Regional changes in neuropeptide levels after 5,7-dihydroxytryptamine-induced serotonin depletion in the rat brain

Summary The levels of five neuropeptides (substance-P, somatostatin, cholecystokinin octapeptide, methionin-enkephalin and dynorphin) were examined in the brain and the spinal cord of rats 2 weeks after intracerebroventricular injection of 5,7-dihydroxytryptamine (5,7-DHT). 5,7-DHT injection caused a significant reduction of the serotonin level in all regions of the brain. The level of each neuropeptide except dynorphin significantly increased in specific regions of the brain after 5,7-DHT treatment without any decrease in their levels in any region. Since, coexistence and interaction between classical neurotransmitters and neuropeptides in the same neurons have been reported, both are indispensable for evaluating pathophysiological state of the brain function associated with abnormal neural transmission. The present findings together with previous reports suggest that neuropeptides act as neurotransmitters and compensate for the impaired function of the serotonergic systems.     

Selective serotonin depletion does not regulate hippocampal neurogenesis in the adult rat brain: differential effects of p-chlorophenylalanine and 5,7-dihydroxytryptamine

Serotonin is suggested to regulate adult hippocampal neurogenesis, and previous studies with serotonin depletion reported either a decrease or no change in adult hippocampal progenitor proliferation. We have addressed the effects of serotonin depletion on distinct aspects of adult hippocampal neurogenesis, namely the proliferation, survival and terminal differentiation of hippocampal progenitors. We used the serotonin synthesis inhibitor p-chlorophenylalanine (PCPA) or the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) to deplete serotonin levels. 5,7-DHT selectively decreased hippocampal serotonin levels, while PCPA resulted in a significant decline in both serotonin and norepinephrine levels. We observed a robust decline in the proliferation and survival of adult hippocampal progenitors following PCPA treatment. This was supported by a decrease in the number of doublecortin-positive cells in the neurogenic niche in the hippocampus. In striking contrast, 5,7-DHT did not alter the proliferation or survival of adult hippocampal progenitors and did not alter the number of doublecortin-positive cells. The terminal differentiation of adult hippocampal progenitors was not altered by either PCPA or 5,7-DHT treatment. An acute increase in serotonin levels also did not influence adult hippocampal progenitor proliferation. These results suggest that selective serotonin depletion or an acute induction in serotonin levels does not regulate adult hippocampal neurogenesis, whereas treatment with PCPA that induces a decline in both serotonin and norepinephrine levels results in a significant decrease in adult hippocampal neurogenesis. Our results highlight the need for future studies to examine the role of other monoamines in both the effects of stress and antidepressants on adult hippocampal neurogenesis.     

5,7-DHT-induced hippocampal 5-HT depletion attenuates behavioural deficits produced by 192 IgG-saporin lesions of septal cholinergic neurons in the rat

Adult Long-Evans male rats sustained injections of 5,7-dihydroxytryptamine into the fimbria-fornix (2.5 microg/side) and the cingular bundle (1.5 microg/side) and/or to intraseptal injections of 192 IgG-saporin (0.4 microg/side) in order to deprive the hippocampus of its serotonergic and cholinergic innervations, respectively. Sham-operated rats were used as controls. The rats were tested for locomotor activity (postoperative days 18, 42 and 65), spontaneous T-maze alternation (days 20-29), beam-walking sensorimotor (days 34-38), water maze (days 53-64) and radial maze (days 80-133) performances. The cholinergic lesions, which decreased the hippocampal concentration of ACh by about 65%, induced nocturnal hyperlocomotion, reduced T-maze alternation, impaired reference-memory in the water maze and working-memory in the radial maze, but had no effect on beam-walking scores and working-memory in the water maze. The serotonergic lesions, which decreased the serotonergic innervation of the hippocampus by about 55%, failed to induce any behavioural deficit. In the group of rats given combined lesions, all deficits produced by the cholinergic lesions were observed, but the nocturnal hyperlocomotion and the working-memory deficits in the radial maze were attenuated significantly. These results suggest that attenuation of the serotonergic tone in the hippocampus may compensate for some dysfunctions subsequent to the loss of cholinergic hippocampal inputs. This observation is in close concordance with data showing that a reduction of the serotonergic tone, by pharmacological activation of somatodendritic 5-HT(1A) receptors on raphe neurons, attenuates the cognitive disturbances produced by the intrahippocampal infusion of the antimuscarinic drug, scopolamine. This work has been presented previously [Serotonin Club/Brain Research Bulletin conference, Serotonin: From Molecule to the Clinic (satellite to the Society for Neuroscience Meeting, New Orleans, USA, November 2-3, 2000)].     

Enhanced spatial discrimination learning in rats following 5,7-DHT-induced serotonergic deafferentation of the hippocampus.

Learning in rats trained in the Stone 14-unit T-maze, a complex, positively reinforced spatial discrimination task was assessed following cytotoxic (5,7-dihydroxytryptamine; 5,7-DHT) deafferentation of the serotonergic inputs to the hippocampus. Serotonergic deafferentation was accomplished by infusing the cytotoxin in to the fornix-fimbria/cingulum bundle. Lesioned rats reached criterion (i.e. learned) in significantly fewer trials and made significantly fewer errors throughout training than either vehicle-injected or sham-operated controls. This represents the first time that the effects of selective chronic destruction of serotonergic inputs to the hippocampus have been investigated. The present results provide, therefore, evidence in support of a neuromodulatory role for serotonin (5-HT) within the rat hippocampus in the mediation of the processes underlying learning and memory for this task. Other studies are, therefore, warranted in order to determine whether hippocampal 5-HT also plays a role in the mediation of the processes underlying learning and memory in other types of tasks.     

Serotonin reuptake inhibitors do not prevent 5,7-dihydroxytryptamine-induced depletion of serotonin in rat brain

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.     

Temporal effects of intrahypothalamic 5,7-dihydroxytryptamine: relationship between serotonin levels and [H]serotonin binding

The relationship between serotonin (5-HT) levels and [³H]5-HT binding in discrete hypothalamic areas was examined in separate groups of animals at various times, following unilateral intrahypothalamic injection of 5,7-dihydroxytryptamine (5,7-DHT). Seven days post-5,7-DHT lesion, 5-HT levels were significantly decreased in both the ipsilateral and contralateral ventromedial and dorsomedial hypothalamic nuclei (VMN, DMN). In the lateral hypothalamic area (LHA), 5-HT levels were significantly decreased only ipsilaterally. Fifty days postlesion, 5-HT levels in the ipsilateral VMN remained significantly below sham, while the DMN and LHA returned to sham values. Seven days after 5,7-DHT there was a significant increase in [³H]5-HT labeling densities in the ipsilateral and contralateral ventromedial hypothalamic area as well as in the ipsilateral LHA. In contrast, in the dorsomedial hypothalamic area there was no increase in [³H]5-HT binding. Fifty days postlesion, no significant differences in [³H]5-HT binding between 5,7-DHT and sham were observed in any areas examined. This data provides further evidence for the regeneration of 5-HT fibers in the hypothalamus and demonstrates that the relationship between [³H]5-HT binding and 5-HT levels varies from one hypothalamic area to another.     

Temporal effects of intrahypothalamic 5,7-dihydroxytryptamine: relationship between serotonin levels and [3H]serotonin binding

The relationship between serotonin (5-HT) levels and [3H]5-HT binding in discrete hypothalamic areas was examined in separate groups of animals at various times, following unilateral intrahypothalamic injection of 5,7-dihydroxytryptamine (5,7-DHT). Seven days post-5,7-DHT lesion, 5-HT levels were significantly decreased in both the ipsilateral and contralateral ventromedial and dorsomedial hypothalamic nuclei (VMN, DMN). In the lateral hypothalamic area (LHA), 5-HT levels were significantly decreased only ipsilaterally. Fifty days postlesion, 5-HT levels in the ipsilateral VMN remained significantly below sham, while the DMN and LHA returned to sham values. Seven days after 5,7-DHT there was a significant increase in [3H]5-HT labeling densities in the ipsilateral and contralateral ventromedial hypothalamic area as well as in the ipsilateral LHA. In contrast, in the dorsomedial hypothalamic area there was no increase in [3H]5-HT binding. Fifty days postlesion, no significant differences in [3H]5-HT binding between 5,7-DHT and sham were observed in any areas examined. This data provides further evidence for the regeneration of 5-HT fibers in the hypothalamus and demonstrates that the relationship between [3H]5-HT binding and 5-HT levels varies from one hypothalamic area to another.     

Effects of 5,7-dihydroxytryptamine depletion of tissue serotonin levels on extracellular serotonin in the striatum assessed with in vivo microdialysis: relationship to behavior.

Effects of i.c.v. administration of 5,7-dihydroxytryptamine (5,7-DHT) on biochemistry and behavior were studied in awake Sprague-Dawley rats. It was found that 5,7-DHT depletion of striatal tissue levels of serotonin (5-HT) does not diminish extracellular levels until substantial depletions occur. This finding is similar to those observed after 6-hydroxydopamine lesions of the brain dopamine systems. Although varying amounts of 5,7-DHT produced serotonin depletions in striatal tissue, decreases in extracellular levels were only observed at tissue depletions greater than 60% compared to saline-injected control subjects. Thus, the effects of serotonin lesions which produce only moderate depletions may not be the result of decreased extracellular serotonin, but instead may be the result of compensatory changes in remaining neurons which maintain normal extracellular serotonin concentrations. Different degrees of striatal serotonin depletion were associated with opposite behavioral effects. Moderate levels of serotonin depletion (50-75%) produced evidence of increased anxiety, while these effects were no longer seen in rats with more severe 5-HT depletions (>75%).     

The influence of serotonin depletion on rat behavior in the Vogel test and brain 3H-zolpidem binding

Summary. The influence of p-chlorophenylalanine (p-CPA) and 5,7-dihydroxytryptamine (5,7-DHT)-induced serotonin depletion on rat behavior as well as on zolpidem's the behavioral effects and binding to some brain areas of zolpidem, was examined with the help of Vogel's punished drinking test and autoradiography, respectively. Moreover, changes in the serotonin levels and turnover rate were studied in the forebrain and brainstem of rats pretreated with various ligands at the benzodiazepine (BDZ) receptors (midazolam, bretazenil, abecarnil, zolpidem). These drugs were given at doses shown previously to significantly disinhibit animal behavior suppressed by punishment in the Vogel test (Nazar et al., 1997). It was found that serotonin decrease in the frontal cortex and hippocampus after p-CPA significantly and inversely correlated with rat behavior controlled by fear in the VT. p-CPA produced an anticonflict activity in the absence of effect on spontaneous drinking, pain threshold and motility of animals. All applied benzodiazepine receptor ligands decreased the 5-HT turnover rate in the frontal cortex and hippocampus, whereas in the brainstem only abecarnil and zolpidem diminished 5-hydroxyindoleacetic acid levels. This part of the study replicated earlier data with neurotoxins and indicated that the anxiolytic-like effect of 5-HT depletion in some models of anxiety did not depend on changes in animal appetitive behavior or stimulus control. Moreover, the fact that all non-selective and selective (zolpidem) agonists of the type 1 benzodiazepine receptors seemed to produce the same anticonflict effect and decreasing 5-HT turnover indicates that this subtype of benzodiazepine receptor may be important for the interaction between brain 5-HT and GABA/BDZ systems. Accordingly, it was found that serotonin decrease enhanced the anticonflict effect of zolpidem in the Vogel test and increased ³H-zolpidem binding to the occipital cortex and substantia nigra. Altogether, the present study provides more arguments for the role of changes in the activity of brain 5-HT innervation in the control of emotional processes. Moreover, it points to the BDZ₁ receptor subtype as a possible target of interaction between brain 5-HT and GABA_A/BDZ systems. Received January 12, 1998; accepted September 2, 1998     

Selective prefrontal serotonin depletion impairs acquisition of a detour-reaching task

We have recently shown that serotonin in the primate orbitofrontal cortex (OFC) contributes to the flexible control of behaviour. 5,7-dihydroxytryptamine-induced 5-HT depletions of OFC impair performance on a serial reversal discrimination task [Clarke et al. (2004)Science, 304, 878-880]. The deficit is characterized by perseverative responding to the previously rewarded stimulus, a deficit similar to that seen following lesions of the intrinsic neurones of the OFC [Dias et al. (1996)Nature, 380, 69-72]. The effect is neurochemically selective as dopaminergic lesions of the OFC, induced by 6-hydroxydopamine, have no effect [Clarke et al. (2006)Cerebral Cortex]. In order to test for the generality of the effect of serotonin on orbitofrontal processing and, in particular, its effects on flexible behaviour, the present study investigated the effects of serotonin depletions of OFC on performance of another task dependent upon an intact OFC, the detour-reaching task [Wallis et al. (2001)European Journal of Neuroscience, 13, 1797-1808]. Successful performance of this task requires inhibition of the animal's prepotent response tendency to reach directly along its line of sight to the reward. Compared with sham-operated controls, we found that lesioned monkeys made significantly more barrier reaches directly along their line of sight to the visible reward during task acquisition. This finding provides further support for the role of prefrontal serotonin in inhibitory control processes specifically in tasks sensitive to OFC dysfunction.     

Pressor effects of dorsal raphe stimulation and intrahypothalamic application of serotonin in the spontaneously hypertensive rat

Electrical stimulation of the dorsal raphe nucleus or direct microinjection of serotonin into the preoptic region of the anterior hypothalamus produces a transient rise in arterial blood pressure in both spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) controls. SHRs are more responsive to raphe stimulation but are somewhat less responsive to serotonin injections when compared to WKYs. The serotonin antagonist metergoline blocks the pressor response to serotonin in both strains. These results suggest that the central serotonin neuronal system plays a similar, but not identical, role in blood pressure modulation in hypertensive and normotensive rats.     

Neural and hormonal consequences of neonatal 5,7-dihydroxytryptamine may not be associated with serotonin depletion

The neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) is often used in neonatal rats to induce specific, rapid, and permanent depletion of brain serotonin (5-HT). One assumed benefit of using this drug in neonates is that it is well-tolerated, with pups exhibiting few side effects normally attributed to 5-HT depletion. Here, we present evidence that 5,7-DHT administered neonatally induces seizure-like behavior, decreases weight gain, and increases plasma corticosterone without depletion of brain 5-HT.     

Selective depletion of somatostatin in rat brain by cysteamine

A single injection of cysteamine (300 mg/kg, subcutaneously) results in a 70–80% decrease in somatostatin levels in the periventricular nucleus where somatostatin-producing neurons are located and the median eminence where somatostatinergic nerve terminals are. The drug seems quite selective: no changes in levels of other neuropeptides — LH-RH, vasopressin, enkephalin, VIP, CCK — were observed in the same animals.     

A general role for serotonin in the control of behavior: studies with intracerebral 5,7-dihydroxytryptamine

Multiple injections of 5,7-dihydroxytryptamine (5,7-DHT) into the rat brainstem reduced forebrain levels of serotonin and 5-hydroxyindoleacetic acid to 3-10% of the levels observed in control rats that had received intrabrainstem injection of a Locke's solution vehicle. This treatment reduced or abolished atropine-resistant cerebral activation (ARCA) in most cases. In rats in which ARCA was impaired or lost, a number of behavioral abnormalities were observed. These included: high levels of locomotion in an open field test; a deficiency in swimming to, and climbing upon, a visible platform in a water-filled tank; deficient social behavior; and impaired performance in a simple test of active avoidance. These deficits were not due to low level motor impairment. The 5,7-DHT-treated rats displayed a circadian rhythm of activity in running wheels. It is proposed that ascending serotonergic projections are an important component in the cerebral control of the Type 1 behavior with which the occurrence of ARCA is closely linked. Since Type 1 behavior includes such motor patterns as walking and manipulation of objects with the limbs, which are essential components of a great variety of behavioral performances, it is to be expected that a loss of ascending serotonergic function will result in a generalized deficit in behavior.     

Morphine induces an increase in extracellular serotonin in the rat diencephalon

The effect of systemic morphine on extracellular serotonin (5-HT) in the diencephalon of unanesthetized, unrestrained rats was investigated by in vivo microdialysis coupled to high performance liquid chromatography with electrochemical detection. Administration of morphine resulted in a dose dependent increase in extracellular 5-HT. Significant increases were first seen at a dose of 5 mg/kg, and a maximal increase occurred at 10 mg/kg. This increase was blocked by pretreatment with naltrexone, indicating that the effect of morphine on 5-HT was mediated by opiate receptors. Morphine also had a significant effect on extracellular 5-hydroxyindoleacetic acid (5-HIAA), leading to a gradual increase across a range of doses. The effect of morphine on 5-HT was compared to effects of morphine on nociception and catalepsy. Increases in 5-HT and 5-HIAA were first detected at doses that were analgesic but too low to elicit catalepsy. Consistent with many reports that opioids cause an increase in synthesis and turnover of 5-HT in the rat forebrain, the present results provide more direct evidence that 5-HT release is increased after morphine administration.     

Water maze impairments after combined depletion of somatostatin and serotonin in the rat

This study examined the effect of both separate and combined depletion of brain somatostatin and serotonin on acquisition of the water maze (WM) task. Naïve male Long-Evans rats received injections of p-chlorophenylalanine (PCPA; 500 mg/kg × 2) to deplete serotonin or cysteamine (90 or 200 mg/kg) to deplete somatostatin, or both treatments prior to spatial and reversal training in the water maze. Some rats first received Morris’ nonspatial pretraining to train them in the behavioral strategies that are required for successful spatial place learning in this task, prior to drug treatment and spatial training. A detailed behavioral analysis indicated that somatostatin or serotonin depletion alone caused little or no impairment in naïve animals. Depletion of both somatostatin and serotonin in naïve rats impaired performance, with differences in the impairments that depended on the dose of cysteamine. Nonspatially pretrained rats were not impaired. Thus, neither somatostatin nor serotonin alone is crucial for the water maze task, but impairments occur if both somatostatin and serotonin are depleted in naïve rats. The results indicate that some of the performance impairment was due to strategies impairment rather than a spatial place learning impairment. Depletion of both somatostatin and serotonin in naïve rats produces results comparable to the spatial navigation deficits seen in some Alzheimer patients and suggests that combinations of antagonist treatments may better model this disorder than single antagonist treatments do.     

Regeneration of serotonergic fibers in the rat hypothalamus following unilateral 5,7-dihydroxytryptamine injection

The time course of degeneration and regeneration of serotonin (5-HT) fibers in the rat hypothalamus was studied with 5-HT immunocytochemistry and [3H]5-HT uptake following unilateral injections of 5,7-dihydroxytryptamine (5,7-DHT) into the dorsolateral hypothalamus. Within 3 days of the lesion, 5-HT fibers in the ipsilateral hypothalamus were swollen and darkly stained for 5-HT. In the contralateral hypothalamus few swollen fibers were apparent and these were generally restricted to the area adjacent to the fornix. Swollen 5-HT fibers were evident in the ipsilateral hypothalamus 3-19 days post-lesion in the medial forebrain bundle (MFB) during which time there was a gradual decrease in their density. In the medial and periventricular areas of the ipsilateral hypothalamus there were essentially no 5-HT fibers 7-30 days post-lesion. Sprouting 5-HT fibers were observed 12-19 days post-lesion. Thirty days post-lesion the density of 5-HT fibers in the MFB appeared normal; however, medial and periventricular areas remained denervated. Fifty days post-lesion there was an apparent bilateral hyperinnervation in the lateral and dorsomedial hypothalamic areas of 5,7-DHT-injected animals as compared to sham-injected animals. The morphological data were paralleled by changes in [3H]5-HT uptake. Seven days post-lesion specific high affinity uptake was reduced to 27% of sham in the ipsilateral hypothalamus and to 53% of sham in the contralateral hypothalamus. By 50 days post-lesion, specific high affinity uptake of [3H]5-HT was 141% of sham in the ipsilateral hypothalamus and 96% of sham in the contralateral hypothalamus.     

Effects of 5,7-dihydroxytryptamine on serotonin1 and serotonin2 receptors throughout the rat central nervous system using quantitative autoradiography

The effects of the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), on serotonin1 (5-HT1) and 5-HT2 receptors were investigated using the high degree of resolution provided by quantitative autoradiography in an effort to determine the synaptic location of these receptors. 5,7-DHT treatment resulted in a decrease in 5-HT1 binding in the dentate gyrus and CA3c/4 of the anterior hippocampus and in the dorsal raphe nucleus, whereas no changes were observed in the posterior hippocampus nor in many other brain structures. 5-HT2 receptors exhibited no changes in any brain area examined in response to 5,7-DHT treatment, despite over 90% serotonin depletion in most of the forebrain nuclei examined. The results indicate that at least some of the 5-HT1 sites labelled by [3H]5-HT in the hippocampus and dorsal raphe nucleus are presynaptic, whereas 5-HT2 receptors are probably postsynaptic. In addition, the distribution profiles of 5-HT1 and 5-HT2 binding sites were compared in the rat central nervous system at various anatomical levels. 5-HT1 binding sites were identified using [3H]5-HT, while 5-HT2 binding sites were labelled with [3H]ketanserin. Both receptor subtypes displayed distinctly different localization patterns, which, in most cases was the inverse of the other pattern. In the brainstem it is significant that 5-HT2 receptors are concentrated in the facial nucleus and the motor nucleus of the trigeminal nerve, areas known to influence head and facial movement. The serotonin-mediated head-shake response occurs when 5-HT2 receptors are activated. In contrast, 5-HT1 receptors are distributed throughout the brainstem and in specific portions of the spinal cord. These areas are thought to control the serotonin behavioral syndrome and this behavior is 5-HT1A-mediated. All raphe nuclei were devoid of 5-HT2 receptors; only 5-HT1 receptor were found in these nuclei. Correlations with serotonin terminal distribution patterns are discussed. The pattern of 5-HT2 receptor distribution was also compared with the pattern of alpha 1 receptors, using [3H]prazosin in order to determine whether [3H]ketanserin significantly labels alpha 1 receptors. Although some similarities exist, overlap of binding did not occur in other nuclei, indicating that alpha 1 contamination of this system is probably negligible.