperbolic model, parallel displacement of the linear indifference function uniquely specifies a change in time discounting. Thus these results indicate that central 5-HT depletion results in an increase in the rate of time discounting for food reinforcers.

Pindolol-insensitive [3H]-5-hydroxytryptamine binding in the rat hypothalamus; identity with 5-hydroxytryptamine7 receptors.

Pindolol-insensitive [3H]-5-hydroxytryptamine ([3H]-5-HT) binding to rat hypothalamic membranes was pharmacologically and functionally characterized to resolve whether this procedure selectively labels 5-HT7 receptors. Consistent with a previous report, 3 microM and not 100 nM pindolol was required to occupy fully 5-HT1A and 5-HT1B receptors. Remaining [3H]-5-HT binding was saturable (KD, 1.59+/-0.21 nM; Bmax, 53.8+/-3.1 fmol x mg protein(-1)). Displacement of [3H]-5-HT with metergoline and 5-CT revealed shallow Hill slopes (<0.5) but seven other compounds had slopes >0.8 and pKi values and the rank order of affinity were significantly correlated (r = 0.81 and 0.93, respectively) with published [3H]-5-HT binding to rat recombinant 5-HT7 receptors. In the presence of pindolol, 5-HT-enhanced accumulation of [32P]-cyclic AMP was unaffected by the 5-HT4 antagonist RS39604 (0.1 microM) or the 5-ht6 antagonist Ro 04-6790 (1 microM) but significantly attenuated by mesulergine (250 nM), ritanserin (450 nM) or methiothepin (200 nM) which have high affinity for the 5-HT7 receptor. Intracerebroventricular pretreatment with the serotonergic neurotoxin 5,7-dihydroxytryptamine, 5,7-DHT, elevated the [3H]-5-HT Bmax 2 fold, indicating that the hypothalamic 5-HT7 receptor is post-synaptic to 5-HT nerve terminals and regulated by synaptic 5-HT levels. These results suggest that, in the presence of 3 microM pindolol, [3H]-5-HT selectively labels hypothalamic binding sites consistent with functional 5-HT7 receptors.

Indoleamine-accumulating cell death and endogenous glial cell reaction induced by 5,7-dihydroxytryptamine in the cat retina.

We investigated the patterns of degenerative changes of indoleamine-accumulating cells (IACs) induced by 5,7-dihydroxytryptamine (5,7-DHT, 100 microg), and the glial reaction to the neurodegenerative changes of IACs in the cat retina by using light-and electron-microscopy. The neurons accumulating 5,7-DHT in the cat retina were a few ganglion cells and displaced amacrine cells located in the ganglion cell layer (GCL), and some amacrine cells in the inner nuclear layer (INL). The cell density (per unit area, 1 mm2) of the 5,7-DHT accumulating cells in the GCL and INL was 910 and 134 cells, respectively. Most 5,7-DHT accumulating cells showed dark degeneration characterized by widening of the cellular organelles at early stage, and by darkening of the cytoplasm at a late stage. In addition, amacrine cells, showing a typical filamentous degeneration, were observed in a few cases. The degenerated neurons were phagocytosed by microglial cells and astrocytes. The immunoreactivity for glial fibrillary acidic protein (GFAP) in Muller cells was increased at early stage, but thereafter abruptly decreased. In a few cases, severe degenerative changes were observed in Miller cells. These results indicate that 5,7-DHT induces severe dark degeneration of IACs, and most degenerated cells could be eliminated by microglial cells and astrocytes in the cat retina.

Effects of 5-HT denervation of the suprachiasmatic nuclei or lesions of the median raphe nucleus on daily torpor in the Djungarian hamster, Phodopus sungorus.

In the Djungarian hamster (Phodopus sungorus), using 5,7-dihydroxytryptamine (a specific neurotoxin), depletion of the serotonin (5-HT) fibers innervating the suprachiasmatic nuclei (SCN) was performed in animals displaying torpor bouts. The characteristics of postoperative torpor bouts in lesioned hamsters were similar to those observed before the denervation. 5-HT innervation of the SCN thus seems unnecessary for the expression and temporal organization of daily torpor. After chemical or thermic lesions of the 5-HT neurons of the anterior median raphe nucleus (MR) in hamsters displaying torpor bouts, the phenomenon of torpor was not prevented. In addition, hamsters lesioned on the day of transfer to short photoperiod (SP) displayed their first torpor bouts after the same duration of SP exposure as intact hamsters. These results suggest that, contrary to what is observed in hibernation, 5-HT neurons of the anterior part of the MR are not involved in the mechanisms governing the occurrence and seasonal timing of daily torpor.

Electrophysiological and histochemical properties of postnatal rat serotonergic neurons in dissociated cell culture.

Serotonin modulates a variety of neural processes, and is present in a subpopulation of neurons in the raphe nuclei. To study their electrophysiological properties, cells from the mesopontine raphe nuclei of the neonatal rat were dissociated and grown for up to 10 weeks in microcultures. Approximately one third of the neurons were identified as serotonergic based on the presence of serotonin immunoreactivity, tryptophan hydroxylase immunoreactivity, or a high affinity monoamine transporter. About 5% of cultured raphe neurons contained tyrosine hydroxylase immunoreactivity, while 25% contained GABA immunoreactivity. However, no neurons contained both serotonin and tyrosine hydroxylase staining, and less than 1% displayed both serotonin and GABA immunoreactivities. Cultured serotonergic neurons did not exhibit pacemaker firing in the presence of alpha 1 adrenergic receptor agonists such as phenylephrine or norepinephrine. Approximately one third were hyperpolarized by serotonin or the selective serotonin1A receptor agonist, (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin. Virtually all serotonergic neurons responded to application of glutamate, kainate, N-methyl-D-aspartate, GABA, and glycine. Depolarizing and hyperpolarizing synaptic potentials blocked by glutamate or GABAA receptor antagonists were frequently observed in both serotonergic and non-serotonergic raphe neurons. Slow inhibitory postsynaptic potentials were evoked by activating single presynaptic serotonergic neurons with a brief intracellular current pulse. The slow inhibitory synaptic potential had a mean latency to onset of 35 +/- 5 ms, a duration of 0.8-2.6 s, and was inhibited by the serotonin1A autoreceptor antagonists, (-)propranolol and spiperone. The rising and falling phases of the inhibitory potential could be fit by single exponential functions with mean time constants of 53 +/- 8 ms and 504 +/- 78 ms, respectively. Serotonin1A receptor-mediated autoinhibition was observed in microcultures containing a solitary serotonergic neuron, and thus constituted synaptic serotonin release, responsiveness, and re-uptake by a single vertebrate neuron. In summary, histochemical and electrophysiological evidence was obtained for catecholaminergic, GABAergic, and glutamatergic non-serotonergic raphe neurons in culture, many of which formed functional synaptic connections with neighboring cells. Additionally, cultured mesopontine serotonergic neurons expressed many of the cytochemical markers, neurotransmitter receptors, and synaptic functions observed in such cells in vivo, but the proportion of neurons sensitive to serotonergic and adrenergic agonists was significantly less than that reported in vivo. For the first time, the kinetics and pharmacology of serotonergic synaptic transmission by a single vertebrate serotonergic raphe neuron were determined, and found to resemble those observed after extracellular stimulation of populations of raphe neurons in slices and in vivo.

Differential effects of 5,7-dihydroxytryptamine-induced serotoninergic degeneration of 5-HT1A receptors and 5-HT uptake sites in the rat brain.

The time-course of 5,7-dihydroxytryptamine-induced lesions (2, 5 and 14 days after i.c.v. injection of 150 micrograms) and the effects of acute reserpine treatment (10 mg/kg, i.p., one or 5 days before scheduled death), were evaluated by autoradiography of [3H]paroxetine binding sites in the rat brain. Reserpine had no significant effect on [3H]paroxetine binding, indicating that the depletion of serotonin is not sufficient per se to alter the serotonin uptake sites in any region. Two days after the 5,7-dihydroxytryptamine lesion, [3H]paroxetine binding was already decreased in the majority of brain regions. In the caudate putamen these binding sites were significantly decreased only 14 days after the lesion, whereas the ventral tegmental area (or the enclosed median forebrain bundle), the dorsal raphe (mainly the ventral portion) and the median raphe maintained their high density of serotonin uptake sites even after 14 days. Results were similar using [3H]citalopram as ligand for the serotonin uptake sites, in the brains of rats lesioned 5 days before death; an exception was the ventral portion of the dorsal raphe, where there was a significant increase with [3H]paroxetine and a decrease with [3H]citalopram binding. In adjacent sections of the same brains we also measured [3H]8-OH-DPAT binding, confirming that it completely disappears in the dorsal raphe after the lesion. Thus, considering the extent of serotonin cell body degeneration, there appears to be a paradoxical mismatch between the excessive loss of [3H]8-OH-DPAT binding and the resistance of [3H]citalopram or [3H]paroxetine binding in the dorsal raphe, suggesting that the two binding sites may undergo adaptive regulation in surviving neurons.

Midbrain dopaminergic neurons from postnatal rat in long-term primary culture.

Midbrain dopamine neurons project extensively throughout the vertebrate forebrain and influence a wide variety of brain functions. These neurons, which are believed to form a major brain reward system, are involved in initiation and control of motor programs, addictive behaviors, and determination of mood. Given their critical role in behavioral function, relatively little is known about their fundamental cellular physiological and pharmacological properties. A long-term dissociated culture system for postnatal rat dopamine neurons was developed to permit both acute and chronic studies of these cells. Dopamine neurons were dissociated from slices of ventral midbrain from neonatal rat pups and maintained in cell culture for several months. The dopaminergic phenotype was confirmed by catecholamine fluorescence and by tyrosine hydroxylase immunocytochemistry. After four weeks in culture, dopamine neurons had cell bodies 10-40 microns in diameter, displayed either fusiform or multipolar morphology, and had processes with varicosities of 0.5-2 microns in diameter. Electrophysiological recordings were made from 71 dopamine neurons identified by 5,7-dihydroxytryptamine fluorescence after six to 67 days in culture. The neurons had resting potentials of -51 +/- 5 mV, broad action potentials with durations of 2.9 +/- 1.3 ms, and the majority of the neurons (65%) displayed anomalous rectification. Most dopamine neurons in culture fired spontaneously in a pacemaker-like manner with a frequency of 2.3 +/- 1.3 Hz, or in a bursting pattern, typically having two to seven action potentials per burst. All neurons tested had glutamate and gamma-aminobutyric acid receptors, and 90% of neurons responded to dopamine or quinpirole with inhibition of firing, suggesting the presence of dopamine autoreceptors. Some neurons were inhibited by concentrations of quinpirole as low as 10 nM. The results show that midbrain dopamine neurons can be maintained in dissociated cell culture for periods of several months. These neurons can be identified prior to electrophysiological recording, and they express many of the physiological characteristics that have been reported for midbrain dopamine neurons in vivo.

Synaptic contacts of serotonin-like immunoreactive and 5,7-dihydroxytryptamine-accumulating neurons in the anuran retina.

The synapses of serotonin-like immunoreactive retinal neurons were studied in Bufo marinus and Xenopus laevis and those of 5,7-dihydroxytryptamine-labelled cells in Xenopus. Immunoreactivity to serotonin was mostly confined to amacrine cells. Synapses formed by profiles of labelled cells were almost uniformly distributed in the inner plexiform layer in both species. Interamacrine synapses were the most frequent, and in some cases two labelled amacrine cell profiles made a gap junction. Some of the labelled amacrine cells synapsed on to presumed ganglion cell dendrites and onto bipolar cell terminals. Labelled bipolar cell terminals synapsed on to non-labelled amacrine cell dendrites and received inputs both from labelled and non-labelled amacrine cells. Labelled bipolar cell profiles were not observed in the outer plexiform layer. After preloading and photoconversion of 5,7-dihydroxytryptamine in the Xenopus retina, labelled bipolar cell dendrites in the outer plexiform layer were observed to be postsynaptic to cone pedicles and less frequently to rods and horizontal cells. In the inner plexiform layer, synapse types formed by labelled bipolar cells were similar to those with serotonin immunoreactivity. The frequency of synapses formed by 5,7-dihydroxytryptamine-labelled amacrine cells increased, compared with serotonin immunocytochemistry. Labelled amacrine cells synapsed mostly with non-labelled amacrine cells, although the ratio of contacts formed by two labelled profiles increased. Synapses from labelled amacrine cell dendrites to non-labelled bipolar cell terminals and from non-labelled bipolar cell terminals to labelled amacrine cell profiles increased in number, while those from labelled amacrine cells to presumed ganglion cell dendrites decreased. The quantitative data obtained by the two approaches enabled us to propose different neuronal circuits for serotonin-synthesizing and -accumulating neurons of the Xenopus retina.

5,7-Dihydroxytryptamine lesions in the fornix-fimbria attenuate latent inhibition.

When animals are preexposed to a stimulus without consequence they are subsequently slower to associate this stimulus with an important event, such as footshock. This retarding effect of stimulus preexposure is called latent inhibition and can be demonstrated in a variety of classical and instrumental paradigms and in a wide range of species, including man. Latent inhibition is disrupted in acute schizophrenics and by amphetamine treatment in both rat and man. The present study investigated the role of hippocampal 5HT terminals in latent inhibition using a conditioned suppression procedure with male Sprague-Dawley rats. Microinjections of 5,7-dihydroxytryptamine in the fornix-fimbria significantly reduced hippocampal indoleamine levels and attenuated latent inhibition of conditioned suppression. This finding supports the hypothesis that the destruction of mesolimbic 5-hydroxytryptamine terminals reduces latent inhibition. This result is discussed in terms of the possible involvement of reduced serotonergic function in schizophrenic attentional disorder. In addition to the predicted lesion effect, biochemical analyses indicated that experimental treatments in the latent inhibition procedure altered neurotransmitter turnover: utilization ratios for 5-hydroxytryptamine and/or dopamine were increased in preexposed relative to nonpreexposed animals in four of the six brain regions sampled.