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Stereotaxic injection has been widely used for direct delivery of compounds or viruses to targeted brain areas in rodents. Direct targeting of serotonergic neurons in the dorsal raphe nucleus (DRN) can cause excessive bleeding and animal death, due to its location below the superior sagittal sinus (SSS). This protocol describes the generation of a DRN serotonergic neuron-lesioned mouse model (>90% survival rate) with stable loss of >70% 5-HT-positive cells in the DRN. The lesion is induced by stereotaxic injection of a selective serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the DRN using an angled approach (30° in the anterior/posterior direction) to avoid injury to the SSS. DRN serotonergic neuron-lesioned mice display anxiety-associated behavior alterations, which helps to confirm success of the DRN lesion surgery. This method is used here for DRN lesions, but it can also be used for other stereotaxic injections that require angular injections to avoid midline structures. This DRN serotonergic neuron-lesioned mouse model provides a valuable tool for understanding the role of serotonergic neurons in the pathogenesis of psychiatric disorders, such as generalized anxiety disorder and major depressive disorder.

Hyperacusis may be defined as diminishing tolerance to moderate and high intensity sounds in people with normal hearing sensitivity. Serotonin plays a critical role in some of auditory tasks including startle reflex and prepulse inhibition. Serotonin deficiency can cause some diseases which can coincide with hyperacusis. The aim of the present study was to investigate the probable influence of serotonergic depletion in nucleus accumbens (NAcc) on the startle reflex. The startle reflexes were examined in Wistar rats (n: 48) in different intensities with and without the background noise. The amplitude of startle reflex significantly increased in NAcc-injected rats without background noise, while this difference disappeared in the presence of background noise in all intensities. These data proposed that the injection of 5, 7-Dihydroxytryptamine (5, 7-DHT) into nucleus accumbens will cause hyperacusis-like behavior, and strengthens the possibility of the role of serotonin and nucleus accumbens in hyperacusis., (Copyright © 2019. Published by Elsevier B.V.)

Background: For centuries, cannabinoids have been known to be effective in pain states. Itch and pain are two sensations sharing a lot in common., Objective: The goal of this research was to observe whether the cannabinoid agonist WIN 55,212-2 reduces serotonin-induced scratching behaviour and whether neurotoxic destruction of descending serotonergic and noradrenergic pathways mediate the antipruritic effect of WIN 55,212-2. Material and methods Scratching behaviour was induced by intradermal injection of serotonin (50 µg/50 µl/mouse) to Balb/c mice. The neurotoxins 5,7-dihydroxytryptamine (5,7-DHT, 50 μg/mouse) and 6-hydroxydopamine (6-OHDA, 20 μg/mouse) are applied intrathecally to deplete serotonin and noradrenaline in the spinal cord. WIN 55,212-2 (1, 3, 10 mg/kg, i.p.) dose-dependently attenuated serotonin-induced scratches. Neurotoxic destruction of neither the serotonergic nor the noradrenergic systems by 5,7-DHT and 6-OHDA, respectively, had any effect on the antipruritic action of WIN 55,212-2., Conclusion: Our findings indicate that cannabinoids dose-dependently reduce serotonin-induced scratching behaviour and neurotoxic destruction of descending inhibitory pathways does not mediate this antipruritic effect.

Aims: Rheumatoid arthritis brings great burdens to the patients. In addition to the highly expensive treatment, they are commonly associated with severe side effects. In such context, the research for safe and affordable treatments is needed., Main Methods: Arthritis was induced by CFA (0.5mg/mL) in female wistar rats. Trypsin was given p.o. (2.95mg/kg; 2mL) 24h after the intra-articular CFA injection. Articular incapacitation was measured daily by counting the paw elevation time (PET; s) during 1-min periods of stimulated walk, throughout the 7-days after intra-articular CFA injection. Articular diameter (AD) was accessed just after each PET measurement, taken the difference between naïve and diseased knee-joint diameter (cm)., Key Findings: The present study showed that orally administered trypsin was able to reduce nociception and edema, effects that could be observed throughout the evaluation period. These effect, however, were not observed in animals underwent subdiaphragmatic vagotomy, suggesting a vagal mediation for trypsin effects. Likewise, these effects were blocked in rats which received intrathecal injection of the neurotoxins 5,7-dihydroxytryptamine or 6-hydroxydopamine, suggesting the involvement of spinal amines from axon terminals., Significance: The present study proposes that oral trypsin may cause vagal activation, followed by the activation of descending inhibitory pathways and such mechanism may lead to a novel approach for the treatment of arthritis., (Copyright © 2016. Published by Elsevier Inc.)

Hyperactivation of the amygdala is implicated in anxiety and mood disorders, but the precise underlying mechanisms are unclear. We previously reported that depletion of serotonin (5-hydroxytryptamine, 5-HT) in the basolateral nucleus of the amygdala (BLA) using the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) potentiated learned fear and increased glutamate receptor (Glu) expression in BLA. Here we investigated the hypothesis that CaMKII facilitates anxiety-like behavior and increased Glu/AMPA receptor subunit A1 (GluA1) expression following depletion of 5-HT in the BLA. Infusion of 5,7-DHT into the BLA resulted in anxiety-like behavior in the open field test (OFT) and increased the phosphorylation of CaMKIIα (Thr-286) in the BLA. Knockdown of the CaMKIIα subunit using adeno-associated virus (AAV)-delivered shRNAi concomitantly attenuated anxiety-like behavior in the OFT and decreased GluA1 expression in the BLA. Our results suggest that the CaMKII signaling plays a key role in low 5-HT-induced anxiety and mood disturbances, potentially through regulation of GluA1 expression in the BLA., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Withdrawal from amphetamine is associated with increased anxiety and sensitivity to stressors which are thought to contribute to relapse. Rats undergoing amphetamine withdrawal fail to exhibit stress-induced increases in serotonin (5-HT) release in the ventral hippocampus and show heightened anxiety-like behaviors. Therefore, we tested the hypothesis that reducing 5-HT levels in the ventral hippocampus is a causal mechanism in increasing anxiety-like behaviors during amphetamine withdrawal. First, we tested whether reducing 5-HT levels in the ventral hippocampus directly increases anxiety behavior. Male rats were bilaterally infused with 5,7-dihydroxytryptamine (5,7-DHT) into the ventral hippocampus, which produced a 83% decrease in ventral hippocampus 5-HT content, and were tested on the elevated plus maze (EPM) for anxiety-like behavior. Reducing ventral hippocampus 5-HT levels decreased the time spent in the open arms of the maze, suggesting that diminished ventral hippocampus 5-HT levels increases anxiety-like behavior. Next, we tested whether increasing 5-HT levels in the ventral hippocampus reverses anxiety behavior exhibited by rats undergoing amphetamine withdrawal. Rats were treated daily with either amphetamine (2.5-mg/kg, i.p.) or saline for 2weeks, and at 2weeks withdrawal, were infused with the selective serotonin reuptake inhibitor paroxetine (0.5μM) bilaterally into the ventral hippocampus and tested for anxiety-like behavior on the EPM. Rats pre-treated with amphetamine exhibited increased anxiety-like behavior on the EPM. This effect was reversed by ventral hippocampus infusion of paroxetine. Our results suggest that 5-HT levels in the ventral hippocampus are critical for regulating anxiety behavior. Increasing 5-HT levels during withdrawal may be an effective strategy for reducing anxiety-induced drug relapse., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Coping is defined as the behavioral and physiological effort made to master stressful situations. The ability to cope with stress leads either to healthy or to pathogenic outcomes. The medial prefrontal cortex (mpFC) and amygdala are acknowledged as having a major role in stress-related behaviors, and mpFC has a critical role in the regulation of amygdala-mediated arousal in response to emotionally salient stimuli. Prefrontal cortical serotonin (5-hydroxytryptamine (5-HT)) is involved in corticolimbic circuitry, and GABA has a major role in amygdala functioning. Here, using mice, it was assessed whether amygdalar GABA regulation by prefrontal 5-HT is involved in processing stressful experiences and in determining coping outcomes. First (experiment 1), bilateral selective 5-HT depletion in mpFC of mice reduced GABA release induced by stress in basolateral amygdala (BLA) and passive coping in the Forced Swimming Test (FST) (experiment 2). Moreover, prefrontal-amygdala disconnection procedure that combined a selective unilateral 5-HT depletion of mpFC and infusion of an inhibitor of GABA synthesis into the contralateral BLA, thereby to disrupt prefrontal-amygdalar serial connectivity bilaterally, showed that disconnection selectively decreases immobility in the FST. These results point to prefrontal/amygdala connectivity mediated by 5-HT and GABA transmission as a critical neural mechanism in stress-induced behavior.

Serotonin neurons of the caudal raphe facilitate ventilatory and sympathetic responses that develop following blood loss in conscious rats. Here, we tested whether serotonin projections to the caudal portion of the dorsomedial brain stem (including regions of the nucleus tractus solitarius that receive cardiovascular and chemosensory afferents) contribute to cardiorespiratory compensation following hemorrhage. Injections of the serotonin neurotoxin 5,7-dihydroxytryptamine produced >90% depletion of serotonin nerve terminals in the region of injection. Withdrawal of ∼21% of blood volume over 10 min produced a characteristic three-phase response that included 1) a normotensive compensatory phase, 2) rapid sympathetic withdrawal and hypotension, and 3) rapid blood pressure recovery accompanied by slower recovery of heart rate and sympathetic activity. A gradual tachypnea developed throughout hemorrhage, which quickly reversed with the advent of sympathetic withdrawal. Subsequently, breathing frequency and neural minute volume (determined by diaphragmatic electromyography) declined below baseline following termination of hemorrhage but gradually recovered over time. Lesioned rats showed attenuated sympathetic and ventilatory responses during early compensation and later recovery from hemorrhage. Both ventilatory and sympathetic responses to chemoreceptor activation with potassium cyanide injection were attenuated by the lesion. In contrast, the gain of sympathetic and heart rate baroreflex responses was greater, and low-frequency oscillations in blood pressure were reduced after lesion. Together, the data are consistent with the view that serotonin innervation of the caudal dorsomedial brain stem contributes to sympathetic compensation during hypovolemia, possibly through facilitation of peripheral chemoreflex responses.

The prefrontal cortex and the nucleus accumbens are two brain sites which are known to be involved in the modulation of the acoustic startle response. In particular, the release of monoaminergic transmitters within these brain sites plays an important role in prepulse inhibition of the startle response which serves as an operational measure of sensorimotor gating. Like for dopamine, it is well established that serotonin transmission plays an important role in prepulse inhibition. However, there are only few studies investigating the effects of local manipulation of serotonin transmission on prepulse inhibition. The aim of the present study was to test whether prepulse inhibition or the startle response itself was affected by serotonergic depletion of either the prefrontal cortex or the nucleus accumbens. Serotonergic depletion was induced by local injections of 5,7-dihydroxytryptamine and verified by ex vivo analysis of transmitter levels by high pressure liquid chromatography. In our behavioural tests, we found that 5,7-dihydroxytryptamine into the prefrontal cortex decreased prepulse inhibition, whereas injections into the nucleus accumbens facilitated prepulse inhibition. The time course of these behavioural effects, as well as the transmitter level changes within the different brain sites was very different. Most interestingly, 5,7-dihydroxytryptamine injections into the nucleus accumbens affect serotonin and dopamine levels in both, nucleus accumbens and prefrontal cortex. Taken together, the present study supports an important role of serotonin in the modulation of prepulse inhibition and baseline startle magnitude. However, the observed changes cannot be attributed to a specific brain site since our data clearly show that local 5,7-dihydroxytryptamine injections also affect transmitter levels in brain sites away from the injection site.

Central serotonin (5-HT) function is thought to be a critical component of behavioral inhibition and impulse control. However, in recent clinical studies, 5-HT manipulations failed to affect stop-signal reaction time (SSRT), which is a fundamental process in behavioral inhibition. We investigated the effect of central 5-HT depletion (intracerebroventricular 5,7-dihydroxytryptamine) in rats on two aspects of behavioral inhibition, SSRT and 'waiting', using the stop-signal task. 5-HT depletion had no effects on SSRT or any other primary measure on the stop-signal task. However, within the same task, there was a deficit in 'waiting' in 5-HT-depleted rats when they were required to withhold from responding in the terminal element of the stop-signal task for an extended period. D-Amphetamine had dose-dependent, but not 5-HT-dependent effects on SSRT. Conversely, the dose that tended to improve, or decrease, SSRT (0.3 mg/kg) impaired the ability to wait, again independently of 5-HT manipulation. These findings suggest that SSRT and 'waiting' are distinct measures of behavioral inhibition, and show that 5-HT is critical for some forms of behavioral inhibition but not others. This has significant implications for the treatment of conditions such as attention deficit and hyperactivity disorder, substance abuse, and affective disorders, in which inhibitory and impulse-control deficits are common.

Rationale: Selective 5-ht(6) receptor antagonists like Ro 04-6790 prolong memory in many rodent preclinical paradigms, possibly by blocking tonic 5-HT-evoked GABA release and allowing disinhibition of cortico-limbic glutamatergic and cholinergic neurones. If this is the case, behavioural responses to Ro 04-6790 should be abolished by depletion of endogenous 5-HT, and selective lesions of dorsal raphé (DR) or median raphé (MR) 5-HT pathways would allow the neuroanatomical substrates underlying the cognitive effects of 5-ht(6) receptor antagonists to be elucidated., Objectives: This study compared the effect of Ro 04-6790 on novel object discrimination (NOD) before and after sham or 5,7-dihydroxytryptamine (5,7-DHT)-induced lesions produced by injection into the lateral ventricles (LV), DR or MR., Materials and Methods: NOD tests used a 4 h inter-trial interval (ITI) and Ro 04-6790 (10 mg kg(-1) i.p.) was administered 20 min before the familiarization trial. Brain region-specific 5-HT depletion was assessed by high performance liquid chromatography with electrochemical detection (HPLC-ED)., Results: Widespread LV or selective MR, but not DR lesions, abolished the ability of Ro 04-6790 to delay natural forgetting. Successful performance of all lesioned rats in subsequent 'drug-free' NOD tests using a 1 h ITI excluded the possibility of any confounding effects on visual acuity or motivation., Conclusions: The ability of Ro 04-6790 to prolong object recognition memory requires blockade of MR 5-HT function. Because DR lesions did not produce the expected depletion of striatal 5-HT an additional contribution of DR inputs to this region cannot be completely excluded.

In this study we have explored differences in animal reactivity to conditioned aversive stimuli using the conditioned fear test (a contextual fear-freezing response), in rats subjected to the selective lesion of the prefrontal cortex serotonergic innervation, and differing in their response to the acute painful stimulation, a footshock (HS--high sensitivity rats, and LS--low sensitivity rats, selected arbitrarily according to their behavior in the 'flinch-jump' pre-test). Local administration of serotonergic neurotoxin (5,7-dihydroxytryptamine) to the dorsomedial part of the prefrontal cortex caused a very strong, structure and neurotransmitter selective depletion of serotonin concentration. In HS rats, the serotonergic lesion significantly disinhibited rat behavior controlled by fear, enhanced c-Fos expression in the dorsomedial prefrontal area, and increased the concentration of GABA in the basolateral amygdala, measured in vivo after the testing session of the conditioned fear test. The LS animals revealed an opposite pattern of behavioral and biochemical changes after serotonergic lesion: an increase in the duration of a freezing response, and expression of c-Fos in the basolateral and central nuclei of amygdala, and a lower GABA concentration in the basolateral amygdala. In control conditions, c-Fos expression did not differ in LS and HS, naïve, not conditioned and not exposed to the test cage animals. The present study adds more arguments for the controlling role of serotonergic innervation of the dorsomedial part of the prefrontal cortex in processing emotional input by other brain centers. Moreover, it provides experimental data, which may help to better explain the anatomical and biochemical basis of differences in individual reactivity to stressful stimulation, and, possibly, to anxiolytic drugs with serotonergic or GABAergic profiles of action.

Background: Regulation of pain states is, in part, dependent upon plastic changes in neurones within the superficial dorsal horn. There is also compelling evidence that pain states are under the control of descending projections from the brainstem. While a number of transcription factors including Methyl-CpG-binding protein 2 (MeCP2), Zif268 and Fos have been implicated in the regulation of dorsal horn neurone sensitization following injury, modulation of their activity by descending controls has not been investigated., Results: Here, we describe how descending controls regulate MeCP2 phosphorylation (P-MeCP2), known to relieve transcriptional repression by MeCP2, and Zif268 and Fos expression in the rat superficial dorsal horn, after CFA injection into the hind paw. First, we report that CFA significantly increased P-MeCP2 in Lamina I and II, from 30 min post injection, with a maximum reached after 1 h. The increase in P-MeCP2 paralleled that of Zif268 and Fos, and P-MeCP2 was expressed in large sub-populations of Zif268 and Fos expressing neurones. Serotonergic depletion of the lumbar spinal cord with 5,7 di-hydroxytryptamine creatinine sulphate (5,7-DHT) reduced the inflammation evoked P-MeCP2 in the superficial dorsal horn by 57%, and that of Zif268 and Fos by 37.5% and 30% respectively. Although 5,7-DHT did not change primary thermal hyperalgesia, it significantly attenuated mechanical sensitivity seen in the first 24 h after CFA., Conclusion: We conclude that descending serotonergic pathways play a crucial role in regulating gene expression in the dorsal horn and mechanical sensitivity associated with an inflammatory pain state.

Intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT) (150 microg; 4.5 microl/ventricle), a serotonergic neurotoxin, significantly decreased spontaneous alternation in Y-maze task and working memory in radial 8 arm-maze task, suggesting effects on short-term memory, without affecting long-term memory, explored by reference memory in radial 8 arm-maze task and step-through latency in multi-trial passive avoidance task. Parachlorophenylalanine (PCPA) (3 days treatment 200 microg, i.c.v.), a serotonin synthesis inhibitor, did not impair step-through-latency in multi-trial passive avoidance task, suggesting no effects on long-term memory. These results suggest that serotonin, among other neurotransmitters, play an important role in cognitive functions, especially short-term memory.

It is well known that the dorsal raphe nucleus (DRN) sends serotonergic and nonserotonergic projections to target regions in the brain stem and forebrain, including the vestibular nuclei. Although retrograde tracing studies have reported consistently that there are differences in the relative innervation of different target regions by serotonergic and nonserotonergic DRN neurons, the relative termination patterns of these two projections have not been compared using anterograde tracing methods. The object of the present investigation was to trace anterogradely the individual serotonergic and nonserotonergic components of the projection from DRN to the vestibular nuclei in rats. To trace nonserotonergic DRN projections, animals were pretreated with the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), and then, after 7 days, the anterograde tracer biotinylated dextran amine (BDA) was iontophoretically injected into the DRN. In animals treated with 5,7-DHT, nonserotonergic BDA-labeled fibers were found to descend exclusively within the ventricular plexus and to terminate predominantly within the periventricular aspect of the vestibular nuclei. Serotonergic DRN projections were traced by injecting 5,7-DHT directly into DRN, and amino-cupric-silver staining was used to visualize the resulting pattern of terminal degeneration. Eighteen hours after microinjection of 5,7-DHT into the DRN, fine-caliber degenerating serotonergic terminals were found within the region of the medial vestibular nucleus (MVN) that borders the fourth ventricle, and a mixture of fine- and heavier-caliber degenerating serotonergic terminals was located further laterally within the vestibular nuclear complex. These findings indicate that fine-caliber projections from serotonergic and nonserotonergic DRN neurons primarily innervate the periventricular regions of MVN, whereas heavier-caliber projections from serotonergic DRN neurons innervate terminal fields located in more lateral regions of the vestibular nuclei. Thus, serotonergic and nonserotonergic DRN axons target distinct but partially overlapping terminal fields within the vestibular nuclear complex, raising the possibility that these two DRN projection systems are organized in a manner that permits regionally-specialized regulation of processing within the vestibular nuclei.

Purpose: Although sound-induced (audiogenic) seizures in the genetically epilepsy-prone rat (GEPR) initially occur independent of the forebrain, repeated audiogenic seizures recruit forebrain seizure circuits in a process referred to as audiogenic kindling. In GEPR-3s, audiogenic kindling results in facial and forelimb (F&F) clonic seizures that are typical of forebrain seizures. However, in GEPR-9s, audiogenic kindling produces posttonic all-limb clonus not usually observed during forebrain seizures. We hypothesized that the more severe brainstem seizures of the GEPR-9 prevent the expression of F&F clonic seizures during audiogenic kindling. Therefore attenuation of audiogenic seizures during audiogenic kindling in GEPR-9s should allow F&F clonic seizures to be expressed. Likewise, intensifying audiogenic seizure severity in GEPR-3s should inhibit audiogenically kindled F&F clonic seizures. We have tested this hypothesis in the present study., Methods: Lesions of the superior colliculus or treatment with low-dose phenytoin were used to suppress audiogenic seizure severity in GEPR-9s. Depletion of brain serotonin was used to increase the seizure severity in GEPR-3s. All GEPRs were then subjected to audiogenic kindling. Behavioral and electrographic seizures were assessed., Results: Suppression of audiogenic seizure severity during audiogenic kindling in GEPR-9s increased the incidence forebrain seizure behavior. Kindled GEPR-9s that continued to display full tonic seizures did not exhibit forebrain convulsions, but did show posttonic clonus and forebrain seizure activity in the EEG. GEPR-3s chronically depleted of brain serotonin, along with displaying tonic brainstem seizures, tended to display less severe forebrain seizures during audiogenic kindling., Conclusions: These findings support the concept that severe brainstem seizures prevent the behavioral expression of forebrain seizures in audiogenically kindled GEPR-9s. It appears that the severe brainstem seizure of the GEPR-9 does not allow the forebrain seizure to manifest its typical behavioral concomitants despite electrographic evidence that spike-wave discharge is occurring in the forebrain.

Although the selective toxicity of 5,7-dihydroxytryptamine (5,7-DHT) is thought to depend on the drug's transport into serotonin (5HT) neurons via the 5HT transporter, few studies have critically examined this postulation. We therefore evaluated if 5,7-DHT-induced reductions in 5HT concentrations and synthesis rate in rat brain are blocked by pretreatment with 5HT-selective reuptake inhibitors. Rats pretreated with desipramine (DMI) (to prevent norepinephrine depletion) received intracerebroventricular injections of 5,7-DHT (5, 50, 100, 200 microg/rat) 30 min after fluoxetine (20 mg/kg ip). Forty-eight hours later, they received m-hydroxybenzylhydrazine 30 min before sacrifice. The concentrations of 5HT and 5-hydroxytryptophan (5HTP, an index of 5HT synthesis) were measured in hypothalamus, cortex and brainstem. Each 5,7-DHT dose produced significant reductions in 5HT and 5HTP concentrations in all regions examined (5 microg reduced 5HT but not 5HTP), effects that were not blocked by fluoxetine. Two other 5HT reuptake blockers (chlorimipramine, alaproclate) also failed to block the 5HT and 5HTP depleting actions of 5,7-DHT. Desipramine blocked 5,7-DHT-induced norepinephrine (NE) depletion. Pretreatment with the 5HT receptor antagonist metergoline, or the 5HT(1A) agonist 8-hydroxy-(di-n-propylamino)tetralin (to slow 5HT neuronal firing rate) also failed to antagonize the 5HT depleting action of 5,7-DHT. Together, the data strongly suggest that the mechanism by which 5,7-DHT depletes the brain of serotonin does not involve 5HT-transporter-mediated concentration of neurotoxin in 5HT neurons, may not involve 5HT receptor interaction, and does not depend on the firing rate of the 5HT neuron.

Serotonergic dysregulation within the prefrontal cortex (PFC) is implicated in many neuropsychiatric disorders, but the precise role of serotonin within the PFC is poorly understood. Using a serial discrimination reversal paradigm, we showed that upon reversal, selective serotonin depletion of the marmoset PFC produced perseverative responding to the previously rewarded stimulus without any significant effects on either retention of a discrimination learned preoperatively or acquisition of a novel discrimination postoperatively. These results highlight the importance of prefrontal serotonin in behavioral flexibility and are highly relevant to obsessive-compulsive disorder, schizophrenia, and the cognitive sequelae of drug abuse in which perseveration is prominent.

The involvement of spinopetal noradrenergic and serotonergic systems in antinociception induced by endomorphin-1 (EM-1) and endomorphin-2 (EM-2) given supraspinally or spinally were investigated in male CD-1 mice. Groups of mice were pretreated intrathecally (i.t.) with 6-hydroxydopamine (6-OHDA, 20 microg) or 5,7-dihydroxytryptamine (5,7-DHT, 50 microg) for 3 days before intracerebroventricular (i.c.v.) or i.t. injection of different doses of EM-1 or EM-2, and the tail-flick response was measured for antinociceptive effects. I.t. pretreatment with 6-OHDA for 3 days, which markedly depleted noradrenaline (NA) contents by more than 90%, but not serotonin (5-HT) in the spinal cord, completely abolished the antinociception induced by i.c.v.-administered EM-1 or EM-2. Intrathecal pretreatment with 5,7-DHT for 3 days, which markedly reduced 5-HT contents by more than 92%, but only reduced NA by 14 - 25% in the spinal cord, also markedly attenuated the antinociception induced by i.c.v.-administered EM-1 or EM-2. However, the antinociception induced by i.t.-administered EM-1 or EM-2 was not affected in either 6-OHDA or 5,7-DHT pretreated mice. It is concluded that NA and 5-HT in the spinal cord are involved in the antinociception induced by supraspinally, but not spinally administered EM-1 and EM-2.

Using the microdialysis method, we investigated whether the levels of serotonin (5-hydroxytryptamine, 5-HT) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTPL), in the locus coeruleus are influenced by tryptophan alone or simultaneous administration of tryptophan and ethanol. Tryptophan (50 mg/kg, i.p.) led to a significant increase in the levels of 5-HIAA, but not 5-HT in the locus coeruleus. However, ethanol (1.25 g/kg) had no effect on the levels of 5-HT and its metabolites. Combined administration of tryptophan and ethanol caused very marked increases in 5-HIAA and 5-HTPL levels in the locus coeruleus. A time lag in the increased 5-HIAA levels between tryptophan alone and tryptophan plus ethanol was observed. Moreover, 5-HIAA levels in the locus coeruleus induced by tryptophan were abolished by microinjection of 5,7-dihydroxytryptamine (150 microg/4 microl) into the dorsal raphe nucleus. Judging from the present results, the serotonergic afferents to the locus coeruleus may originate for about 20-30% from cell bodies located in the dorsal raphe nucleus. Teeth-chattering was significantly detected in the tryptophan plus ethanol-treated rats when compared with the tryptophan-treated rats, but not in the saline-treated controls. These results may suggest that the increased levels of 5-HIAA and 5-HTPL in the locus coeruleus induced by tryptophan are potentiated by ethanol, and that these levels are partly responsible for behavioral activation.

The serotonergic system is implicated in learning and memory and its disorder, e.g. after 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") abuse. This study examined the effects of widespread depletion of serotonin (5-HT) using intraventricular injection of 5,7-dihydroxytryptamine (5,7-DHT) on the learning of a working memory task in the dark agouti (DA) rat. The lesion impaired acquisition but not later performance of a nonspatial working memory rule, as measured using nonmatch to sample object recognition in the Y-maze. The lesion had a marginal effect on choice completion times over the course of testing. However, nonspecific effects did not provide a good account of the reduction in choice accuracy as this persisted when completion times were taken into account statistically. Similarly, in a second experiment, the same lesioned rats were slowed in the acquisition of spatial alternation in the T-maze. However, in the open field, there were no comparably long-lasting effects of the serotonergic depletion on line crossings and defecation, only a transient reduction in activity on the first day.Together, these data suggest that the serotonergic system is important in the acquisition of working memory tasks in the rat and that this outcome was unlikely to be the result of nonspecific effects of the lesion.

Regulatory mechanisms in the basolateral nucleus of the amygdala (BLA) serves as a filter for unconditioned and conditioned aversive information that ascend to higher structures from the brainstem whereas the central nucleus (CeA) is the main output for the resultant defense reaction. We have shown that neural substrates in the inferior colliculus are activated by threatening stimuli of acoustic nature and have important functional links with the amygdala. In this work, we examined the influence of lesions with 5,7-dihydroxytryptamine (5,7-DHT) of these nuclei of amygdala on the aversive responses induced by electrical stimulation of the inferior colliculus. Thus, rats were implanted with an electrode in the CeA of the inferior colliculus for the determination of the thresholds of alertness, freezing and escape responses. Each rat also bore a cannula implanted in the BLA or CeA for injection of 5,7-DHT (8.0 microg/0.8 microl) or its vehicle. The data obtained show that CeA lesions increase the thresholds of aversive responses whereas BLA lesions decrease the thresholds of these responses. From this evidence it is suggested that defensive behavior induced by activation of the neural substrates of aversion in the inferior colliculus seems to depend on the integrity of the amygdala. BLA regulates the input and CeA functions as the output for these aversive states generated at brainstem level. It is likely that aversive information ascending from the inferior colliculus may receive either inhibitory or excitatory influences of 5-HT mechanisms in the BLA or CeA, respectively., (Copyright 2002 Elsevier Science Inc.)

In a previous experiment [Eur J Neurosci 12 (2000) 79], combined intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7-DHT; 150 microg) and 192 IgG-saporin (2 microg) in female rats produced working memory impairments, which neither single lesion induced. In the present experiment, we report on an identical approach in male rats. Behavioral variables were locomotor activity, T-maze alternation, beam-walking, Morris water-maze (working and reference memory) and radial-maze performances. 192 IgG-saporin reduced cholinergic markers in the frontoparietal cortex and the hippocampus. 5,7-DHT lesions reduced serotonergic markers in the cortex, hippocampus and striatum. Cholinergic lesions induced motor deficits, hyperactivity and reduced T-maze alternation, but had no other effect. Serotonergic lesions only produced hyperactivity and reduced T-maze alternation. Beside the deficits due to cholinergic lesions, rats with combined lesions also showed impaired radial-maze performances. We confirm that 192 IgG-saporin and 5,7-DHT injections can be combined to produce concomitant damage to cholinergic and serotonergic neurons in the brain. In female rats, this technique enabled to show that interactions between serotonergic and basal forebrain cholinergic mechanisms play an important role in cognitive functions. The results of the present experiment in male rats are not as clear-cut, although they are not in obvious contradiction with our previous results in females.

Because some bulbospinal respiratory premotor neurons have bilateral projections to the phrenic nuclei, we investigated whether changes in contralateral phrenic motoneuron function would occur after unilateral axotomy via C(2) hemisection. Phrenic neurograms were recorded under baseline conditions and during hypercapnic and hypoxic challenge in C(2) hemisected, normal, and sham-operated rats at 1 and 2 months after injury. The rats were anesthetized, vagotomized, and mechanically ventilated. No group differences were seen in contralateral neurograms at 1 month after injury. At 2 months, however, there was a statistically significant decrease in respiratory rate (RR) at normocapnia, an elevated RR during hypoxia, and an attenuated increase in phrenic neurogram amplitude during hypercapnia in the C(2)-hemisected animals. To test whether C(2) hemisection had induced a supraspinal change in respiratory motor drive, we recorded ipsilateral and contralateral hypoglossal neurograms during hypercapnia. As with the phrenic motor function data, no change in hypoglossal output was evident until 2 months had elapsed when hypoglossal amplitudes were significantly decreased bilaterally. Last, the influence of serotonin-containing neurons on the injury-induced change in phrenic motoneuron function was examined in rats treated with the serotonin neurotoxin, 5,7-dihydroxytryptamine. Pretreatment with 5,7-dihydroxytryptamine prevented the effects of C(2) hemisection on contralateral phrenic neurogram amplitude and normalized the change in RR during hypoxia. The results of this study show novel neuroplastic changes in segmental and brainstem respiratory motor output after C(2) hemisection that coincided with the spontaneous recovery of some ipsilateral phrenic function. Some of these effects may be modulated by serotonin-containing neurons.

The concentrations of 5,7-dihydroxytryptamine (5,7-DHT) and serotonin (5-HT) were measured in brainstem, hypothalamus and cerebral cortex 0, 2, 6, 12, and 24 hours following the bilateral, lateral ventricular injection of 5,7-DHT (100 microg/each ventricle) into adult male rats. At 6 hours, 5,7-DHT levels had decreased 99% from 0 hr values in all brain regions. Thereafter, 5,7-DHT levels continued to decline in cortex, but not in hypothalamus or brainstem; at 24 hr, but not 48 hr, 5,7-DHT peaks were still measurable in each brain region examined. Serotonin levels in all three regions also fell markedly by 2-6 hours after 5,7-DHT administration. At 24 hours, hypothalamus and brainstem 5HT levels had declined >70% and cerebral cortex approximately 50% below control values. The relevance of these findings to the protective action of monoamine reuptake blockers is discussed.

The ability of serotonin (5-HT) to influence striatal glutamatergic transmission was examined by determining changes over time in glutamate extracellular levels, transporter expression and synaptosomal uptake in rats with lesion of serotonergic neurones. By 8 days after intraraphe injections of 5,7-dihydroxytryptamine, producing 80% decreases in striatal tissue 5-HT levels, no changes were observed in the glutamatergic transmission. When 5-HT depletion was almost complete (21 days post-lesion), high affinity glutamate uptake in striatal synaptosomal preparations was significantly increased (156% of control), although no changes in striatal GLT1, GLAST and EAAC1 mRNAs, and GLT1 protein were detected by in situ hybridization and immunohistochemistry. Meanwhile, the serotonin lesion produced large increases in basal extracellular levels of glutamate and glutamine (364% and 259%, respectively) determined in awake rats by in vivo microdialysis, whereas no change was observed in dopamine levels as compared with control rats. High potassium depolarization as well as L-trans-pyrrolidine-2,4-dicarboxylate, also induced larger increases in extracellular levels of glutamate in lesioned rats than in controls. Finally, similar changes in glutamate transmission were observed by 3 months post-lesion. These results suggest that 5-HT has a long lasting and tonic inhibitory influence on the striatal glutamatergic input, without affecting the basal dopaminergic transmission.

These experiments examined the impact of extensive depletions of forebrain 5-hydroxytryptamine (5-HT; serotonin) levels on prepulse inhibition (PPI) of the acoustic startle reflex in rats. In Experiment 1, injection of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the dorsal and median raphe nuclei disrupted PPI. This deficit was observed beginning 2 days after lesioning and was still apparent 8 weeks later. Basal startle reactivity was not altered. The 5-HT(1A) receptor agonist 8-OH-DPAT (0.1 mg/kg) and the dopamine receptor agonist apomorphine (1mg/kg) also disrupted PPI; the effect of 8-OH-DPAT, but not apomorphine, was potentiated in 5-HT-depleted rats. Basal startle reactivity was enhanced by 8-OH-DPAT in sham-lesioned rats but not in 5,7-DHT-lesioned rats. In Experiment 2, a second method for depleting 5-HT was used. The tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA) also disrupted PPI without altering basal startle reactivity. Again, 8-OH-DPAT disrupted PPI in control animals; this effect was not altered in PCPA-treated rats but the increase in basal startle reactivity induced by 8-OH-DPAT was not observed in PCPA-treated rats. Taken together with the results of previous experiments involving drugs that enhance 5-HT neurotransmission it appears that both increases and decreases in 5-HT activity disrupt PPI.

Several serotonergic drugs are effective for anxiety disorders, but underlying mechanisms are unclear, and findings in experimental animals are difficult to reconcile with human data. It has been proposed that differential effects of serotonin within specific anatomical systems may account for these difficulties, and the amygdala has been suggested as one of the structures involved. To examine this hypothesis, the neurotoxin 5,7-dihydroxytryptamine was administered locally in rat amygdala. Within the amygdala, serotonin was depleted by approximately 80%, with other transmitters unaffected, and serotonin transporter labelling was decreased by approximately 85%. Cortical areas near the lesion site were also affected, although to a lesser degree. Other forebrain areas were unaffected. Lesions resulted in a specific anti-conflict effect in a punished drinking test, but did not influence elevated plus-maze behavior (under baseline conditions and after restraint stress), locomotor activity or ethanol intake. These data suggest that the punished drinking test and the elevated plus-maze may activate different components of fear circuitry, and that the serotonergic input to the amygdala specifically participates in fear-related behavioral suppression mediated by this structure.

The primary visual cortex shows synaptic plasticity during a postnatal "critical period," and its plasticity declines with development. Indeed, we found a developmental decrease in the induction of long-term potentiation (LTP) in the rat visual cortex. In visual cortex slices obtained from 2- to 3-week-old rats, tetanic stimulation (100 Hz for 1 sec, twice at an interval of 30 sec) of the white matter reproducibly induced LTP of field potentials in layer II/III. However, in slices from 5-week-old rats, the same tetanic stimulation failed to induce LTP. We hypothesized that endogenous serotonin (5-HT) is responsible for the developmental decrease in visual cortex LTP, because the induction of visual cortex LTP was suppressed by the addition of exogenous 5-HT (10 microm) and because the amount of 5-HT in the visual cortex increased during development. To test this hypothesis, we investigated the effect of methysergide, a 5-HT receptor antagonist, on the induction of visual cortex LTP. When visual cortex slices from 5-week-old rats were perfused with 50 microm methysergide, tetanic stimulation of the white matter induced robust LTP in layer II/III. Furthermore, serotonergic neurons were lesioned by intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT). LTP was induced in visual cortex slices from 5,7-DHT-treated, 5-week-old rats. These results suggest that the induction of visual cortex LTP in 5-week-old rats is suppressed by endogenous 5-HT. 5-HT may be a factor that determines a critical period for synaptic plasticity in the rat visual cortex.

We have been exploring the role of serotonin in fighting behavior in lobsters using a specific model of agonistic behavior, the establishment of hierarchical relationships between pairs of socially naive juvenile lobsters. We selected this model because the behavior is easily evoked, readily quantifiable, and the effects of experience are eleminated by using socially naive animals. In these studies we injected a specific neurotoxin, 5,7-dihydroxytryptamine, into juvenile lobsters over a 4-week period and then measured the effects on fighting behavior. This treatment reduces the levels of serotonin in the nervous system and immunocytochemical studies show a dramatic reduction in neuropil staining for the amine. Control animals received vehicle injection alone. All injected animals were paired against larger or smaller non-injected opponents, and three successive 30-min fights were carried out and statistically analyzed. The results were surprising: As with elevations of serotonin, reduced levels of serotonin increased the amount of time animals engaged in fighting behavior. No significant effects were seen on who initiated encounters, who retreated first, or who the eventual winner would be. Thus, in this model, elevation or reduction of serotonergic function increases the tendency of animals to engage in agonistic encounters.

The locomotor effects of acute amphetamine treatment (1 mg/kg, i.p.) were assessed in Long-Evans rats after 5,7-dihydroxytryptamine (5, 7-DHT) injections into the fimbria-fornix/cingular bundle (FiFx/CB; 4 microg/side), or the dorsal and median raphe (Raphe; 10 microg). In control rats, amphetamine induced a significant increase of home-cage activity for about 2 h. This effect was similar in Raphe rats, but was absent in FiFx/CB rats. The raphe lesions reduced serotonin concentrations by 50% in the dorsal hippocampus, 75% in the ventral hippocampus and 58% in the fronto-parietal cortex. After FiFx/CB lesions, the reduction amounted 50, 61 and only 25%, in each of these regions, respectively. In the fronto-partietal cortex, dopamine concentration was significantly decreased in Raphe (-27%) and FiFx/CB rats (-65%). The results suggest that a serotonergic denervation of the hippocampus by injections of 5,7-DHT into the FiFx/CB pathways hampers the stimulating effects of amphetamine on locomotor activity. This effect might be related to the reduced dopaminergic tone in the fronto-parietal cortex.

Transplants of fetal midbrain raphe neurons into the adult brain have been shown to promote recovery of complex behavioral deficits in several experimental models, but the mechanisms underlying these effects are only partially understood. In the present study, we have used a well-characterized model system to ascertain whether midbrain raphe graft can display behaviorally relevant changes in transmitter release and/or metabolism. Fetal mesencephalic raphe neurons were grafted unilaterally into the hippocampus previously deprived of its innate serotonergic innervation by intraventricular injections of 5,7-dihydroxytryptamine. The contralateral hippocampus remained as a nongrafted, lesioned control. Microdialysis probes were implanted in the hippocampus 5-7 months postgrafting. Under baseline conditions, extracellular levels of serotonin were similar to normal in the grafted hippocampi, but undetectable on the contralateral, nongrafted side. Levels of the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were markedly higher than normal in the grafted hippocampi, but dramatically reduced on the contralateral nongrafted side. Handling stimulation (gentle stroking of a rat's fur and tail for 15 min) induced a 64% increase in serotonin output in the intact rats and a small but significant 12% increase in the grafted animals. Non-noxious tail-pinch (15 min) enhanced serotonin release by 86% in the intact rats and 28% in the grafted ones. Extracellular 5-HIAA levels remained unchanged during both handling and tail-pinch in both the intact and the grafted rats. Forced immobilization of the rats for 15 min induced a transient 124% increase in extracellular serotonin levels in the intact rats and a significant 19% increase in the grafted animals, whereas swimming in temperate water (25-30 degrees C; 15 min) induced no detectable changes in serotonin output in any of the groups. 5-HIAA levels remained unchanged during forced immobilization, but were significantly reduced during the swimming session in both the intact (-38%) and grafted (-15%) animals. The present results indicate that median raphe grafts can become functionally integrated in the denervated host hippocampus and respond by altered indole output when the animal is exposed to different types of environmental challenges., (Copyright 2000 Academic Press.)

Intercollicular decerebration in animals induces sustained facilitation of muscle tone of the limbs and this animal model has been used to assess centrally acting muscle relaxants. We have examined the involvement of central and spinal cord serotonergic pathways in the onset of excessive muscle tone in an intercollicularly decerebrated rat. Descending serotonergic pathways are known to modulate, directly or indirectly, the excitability of spinal cord motoneurons and it is inferred that serotonin (5-HT) plays an important role in locomotion. Alteration of muscle tone has been investigated in 5-HT-depleted rats with a neurotoxin, 5, 7-dihydroxytryptamine (5,7-DHT) after pretreatment with desipramine. Intracerebroventricular (i.c.v.) administration of 5,7-DHT reduced 5-HT content in the forebrain to 50.5% and that in the spinal cord to 10.5%, while intrathecal (i.t.) administration of 5,7-DHT decreased 5-HT content in the spinal cord to 8.9% without causing any change in the forebrain. In contrast, noradrenaline or dopamine content was not affected by the neurotoxin in both tissues. These treatments significantly attenuated the muscle tone in the animal models. Moreover, the measurement of 5-HT and 5-hydroxyindoleacetic acid content in intact rats after decerebration showed that facilitation of the 5-HT turnover in the spinal cord, but not in the forebrain, was enhanced compared with sham-operated rats. These findings suggest that the descending serotonergic pathways are essential to induce excessive muscle tone in the intercollicular decerebrated rats and that 5-HT antagonists might be candidates for centrally acting muscle relaxants.

Rationale: The cholinergic system is considered to be essential for attention and the degeneration of the cholinergic system in Alzheimer's disease (AD) correlates with the cognitive decline seen in AD patients. The serotonergic system also degenerates in AD, but its role in the modulation of cognitive functions, especially attention, is somewhat unclear., Objectives: The present study investigated possible differences between cholinergic muscarinic and nicotinic receptor mediated mechanisms, the role of serotonin (5-HT) and the interaction between the cholinergic and serotonergic systems in the modulation of attention and response control., Methods: The influences of cholinergic receptor blockade and 5-HT lesions on the performance of rats in the five-choice serial reaction time task were assessed. The 5-HT lesions were neurochemically verified., Results: The neurochemical analysis indicated that the levels of 5-HT and 5-hydroxyindoleacetic acid were reduced quite specifically in the hippocampi, parieto-occipital and frontal cortices, and in the striatum of both p-chloroamphetamine (pCA) and 5, 7-dihydroxytryptamine (5,7-DHT) lesioned rats. The behavioural results showed that the pCA lesion caused a transient increase in impulsivity whereas the 5,7-DHT lesion temporarily reduced the motor activity and slightly impaired choice accuracy. Furthermore, the blockade of central muscarinic receptors by scopolamine (0.075 and 0. 150 mg/kg), but not nicotinic receptors by mecamylamine (1.0 or 3.0 mg/kg), impaired the choice accuracy, whereas the blockade of both muscarinic and nicotinic receptors interfered with motor activity, though possibly via peripheral mechanisms. Interestingly, mecamylamine (3.0 mg/kg) reduced impulsivity, whereas scopolamine slightly increased it. Serotonergic lesions did not make the rats more susceptible to the effects of cholinolytics on choice accuracy., Conclusions: 5-HT system is not essential for the modulation of attention, but it is important in the modulation of response control. Central muscarinic receptors are important in the modulation of attention, whereas central nicotinic receptors may be more essential in response control. The results do not support there being an interaction between the serotonergic and the cholinergic systems in the modulation of attention.

Objectives: These experiments investigated the effects of selective serotonin (5-HT) depletion on intravenous self-administration of d-amphetamine., Methods: Depletion of brain 5-HT levels was induced by injecting the serotonergic neurotoxin 5,7-dihydroxytryptamine (5, 7-DHT) into the dorsal and median raphe nuclei. Rats were then trained to self-administer d-amphetamine according to various schedule and access conditions via chronically indwelling intravenous catheters., Results: Large reductions of brain 5-HT did not alter responding for a training dose of 120 microgram/kg d-amphetamine delivered according to a fixed ratio 1 schedule during 3-h sessions. When the dose of d-amphetamine was altered (0, 3.75, 7. 5, 15, 30, 60 microgram/kg per infusion) a characteristic inverted U-shaped dose response function was obtained. The 5-HT depleted rats showed increased responding for the lower doses of d-amphetamine, with a large significant increase in responding for the 7.5 microgram/kg dose. In these same rats, the suppressive effect of 10 mg/kg fluoxetine on d-amphetamine (60 microgram/kg) self-administration was prevented. The 5,7-DHT lesion also did not alter responding for d-amphetamine (120 microgram/kg) in longer (8 h) daily access sessions. Responding for d-amphetamine delivered on a progressive ratio schedule, in which response requirements increased for each successive infusion of d-amphetamine, was also determined in 5-HT depleted rats. The number of d-amphetamine infusions was not different from the number of infusions earned by sham-lesioned rats across a range of doses of d-amphetamine (7.5-60 microgram/kg). In a final experiment, spontaneous acquisition of self-administration of low doses of d-amphetamine (10 and 30 microgram/kg) was measured in 5-HT depleted and control rats. Again, self-administration behaviour in the 5-HT depleted rats did not differ from controls., Conclusions: These results provide no evidence that reducing 5-HT function alters the primary reinforcing effects of self-administered amphetamine. The increase in self-administration of a low dose of amphetamine observed in experiment 1 probably involves some other process such as increased resistance to extinction.

The possible involvement of the serotonergic neuronal system in aversive motivation produced by phencyclidine [1-(1-phenylcyclohexyl)piperidine; PCP] was investigated using a place-conditioning paradigm in rats. PCP (4 mg/kg, i.p.) produced place aversion in this task as reported previously (Kitaichi K, Noda Y, Hasegawa T, Furukawa H, Nabeshima T. Acute phencyclidine induces aversion, but repeated phencyclidine induces preference in the place conditioning test in rats. Eur J Pharmacol 1996;318:7-9). The blockade of serotonin2A (5-HT2A) receptors using the antagonist ritanserin (3 and 10 mg/kg, p.o.) significantly attenuated this aversive property of PCP whereas lesions of serotonergic neurons using 5,7-dihydroxytryptamine (5,7-DHT, 100 microg/animal, i.c.v.) failed to affect it. Repeated PCP treatment (10 mg/kg, i.p. for 14 days), which is enough to diminish the stereotyped 5-HT2A receptor-mediated head-twitch behavior, also decreased the place aversion. These results suggest that the serotonergic neuronal system, specifically the 5-HT2A receptor, may play a critical role in producing PCP-induced place aversion.

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 5,7-dihydroxytryptamine (5,7-DHT) on striatal levels of dopamine (DA), 5-hydroxytryptamine (5-HT), and their metabolites, as well as on locomotor activity were investigated in C57BL/6 mice. The results showed that MPTP significantly increased locomotor activity and decreased striatal DA levels. However, injection of the serotonergic neurotoxin 5,7-DHT in the striatum, either alone or following high doses of MPTP, significantly decreased locomotor activity, and concomitantly decreased striatal levels of 5-HT and 5-HIAA. This study suggests that the increased locomotor activity may be due to increased striatal serotonergic activity which overcompensates for the DA deficiency. The locomotor hypoactivity, induced by 5,7-DHT, might be due to the decreased striatal levels of 5-HT and 5-HIAA.

This experiment examined the effect of destroying the ascending 5-hydroxytryptaminergic (5-HTergic) pathways on timing and switching behaviour in the free-operant psychophysical procedure. Rats received injections of 5,7-dihydroxytryptamine into the dorsal and median raphe nuclei or sham lesions. They were trained to press levers for sucrose reinforcement; sessions consisted of fifty 50-s trials in which reinforcers were available on a variable-interval 30-s schedule. In the first 25 s, of each trial, reinforcement was only available for responses on lever A; in the last 25 s, it was available only for responses on lever B. In phase 1 (70 sessions) repetitive switching between the levers was prevented by withdrawal of lever A after the first response on lever B in each trial; in phase 2 (40 sessions) this constraint on switching was removed; in phase 3 (40 sessions) the constraint was reinstated. Data were collected from probe trials (four per session) in which no reinforcers were delivered, during the last ten sessions of each phase. In all phases, both groups showed declining response rates on lever A and increasing response rates on lever B as a function of time from the onset of the trial. Response rate on lever B, expressed as percentage of overall response rate, could be described by a two-parameter logistic function. Removal of the constraint on switching reduced the slope of the function without changing the indifference point (time corresponding to 50% responding on lever B). The parameters of the timing function did not differ between the groups in any of the phases. However, the lesioned group showed a greater enhancement of switching rate during phase 2 than the control group. The levels of 5-HT and 5-hydroxyindoleacetic acid were reduced in the brains of the lesioned rats, but the levels of noradrenaline and dopamine were not altered. The results provide further evidence for the involvement of the ascending 5-HTergic pathways in switching between response alternatives, but cast doubt on our previous suggestion that the effects of 5-HT depletion on temporal differentiation of behaviour are mediated by facilitated switching.

Drugs that enhance serotonergic neurotransmission reduce food intake by directly or indirectly activating serotonergic receptors. In contrast drugs that inhibit serotonergic neurotransmission such as the 5-HT1A agonist 8-hydroxy-2-(di-n-propyl-amino)tetralin (8-OH-DPAT) stimulate food intake. The present study examined the effects of 8-OH-DPAT on the feeding suppressant action of the indirect 5-HT agonists fenfluramine (FEN; 0.63-2.5 mg/kg) and fluoxetine (FLU; 2.5-10 mg/kg), as well as the 5-HT1B/2C agonist 1-(3-trifluoromethylphenyl)piperazine (TFMPP; 0.5-2 mg/kg). 8-OH-DPAT (62.5-250 microg/kg) was administered 5 min prior to FEN, FLU or TFMPP, injected 30 min before food access. While FEN, FLU and TFMPP dose-dependently reduced 2 h food intake, 8-OH-DPAT stimulated eating behavior. 8-OH-DPAT (62.5-250 microg/kg) pretreatment reversed the anorectic action of FEN (1.25 mg/kg) and FLU (5 mg/kg) but not TFMPP (1 mg/kg). Separate groups of rats were injected with 5,7-dihydroxytryptamine (5,7-DHT; 3 microg free base) into both the dorsal and median raphe, which resulted in extensive 5-HT depletion in hypothalamus (80%), striatum and hippocampus (90%). In both 5, 7-DHT and vehicle-injected rats, FEN (1.25 mg/kg) and FLU (5 mg/kg) suppressed feeding. In 5,7-DHT treated rats, however, the ability of 8-OH-DPAT (125 microg/kg) to block FEN and FLU induced anorexia was attenuated. That is, 8-OH-DPAT pretreatment did not reverse the feeding inhibitory effects of either FEN or FLU. Further, the ability of FEN and FLU to suppress food intake was not altered by the 5,7-DHT lesion. These findings suggest that the reversal of FEN and FLU anorexia by 8-OH-DPAT is partially dependent on the integrity of brain 5-HT systems since their disruption compromises the ability of this 5-HT1A agonist to antagonize the feeding suppressant action of either FEN or FLU. However, the ability of treatments which impair 5-HT neurotransmission to reverse FEN and FLU induced suppression of food intake may depend upon whether this impairment is acute and reversible (8-OH-DPAT), or chronic and irreversible (5,7-DHT)., (Copyright 1998 Published by Elsevier Science B.V. All rights reserved.)

Quantitative autoradiography was used to examine possible adaptive changes in serotonin 5-HT1B/1D and 5-HT2A/2C receptor binding sites in adult rat basal ganglia, after partial or severe lesions of serotonergic neurons produced by intraraphe injections of variable amounts of 5,7-dihydroxytryptamine. In controls, the 5-HT1B/1D sites labeled with S-CM-G[125I]TNH2 were evenly distributed in the core and the shell of the nucleus accumbens. The density of 5-HT1B/1D sites was higher in the ventral than dorsal part of the striatum and no regional differences were detected along the rostrocaudal axis of the structure. The 5-HT2A/2C sites labeled with [125I]DOI were preferentially distributed in the mediodorsal striatum and higher densities were detected in the shell than core of the nucleus accumbens. Following 5,7-dihydroxytryptamine injections, there were no changes in binding of either receptor subtype after partial lesions entailing 80-90% 5-HT depletions. After severe 5-HT depletions (over 95%), large increases in 5-HT1B/1D binding were observed in the substantia nigra (78%), but no changes took place in the globus pallidus. Increases in 5-HT1B/1D binding were also detected in the shell of the nucleus accumbens (27%). Similar sized increases in 5-HT2A/2C binding (22%) were restricted to the medial striatum. The present results suggest a preferential association between 5-HT1B/1D receptors and the striatonigral neurons containing substance P, as indicated by the striatal distribution of these receptors and their selective increases in the substantia nigra after severe 5-HT deprivation. We recently proposed a similar relationship between the 5-HT4 receptors and the striatopallidal neurons containing met-enkephalin. Moreover, the increases in 5-HT1B/1D binding in the substantia nigra and in the shell of the nucleus accumbens reinforce the view of an implication of this receptor subtype in motor functions. In contrast, the prominent increases in 5-HT2A/2C binding after severe 5-HT deprivation as restricted to the medial region of the striatum and suggest up-regulation of most probably 5-HT2C receptors in a region implicated in cognitive functions., (Copyright 1998 Elsevier Science B. V.)

The immediate and long-term effects of the selective serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) on rat striatal serotonergic neurons were examined after its intracerebroventricular administration using in vivo voltammetry. Extracellular concentration of 5-hydroxyindoles increased immediately following intracerebroventricular 5,7-DHT injection (200 microg in 24 microl, 18 min), peaked at 1.5-2 h, and returned to normal by 4 h. 5,7-DHT diffused to the contralateral striatum in detectable amounts 9 to 12 min after the start of injection and returned to basal levels by 1.5 h. Three to 6 days after 5,7-DHT lesions, 5-hydroxytryptophan administration produced an increase in striatal 5-hydroxyindoles that was greater than that produced in pre-lesioned rats. This effect was maximal at 14 to 17 days post-lesion, and remained even after 50 days. The short-term effect of 5,7-DHT may be attributable to increased serotonin release, inhibition of uptake, or monoamine oxidase inhibition. The long-term effect of 5,7-DHT lesions may attributable to increased synthesis of serotonin or decreased reuptake in remaining serotonergic neurons.

To clarify the serotonergic mechanisms involved in the protection against ischemic neuronal damage, ZD-211 (citalopram HBr), a serotonin (5-hydroxytryptamine; 5-HT) re-uptake inhibitor, or buspirone, a 5-HT1A agonist, was locally administered into the hippocampus of gerbils. Additionally, to clarify the role of the 5-HT nervous system in the hippocampus during ischemic neuronal damage, animals were subjected to the local administration of 5,7-dihydroxytryptamine (5,7-DHT), a 5-HT neurotoxin, before ischemia challenge. Gerbils received intrahippocampal administration of ZD-211 (200 nmol/animal) or buspirone (20 nmol/animal) before 5-min ischemia. 5,7-DHT was intrahippocampally administered 7 days before a 2-min non-lethal ischemia challenge. In vehicle-treated animals subjected to 5 min of ischemia, almost all hippocampal CA1 pyramidal neurons were lost. The treatment with ZD-211 or buspirone showed a significant protective effect, and the number of neurons was significantly increased compared to vehicle-treated animals. Pretreatment with NAN-190, a 5-HT1A antagonist, completely abolished the protective effect of ZD-211 or buspirone. In the 5,7-DHT-treated animals, the number of neurons was significantly reduced following 2 min of ischemia compared to vehicle-treated animals in which this period of ischemia is non-lethal. Thus, intrahippocampal treatment with ZD-211 or buspirone can protect neuronal damage following transient ischemia in gerbils. These effects of ZD-211 and buspirone were mediated through the 5-HT1A receptor in the hippocampus. Furthermore, the destruction of the 5-HT nervous system in the hippocampus aggravated ischemic neuronal damage. Therefore, this study showed that the enhanced activity of the 5-HT nervous system in the hippocampus may protect against neuronal damage following cerebral ischemia.

The 5-HT2 receptor antagonist, ritanserin, reduces alcohol intake in rats and the nucleus accumbens (NAC) has been proposed as a site of action for the drug. Recent microdialysis studies have shown that acute subcutaneous (SC) administration of ritanserin increases extracellular 5-HT levels in the NAC. The present study evaluated, in genetically heterogeneous rats with developed preference for 3% ethanol, whether the attenuation of ethanol intake induced by ritanserin might be related to its effect on the synaptic availability of 5-HT in the NAC. Damaging 5-HTergic neurons by intracerebroventricular infusion of 5,7-dihydroxytryptamine (5,7-DHT) abolished the effect of ritanserin on ethanol consumption. Injections of the 5-HT3 receptor antagonist MDL 72222 into the NAC significantly reduced the inhibitory effect of SC injection of ritanserin, 1 mg/kg, and completely abolished the effect of ritanserin, 0.1 mg/kg. Subcutaneous injections of MDL 72222, 0.3 mg/kg 3 times/day, suppressed the effect of SC ritanserin, 0.1 mg/kg. The present findings, together with those of previous experiments showing that the tryptophan hydroxylase inhibitor p-chlorophenylalanine abolishes the effect of ritanserin, support the hypothesis that its effect on ethanol intake may be due to increased synaptic availability of 5-HT into the NAC.

We previously found different effects on behavior, serotonin (5-HT) concentrations, 5-HT uptake sites, and 5-HT1A binding sites of neonatal 5,7-dihydroxytryptamine (5,7-DHT) lesions depending on the route of 5,7-DHT injection. To study the impact of early lesions on 5-HT1B sites as putative 5-HT terminal autoreceptors, we labelled them autoradiographically with [3H]5-HT 4 months after intraperitoneal (i.p.) or intracisternal (i.c.) 5,7-DHT injection during the first postnatal week and quantitated specific binding in 22 brain regions. Changes were confined to the subiculum and substantia nigra, regions with the most 5-HT1B-specific binding and projection areas of structures with high mRNA expression. Both routes of 5,7-DHT injection were associated with increases in specific binding in subiculum (24% for i.p. and 47% for i.c. route). In contrast, there was a 32% increase in specific binding in the substantia nigra in rats with lesions made i.c. but not i.p. No significant differences were found in nucleus accumbens, caudate-putamen or other brain areas. In saturation homogenate binding studies of 5-HT1B sites using [125I]iodocyanopindolol 1 month after i.p. injections, neonatal 5,7-DHT lesions did not significantly alter Bmax or Kd in the neocortex, striatum, diencephalon or brainstem. These data indicate the differential effects of the route of neonatal 5,7-DHT injections on plasticity of 5-HT1B receptor recognition sites and suggest the presence of a subpopulation of post-synaptically located 5-HT1B sites which increases in response to denervation. The data also suggest that sprouting of 5-HT neurons after neonatal 5,7-DHT lesions does not involve 5-HT1B sites.

The acute and chronic treatment with 5-HTP was reported to block the aggressive reaction induced by food competition in undernourished dominant pigeons. Such treatment was not effective, however, in submissive pigeons. In this report we describe the effect of the neurotoxine 5, 7-DHT on aggression, defense, emotion, feeder control, and other behaviors in dominant and submissive male pigeons maintained at 80% of their normal weight. These were ranked through daily food competition trials. To obtain the prelesion scores dominant (n = 15) and submissive pigeons (n = 16) were submitted to a daily trial against a different intermediate subject (six trials). Then, the dominant and submissive groups were subcutaneously injected with Desipramine (25 micrograms/kg), anesthetized at a 60 min interval and injected with 5, 7-DHT into the left lateral ventricle (25 micrograms/25 microliters 0.9% NaCl). After a 30-day interval, both lesioned dominants and submissives were confronted to a different untreated intermediate subject over six daily sessions (postlesion scores). Differences between pre- and postlesion scores for all behavior studied were not found in dominant subjects. In submissive subjects, however, the postlesion scores of total aggression, defensive behavior, and emotional behavior were significantly higher than pre lesion scores. When brain 5-HT was assayed 60 days after injection about 34% depletion was found for both groups of pigeons. These findings suggest that the behavioral response to brain 5-HT denervation in pigeons is related to the behavioral characteristics of the subject previous to the lesion.

The effects of chronically ablating the serotoninergic inputs to various regions of the rat brain on the ability to solve a feature-negative discrimination was measured. After intracerebroventricular administration of the specific neurotoxin 5,7-dihydroxytryptamine, the rats exhibited an impaired capacity to solve such a discrimination, irrespective of whether auditory or visual stimuli were used. Further behavioural analysis revealed that this effect was not due to a reduced capacity to form excitatory associations, since both groups responded equally to reinforced stimuli. By contrast, the lesion more likely resulted in a failure to endow the non-reinforced stimuli with inhibitory properties. This suggestion was supported by the observation that, in a retardation test, the conditioned inhibitor aroused less inhibition in the lesioned group than in vehicle-injected controls. Furthermore, the conditioned inhibitor failed to pass a summation test in lesioned animals, again indicating that their hampered ability to master the discrimination was the result of an impairment in the formation of inhibitory associations. It is concluded that destruction of central 5-hydroxytryptamine-containing pathways impairs the functioning of brain areas underlying inhibitory associative learning.

To further assess the role of 5-HT in the modulation of audiogenic seizures (AGS) in the Genetically Epilepsy-Prone Rat (GEPR), changes in AGS severity after widespread chronic depletion of brain 5-HT by intracerebroventricular administration of 5,7-dihydroxytryptamine (5,7-DHT) were examined in moderate seizure GEPRs (GEPR-3s). Following treatment with 5,7-DHT (150 micrograms/30 microliters), a significant increase in seizure severity was observed at 2, 3 and 4 weeks as compared to vehicle-injected controls. The increase in seizure severity was evidenced by a significant increase in the incidence of tonic convulsions in 5,7-DHT treated animals (53% in treated animals compared to 0% in vehicle treated controls) over the testing period. Interestingly, the latency to wild running was increased in 5,7-DHT treated GEPRs, suggesting that depletion of brain 5-HT may slow initiation of AGS. Neurochemical analysis revealed marked depletion of 5-HT in the cortex (-96%), hippocampus (-94%), thalamus (-80%), hypothalamus (-62%), midbrain (-51%) and pons-medulla (-52%) in animals that received 5,7-DHT. However, no significant reductions in brain norepinephrine content were observed in any of the regions assayed due to the pretreatment of all animals with protriptyline. The present findings lend further support for an inhibitory action of brain 5-HT on audiogenic seizures in GEPRs.

1. 5-Hydroxytryptamine (5-HT) plays a role in the regulation of noradrenergic neurones in the brain, but the precise mechanism of regulation of noradrenaline (NA) release by 5-HT1A receptors has not been defined. The present study describes the effect of a highly potent and selective 5-HT1A receptor agonist, 5-(3-[[(2S)-1,4-benzodioxan-2-ylmethyl)]amino]propoxy)-1,3-b enzodioxole HC1 (MKC-242), on NA release in the hypothalamus using microdialysis in the freely moving rat. 2. Subcutaneous injection of MKC-242 (0.5 mg kg-1) increased extracellular levels of NA and its metabolite, 3-methoxy-4-hydroxyphenylglycol, in the hypothalamus and hippocampus. 3. The 5-HT1A receptor agonists, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (0.2 mg kg-1) and buspirone (3 mg kg-1) mimicked the effect of MKC-242 in increasing NA release in the hypothalamus. 4. The effects of MKC-242 and 8-OH-DPAT in the hypothalamus were antagonized by pretreatment with WAY100135 (10 mg kg-1), a silent 5-HT1A receptor antagonist. 5. Local administration of 8-OH-DPAT (10-100 microM), citalopram (1 microM), a 5-HT reuptake inhibitor, and MDL72222 (10 microM), a 5-HT3 receptor antagonist, into the hypothalamus, had no effect on NA release. 6. Intracerebroventricular injection with 5,7-dihydroxytryptamine caused a marked reduction in brain 5-HT content, but the treatment affected neither basal NA levels nor the MKC-242-induced increase in NA release. 7. The effect of MKC-242 in increasing NA release was not attenuated by repeated treatment with the drug (0.5 mg kg-1, once a day for 2 weeks). 8. The present results suggest that activation of postsynaptic 5-HT1A receptors increases NA release in the hypothalamus.