Serotonergic Afferents Research Articles

Serotonergic connections to the ventral oral pontine reticular nucleus: implication in paradoxical sleep modulation.

Cholinergic microstimulation of the ventral part of the oral pontine reticular nucleus (vRPO) in cats generates and maintains paradoxical sleep. The implication of rostral raphe nuclei in modulating the sleep-wakefulness cycle has been based on their serotonergic projections to the pontine structures responsible for the induction of paradoxical sleep. However, serotonergic neurons have also been described in brainstem structures other than the raphe nuclei. The aim of the present work is to trace the origin of the serotonergic afferents to the vRPO and to the locus coeruleus alpha and perilocus coeruleu alpha nuclei, closely related with different paradoxical sleep events. Anterograde and retrograde horseradish peroxidase conjugated with wheat germ agglutinin tracer injections in these nuclei in cats were combined with serotonin antiserum immunohistochemistry. Our results demonstrate that reciprocal connections linking the rostral raphe nuclei to those oral pontine nuclei are scarce. The percentage of double-labeled neurons after injections in the vRPO averaged 18% in rostral raphe nuclei, while a level of 82% was estimated in mesopontine tegmentum structures other than the raphe nuclei. These results showed that the main source of serotonin to the vRPO, implicated in generation and maintenance of paradoxical sleep, arises from these mesopontine tegmentum structures. This indicates that the serotonin modulation of paradoxical sleep could be the result of activation in non-raphe mesopontine tegmentum structures. The existence of a complicated network in the vRPO, which maintains a balance between different neurotransmitters responsible for the generation and alternance of paradoxical sleep episodes, is discussed.

Compensatory responses in the aging hippocampal serotonergic system following neurodegenerative injury with 5,7-dihydroxytryptamine.

This study utilized a multidisciplinary approach to examine injury-induced compensatory responses in the aging hippocampal serotonin transporter (5-HTT), a membrane protein implicated in a variety of neurodegenerative disorders. Age-dependent cellular, anatomical, and physiological changes of the 5-HTT were evaluated in female Fischer 344 rats (2 and 17 months) following denervation of the serotonergic afferents (fimbria-fornix and cingulum bundle) to the dorsal hippocampus using the neurotoxicant 5,7-dihydroxytryptamine (5,7-DHT). Seven days following 5,7-DHT administration, a uniform loss of the hippocampal 5-HTT immunoreactivity was observed in both age groups. However, at 21 days 5-HTT immunoreactivity in young 5,7-DHT-treated animals was similar to control levels, indicative of recovery, while older animals exposed to 5,7-DHT did not show recovery of hippocampal 5-HTT expression. 5-HTT binding site density, as determined by quantitative autoradiography ([3H]citalopram), supported the immunohistochemical results by demonstrating a recovery of 5-HTT binding sites in young, but not old animals, at 21 days following the lesion (P < 0.001). Furthermore, cellular electrophysiological function of hippocampal CA1 pyramidal neurons in 3- and 18-month-old F344 rats at 21 days following 5,7-DHT or vehicle treatment were assessed using in vivo microiontophoretic application of serotonin (5-HT). Independent of changes in sensitivity to the inhibitory effects of 5-HT application, the time to recovery of cell firing following application of 5-HT was significantly increased in the 18-month 5,7-DHT group compared to the 18-month vehicle and 3-month 5,7-DHT groups (60 and 59% increases, respectively; P < 0.05). Overall, these series of studies comprise a model which can be used to identify cellular events underlying both the formation of injury-induced compensatory processes in younger animals and the lack thereof with advancing age.

Dependence of serotonin release in the locus coeruleus on dorsal raphe neuronal activity.

The serotonergic innervation of the locus coeruleus paetly derives from the dorsal raphe nucleus (DRN). Using the push-pull superfusion technique, we investigated whether and to what extent the release of serotonin and the extracellular concentration of its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the locus coeruleus are influenced by the neuronal activity of the DRN. In anaesthetized rats, a push-pull cannula was inserted into the locus coeruleus, which was continuously superfused with artificial cerebrospinal fluid (aCSF). Serotonin and 5-HIAA levels in the superfusate were determined by HPLC combined with electrochemical detection. Electrical stimulation (5 Hz, 300 microA, 1 ms) of the DRN for 5 min, or its chemical stimulation by microinjection of glutamate (3.5 nmol, 50 nl), led to an increased release of serotonin in the locus coeruleus and to a slight (2 mmHg) decrease in blood pressure. Superfusion of the locus coeruleus with tetrodotoxin (1 microM) abolished the increase in the release rate of serotonin evoked by electrical stimulation of the DRN, while the slight fall in blood pressure was not influenced. Thermic lesion (75 degrees C, 1 min) of the DRN elicited a pronounced decline in serotonin release rate within the locus coeruleus, the maximum decrease being 52%. The decrease in the release of serotonin was associated with a long-lasting rise in blood pressure. Microinjection of the serotonin neurotoxin 5,7-dihydroxytryptamine (5 microg, 250 nl) into the DRN led to an initial increase in the serotonin release rate that coincided with a short-lasting fall in blood pressure. Subsequently, the release of serotonin was permanently reduced and was associated with hypertension. Microinjection of the 5-HT1A receptor agonist (+/-)-8-hydroxy-dipropylaminotetralin (8-OH-DPAT; 7.5 nmol, 50 nl) into the DRN led to a long-lasting reduction of the release rate of serotonin in the locus coeruleus. Microinjection of 8-OH-DPAT into the DRN also slightly lowered blood pressure (3 mmHg). Neither stimulations nor lesion of the DRN, nor microinjection of 8-OH-DPAT into this raphe nucleus, altered the extracellular concentration of 5-HIAA. Judging from the present biochemical results it appears that the serotonergic afferents to the locus coeruleus originate to more than 50% from cell bodies located in the DRN. The neuronal serotonin release in the locus coeruleus is modulated by 5-HT1A receptors lying within the DRN. Changes in blood pressure and release of serotonin elicited by stimulating or lesioning the DRN point to the importance of serotonergic neurons extending between this raphe nucleus and the locus coeruleus in central cardiovascular control.

5-HT1BReceptor–Mediated Presynaptic Inhibition of Retinal Input to the Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) receives glutamatergic afferents from the retina and serotonergic afferents from the midbrain, and serotonin (5-HT) can modify the response of the SCN circadian oscillator to light. 5-HT1B receptor-mediated presynaptic inhibition has been proposed as one mechanism by which 5-HT modifies retinal input to the SCN (Pickard et al., 1996). This hypothesis was tested by examining the subcellular localization of 5-HT1B receptors in the mouse SCN using electron microscopic immunocytochemical analysis with 5-HT1B receptor antibodies and whole-cell patch-clamp recordings from SCN neurons in hamster hypothalamic slices. 5-HT1B receptor immunostaining was observed associated with the plasma membrane of retinal terminals in the SCN. 1-[3-(Trifluoromethyl)phenyl]-piperazine HCl (TFMPP), a 5-HT1B receptor agonist, reduced in a dose-related manner the amplitude of glutamatergic EPSCs evoked by stimulating selectively the optic nerve. Selective 5-HT1A or 5-HT7 receptor antagonists did not block this effect. Moreover, in cells demonstrating an evoked EPSC in response to optic nerve stimulation, TFMPP had no effect on the amplitude of inward currents generated by local application of glutamate. The effect of TFMPP on light-induced phase shifts was also examined using 5-HT1B receptor knock-out mice. TFMPP inhibited behavioral responses to light in wild-type mice but was ineffective in inhibiting light-induced phase shifts in 5-HT1B receptor knock-out mice. The results indicate that 5-HT can reduce retinal input to the circadian system by acting at presynaptic 5-HT1B receptors located on retinal axons in the SCN.

The potentiation of amphetamine-induced hyperlocomotion by fimbria-fornix lesions in rats is abolished by intrahippocampal grafts rich in serotonergic neurons

Three-month old Long-Evans female rats were submitted to aspirative lesions of the fimbria-fornix and intrahippocampal grafts of a cell suspension prepared from a region of the fetal brain including the septum and the diagonal band of Broca (rich in cholinergic neurons) or the raphe (rich in serotonergic neurons). A group of lesioned rats was grafted with both suspensions mixed. Lesion-only and sham-operated rats served as controls. Four months after the lesions, all rats were tested daily for locomotor activity in their home cage, 1 day without being injected, 2 days with an injection of NaCl and 5 days with an injection of 1 mg/kg (i.p.) d-amphetamine. The effects of the lesions and grafts were assessed by measuring the accumulation of [3H]-choline or [3H]-5-hydroxytryptamine (5-HT) by hippocampal slices, and the electrically-evoked release of tritium. Amphetamine injections produced hyperlocomotion which was potentiated by the lesion. This lesion-induced potentiation was also found in rats with septal grafts, but not in those with raphe or co-grafts. The uptake and electrically-evoked release of [3H]-acetylcholine or [3H]-5-HT were reduced in hippocampal slices from lesion-only rats. In rats which received grafts of septal cells or co-grafts, but not in those with raphe grafts, uptake and release of [3H]-acetylcholine were close to normal. Uptake and release of [3H]-5-HT were close to normal in rats with raphe grafts or with co-grafts, but not in those with septal grafts. Altogether, these data suggest that damage to the serotonergic afferents of the hippocampus might play some role in the potentiation of amphetamine-induced hyperlocomotion associated with fimbria-fornix lesions.

Distribution of the 5-HT 5A serotonin receptor mRNA in the human brain

The 5-HT 5A receptor is a member of a new subfamily of serotonin [5-hydroxytryptamine (5-HT)] receptors recently cloned from the human and rodent brain. The role of this receptor in normal brain functions as well as its possible involvement in pathological states is still to be determined. We therefore studied the regional distribution and cellular localization of 5-HT 5A receptor mRNA in human brain sections from autopsy samples by in situ hybridization histochemistry, in order to obtain anatomical information which might be useful in formulating hypotheses on possible functions subserved by this receptor in the central nervous system (CNS). Our results showed that the main sites of 5-HT 5A mRNA expression were the cerebral cortex, hippocampus and cerebellum. In the neocortical regions, the 5-HT 5A receptor mRNA was mainly distributed in the layers II–III and V–VI. In the hippocampus, the dentate gyrus and the pyramidal cell layer of the CA1 and CA3 fields expressed 5-HT 5A mRNA at high levels. The broad distribution in the neocortex and hippocampus supports the view that the 5-HT 5A receptor in these areas might be implicated in high cortical and limbic functions. The 5-HT 5A mRNA was widely distributed in the cerebellum where it was highly expressed in the Purkinje cells, in the dentate nucleus and, at a lower level, in the granule cells. Since the cerebellum receives diffuse serotonergic afferents, this finding suggests that the 5-HT 5A receptor may have an important role in mediating the effects of 5-HT on cerebellar functions.

Sprouting of the serotonergic afferents into striatum after selective lesion of the dopaminergic system by MPTP in adult mice

Neonatal destruction of the nigrostrial dopaminergic (DA) system with 6-hydroxydopamine leads to serotonergic (5-HT) hyperinnervation of the striatum. However, it is not clear whether this occurs in adult animals. We investigated whether serotonergic sprouting occurs in adult mice subjected to bilateral lesion of the DA system by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The effects of the lesion were evaluated using a new rotarod test and immunohistochemistry. One hundred days after the last MPTP lesion, there was a clear bilateral serotonergic hyperinnervation throughout the striatum. Additionally, those mice showing the highest reductions in striatal tyrosine hydroxylase (TH) immunoreactivity and in rotarod performance showed the highest density of serotonergic innervation (116% increase). The functional consequences of this process in Parkinson's disease and secondary parkinsonism remain to be clarified.

Colocalization of substance P or enkephalin in serotonergic neuronal afferents to the hypoglossal nucleus in the rat

Colocalization of substance P or enkephalin in serotonergic neuronal afferents to the hypoglossal nucleus in the rat

Serotonergic afferents mediate activity-dependent entrainment of the mouse circadian clock.

The circadian pacemaker located in the suprachiasmatic nuclei (SCN) of the hypothalamus receives serotonergic afferents from the midbrain raphe nuclei, but the functional role of this projection is unclear. In rodents, locomotor activity increases serotonin content in the SCN, and serotonergic agonists phase shift the circadian clock in a manner closely similar to voluntary bouts of vigorous exercise, suggesting that serotonergic afferents could be part of the activity-dependent entrainment mechanism. We investigated this possibility by selectively lesioning serotonin terminals within and adjacent to the SCN by local microinjection of 5,7-dihydroxytryptamine in mice pretreated with desipramine. This treatment decreased serotonin content 96 +/- 1% and 5-hydroxyindole-3-acetic acid content below levels of detection (nearly 100%) but did not decrease norepinephrine content or neuropeptide Y immunoreactivity in the SCN. These lesions did not alter subsequent running activity levels, yet rendered mice unable to synchronize to a regularly scheduled 2-h wheel running paradigm that entrained sham-lesioned controls. Serotonin afferents are thus necessary for activity-dependent entrainment in the mouse.

O-18-267 - Transplantation of dopaminerglc neurons in a rat model of Parkinson’s disease: Long-term functional restauration of the nigrostriatal pathway

Extensive lesions of the fimbria-fornix pathways and the cingular bundle deprive the hippocampus of a substantial part of its cholinergic, noradrenergic and serotonergic afferents and, among several other behavioural alterations, induce lasting impairment of spatial learning and memory capabilities. After a brief presentation of the neuroanatomical organization of the hippocampus and the connections relevant to the topic of this article, studies which have contributed to characterize the neurochemical and behavioural aspects of the fimbria-fornix lesion “syndrome” with lesion techniques differing by the extent, the location or the specificity of the damage produced, are reviewed. Furthermore, several compensatory changes that may occur as a reaction to hippocampal denervation (sprouting, changes in receptor sensitivity and modifications of neurotransmitter turnover in spared fibres) are described and discussed in relation with their capacity (or incapacity) to foster recovery from the lesion-induced deficits. According to this background, experiments using intrahippocampal or “parahippocampal” grafts to substitute for missing cholinergic, noradrenergic or serotonergic afferents are considered according to whether the reported findings concern neurochemical and/or behavioural effects. Taken together, these experiments suggest that appropriately chosen fetal neurons (or other cells such as, for instance, genetically-modified fibroblasts) implanted into or close to the denervated hippocampus may substitute, at least partially, for missing hippocampal afferents with a neurochemical specificity that closely depends on the neurochemical identity of the grafted neurons. Thereby, such grafts are able not only to restore some functions as they can be detected locally, namely within the hippocampus, but also to attenuate some of the behavioural (and other types of) disturbances resulting from the lesions. In some respects, also these graft-induced behavioural effects might be considered as occurring with a neurochemically-defined specificity. Nevertheless, if a graft-induced recovery of neurochemical markers in the hippocampus seems to be a prerequisite for also behavioural recovery to be observed, this neurochemical recovery is neither the one and only condition for behavioural effects to be expressed, nor is it the one and only mechanism to account for the latter effects. © 1997 Elsevier Science Ltd. All Rights Reserved.

THE FIMBRIA-FORNIX/CINGULAR BUNDLE PATHWAYS: A REVIEW OF NEUROCHEMICAL AND BEHAVIOURAL APPROACHES USING LESIONS AND TRANSPLANTATION TECHNIQUES

Extensive lesions of the fimbria-fornix pathways and the cingular bundle deprive the hippocampus of a substantial part of its cholinergic, noradrenergic and serotonergic afferents and, among several other behavioural alterations, induce lasting impairment of spatial learning and memory capabilities. After a brief presentation of the neuroanatomical organization of the hippocampus and the connections relevant to the topic of this article, studies which have contributed to characterize the neurochemical and behavioural aspects of the fimbria-fornix lesion “syndrome” with lesion techniques differing by the extent, the location or the specificity of the damage produced, are reviewed. Furthermore, several compensatory changes that may occur as a reaction to hippocampal denervation (sprouting, changes in receptor sensitivity and modifications of neurotransmitter turnover in spared fibres) are described and discussed in relation with their capacity (or incapacity) to foster recovery from the lesion-induced deficits. According to this background, experiments using intrahippocampal or “parahippocampal” grafts to substitute for missing cholinergic, noradrenergic or serotonergic afferents are considered according to whether the reported findings concern neurochemical and/or behavioural effects. Taken together, these experiments suggest that appropriately chosen fetal neurons (or other cells such as, for instance, genetically-modified fibroblasts) implanted into or close to the denervated hippocampus may substitute, at least partially, for missing hippocampal afferents with a neurochemical specificity that closely depends on the neurochemical identity of the grafted neurons. Thereby, such grafts are able not only to restore some functions as they can be detected locally, namely within the hippocampus, but also to attenuate some of the behavioural (and other types of) disturbances resulting from the lesions. In some respects, also these graft-induced behavioural effects might be considered as occurring with a neurochemically-defined specificity. Nevertheless, if a graft-induced recovery of neurochemical markers in the hippocampus seems to be a prerequisite for also behavioural recovery to be observed, this neurochemical recovery is neither the one and only condition for behavioural effects to be expressed, nor is it the one and only mechanism to account for the latter effects. © 1997 Elsevier Science Ltd. All Rights Reserved.

Serotonergic function of aging hippocampal CA3 pyramidal neurons: Electrophysiological assessment following administration of 5,7-dihydroxytryptamine in the fimbria-fornix and cingulum bundle

Serotonergic (5-hydroxytryptamine; 5-HT) neurotransmission has been implicated in the regulation of cognitive function and this neurotransmitter system may underlie selective neuronal degeneration found in the aging hippocampus. Age-dependent changes in 5-HT function of hippocampal CA3 subfield pyramidal neurons were evaluated in female Fischer 344 rats (2 and 17 months) following denervation of the serotonergic afferents to the dorsal hippocampus using the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). Vehicle (ascorbic saline) or 5,7-DHT was administered bilaterally in the fimbria-fornix/cingulum bundle and dorsal pyramidal cell responses to microiontophoretic application of 5-HT, the 5-HT1A agonist (+/-)-8-hydroxy-2-(di-N-propylamino) tetralin, the 5-HT1A antagonist WAY 100,135 and N-methyl-D-aspartate were recorded at 3 weeks post-lesion. Independent of changes in sensitivity to the inhibitory effects of 5-HT with aging, the time to recovery of cell firing following application of 5-HT was significantly increased in the 18 months 5,7-DHT group compared to the 18 month Vehicle and 3 month 5,7-DHT groups (3.3- and 2.6-fold, respectively). These results demonstrate that serotonergic neurotransmission is altered with aging following a selective neurotoxic insult to the hippocampus.

5HT1BReceptor Agonists Inhibit Light-Induced Phase Shifts of Behavioral Circadian Rhythms and Expression of the Immediate–Early Genec-fosin the Suprachiasmatic Nucleus

The suprachiasmatic nucleus (SCN) is a circadian oscillator and a critical component of the mammalian circadian system. It receives afferents from the retina and the mesencephalic raphe. Retinal afferents mediate photic entrainment of the SCN, whereas the serotonergic afferents originating from the midbrain modulate photic responses in the SCN; however, the serotonin (5HT) receptor subtypes in the SCN responsible for these modulatory effects are not well characterized. In this study, we tested the hypothesis that 5HT1B receptors are located presynaptically on retinal axon terminals in the SCN and that activation of these receptors inhibits retinal input. The 5HT1B receptor agonists TFMPP and CGS 12066A, administered systemically, inhibited light-induced phase shifts of the circadian activity rhythm in a dose-dependent manner at phase delay and phase advance time points. This inhibition was not affected by previous systemic application of either the selective 5HT1A receptor antagonist (+)WAY 100135 or by the 5HT2 receptor antagonist mesulergine, whereas pretreatment with the nonselective 5HT1 antagonist methiothepin significantly attenuated the effect of TFMPP. TFMPP also produced a dose-dependent reduction in light-stimulated Fos expression in the SCN, although a small subset of cells in the dorsolateral aspect of the caudal SCN were TFMPP-insensitive. TFMPP (1 mM) infused into the SCN produced complete inhibition of light-induced phase advances. Finally, bilateral orbital enucleation reduced the density of SCN 5HT1B receptors as determined using [125I]-iodocyanopindolol to define 5HT1B binding sites. These results are consistent with the interpretation that 5HT1B receptors are localized presynaptically on retinal terminals in the SCN and that activation of these receptors by 5HT1B agonists inhibits retinohypothalamic input.

Laminar development of the mouse barrel cortex: effects of neurotoxins against monoamines.

The rodent somatosensory cortex is characterized by a unique cellular organization in the field of representation of the whiskers, called the barrelfield, which develops in layer IV during the 1st postnatal week in parallel with the establishment of the thalamo-cortical connections. This area is transiently densely innervated by serotonergic afferents during this period. Serotonin depletion delays the formation of barrels in the rat somatosensory cortex. However, no information is available to date on the time-course of the laminar differentiation of the cortex after monoaminergic depletion and the relative contribution of different monoaminergic inputs to this process. To address these issues, newborn mice were treated with selective neurotoxins (6-hydroxydopamine or 5,7-dihydroxytryptamine) at birth to destroy the catecholaminergic and monoaminergic cortical innervation, respectively. The parietal cortex of these animals was examined in Nissl-stained coronal sections prepared on different days of postnatal development (between P2 and P30). Compared with the controls, delayed growth and differentiation of the cortical layers II-IV were observed in the treated animals, most prominently between P2 and P16. From the 3rd postnatal week, no cytoarchitectonic difference could be detected. Although neonatal depletion of the cortical monoaminergic innervation does not affect the laminar organization of the adult mouse barrelfield, it significantly delays the time-course of development of several cortical layers. This delay generates a mismatch in the degree of maturation between cortical neurons and their afferents at a time when neuronal interactions are critical for the establishment of local circuitry.

Comparison of [3H]paroxetine and [3H]cyanoimipramine for quantitative measurement of serotonin transporter sites in human brain

Previous studies have demonstrated that [3H]paroxetine and [3H]cyanoimipramine ([3H]CN-IMI) are highly selective ligands for the serotonin (5-HT) transporter. Using membrane preparation from the putamen, we confirmed that in human brain [3H]paroxetine labeled with high affinity one class of site associated with the 5-HT transporter. [3H]CN-IMI labeled two classes of sites in human brain. The one displaceable by 5-HT and with high affinity to 5-HT uptake inhibitors accounts for about 60% of the [3H]CN-IMI binding and, presumably, is associated with the 5-HT transporter. From the competition experiments, citalopram was selected to define [3H]CN-IMI binding to the 5-HT transporter in tissue sections because of its high selectivity to 5-HT transporter sites. A good correlation of the regional distribution patterns for [3H]CN-IMI and [3H]paroxetine was found using quantitative autoradiography. However, [3H]paroxetine underestimated high concentrations of the 5-HT transporter comparing to [3H]CN-IMI. This is likely to be due to the higher specific activity of [3H]CN-IMI. There is a good correlation between the regional distribution of 5-HT transporter sites labeled with either [3H]paroxetine or [3H]CN-IMI and the density of serotonergic innervation. This suggests that the brain areas that receive numerous serotonergic afferents, such as the hypothalamus and basal forebrain, might be common targets of these antidepressant drugs. Pharmacologic similarity of the sites labeled by both ligands as well as their similar distribution in the brain suggests that both antidepressant drugs interact with the same protein, thereby eliciting a similar neurochemical response.

Serotonergic afferents of the pigeon accessory optic nucleus

Injections of a retrograde tracer into the accessory optic nucleus of the basal optic root (nBOR) of the pigeon, combined with 5-HT immunohistochemistry, revealed that serotonergic projections to the nBOR appeared to originate mainly from the median (MR) and paramedian (PMR) raphe nuclei. These projections were confirmed by the significant decrease in 5-HT immunoreactivity observed in nBOR after lesions in MR and PMR. These data characterize distinct sources of 5-HT innervation to the pigeon nBOR and suggest that those afferents could represent part of a modulatory system that contributes to the role of the nBOR in optokinetic mechanisms.

The density and distribution of serotonergic appositions onto identified neurons in the rat rostral ventromedial medulla

Neurons in the rostral ventromedial medulla (RVM) contribute to the modulation of nociceptive transmission and to the analgesic effects of opioids. The RVM contains serotonergic terminal arbors, serotonergic neurons and several types of serotonin (5-HT) receptors. Limited evidence suggests that 5-HT acting within RVM decreases nociceptive responsiveness and contributes to opioid analgesia. The present study examines the density and distribution of serotonergic afferents onto physiologically identified neurons in the RVM. In anesthetized rats, RVM neurons were characterized by their response to noxious stimulation as either on (excited), off (inhibited) or neutral (unaffected) cells. Tissue containing intracellularly labeled RVM neurons was processed for 5-HT immunocytochemistry. Five off, five on, and three serotonergic neutral cells were examined with the confocal microscope for appositions between 5-HT immunoreactive (5-HT-IR) processes and intracellularly labeled processes. Serotonergic neutral cells had the highest density of 5-HT-IR appositions. The density of 5-HT-IR appositions onto off cells was slightly lower. On cells demonstrated the lowest density of 5-HT-IR appositions. These results indicate that 5-HT contributes to nociceptive modulation by direct actions on the activity of RVM cells. Because the RVM has several sources of serotonergic input and a number of different 5-HT receptor subtypes, further understanding of the role of RVM 5-HT afferents will require pharmacological studies to determine the action of 5-HT on each cell class and anatomical studies to determine the brainstem origin of serotonergic input to each cell class.

Monoamines and nitric oxide are employed by afferents engaged in midline thalamic regulation

The neurochemical identities of afferents to the midline thalamus were investigated in chloral hydrate-anesthetized adult Sprague-Dawley rats. The retrograde tracers, FluoroGold or cholera toxin B subunit, were centered on the paraventricular thalamic nucleus (n.Pvt), a periventricular member of the diffuse thalamocortical projection system that is reciprocally linked with visceral areas of cerebral cortex and implicated in food intake and addictive behavior. Tissues were processed with antisera raised against 5-HT, the catecholamine-synthesizing enzymes, tyrosine hydroxylase or phenylethanolamine N-methyltransferase or the cholinergic anabolic enzyme, ChAT. Serotonergic afferents principally derive from dorsal and median constituents of the mesopontine raphé. Previously unrecognized sources of catecholaminergic afferents were detected. Adrenergic afferents were traced to neurons in the C1 and C3 areas of rostral medullary reticular formation and periventricular gray, respectively, and the C2 area corresponding to the dorsal general viscerosensory field of nucleus tractus solitarii. Noradrenergic afferents arise principally from neurons in the locus ceruleus and A5 area. Dopaminergic projections to the n.Pvt derive from the A14, A13 and A11 cell groups in diencephalon. Afferents presumed to generate nitric oxide (NO) as a diffusible membrane-permeant transcellular signal were detected by processing retrogradely labeled tissues histochemically for NADPH-diaphorase, a molecule associated with nitric oxide synthase. NO in the n.Pvt is generated predominantly by noncholinergic neurons in the lateral hypothalamic area and mesopontine tegmentum. In striking contrast, extensive interactions were predicted between NO and ACh in the central medial and other loci in the nondiscriminative thalamus. We conclude that the n.Pvt is a site of interaction of NO and monoaminergic afferents derived from nuclei implicated in sensory gating, regulation of electrocortical neural activity and behavior. Taken collectively, our data predict that the labile transcellular messenger NO may enable structurally differentiated subnuclei of the diffuse thalamocortical projection system to act in concert as a functionally unified unit.

A serotonin neurotoxin attenuates the phase-shifting effects of triazolam on the circadian clock in hamsters

Several lines of evidence suggest the potential involvement of serotonergic pathways in mediating the effects of activity-inducing stimuli on the circadian clock in rodents. The aim of the present 3 experiments was to examine the effects of the serotonergic neurotoxin, p-hloroamphetamine (PCA, 10 mg/kg) on: (1) the monoamine levels of the hypothalamus, frontal cortex and hippocampus in the hamster; (2) the phase shifts in the circadian rhythm of locomotor activity of hamsters in response to treatment with the short-acting benzodiazepine, triazolam (7.5 mg/kg); and (3) the magnitude of the acute increase in locomotor activity associated with triazolam administration in this species. The administration of PCA to hamsters caused changes of specific monoaminergic systems in the hypothalamus, that were limited to a selective decrease in serotonin levels 7 days post-treatment. The phase shifts of the circadian clock in response to triazolam treatment at CT 6 were considerably attenuated following the administration of the 5-HT neurotoxin. The total amount and the profiles of triazolam-induced wheel-running and general cage activity between CT 6 and CT 12 were not significantly affected by the PCA treatment. The finding that a 5-HT neurotoxin can attenuate the phase-shifting effects of triazolam in hamsters, without interfering with its activity-inducing properties, suggests that serotonergic afferents might be involved in the mechanism for non-photic phase-shifting of the circadian system.

Increased glucocorticoid receptor gene expression in the rat hippocampus following combined serotonergic and medial septal cholinergic lesions

Glucocorticoid excess is associated with hippocampal neuronal dysfunction and loss, mainly affecting CA1. Degeneration of both cholinergic and serotonergic (5-HT) hippocampal afferents is prominent in aged rats and Alzheimer's disease. Lesions of these individual pathways alter hippocampal expression of mineralocorticoid (MR) and glucocorticoid (GR) receptor mRNAs; both transcripts are increased by cholinergic lesions, but markedly decreased by serotonergic denervation. In the present study we found that combined medial septal cholinergic and central 5-HT lesions increase hippocampal GR mRNA expression, specifically in CA1 and CA2 subfields, whereas MR mRNA expression was similar to controls. Thus the effects of the cholinergic lesion, at least upon GR gene expression, appear to predominate while the effects of the lesions upon MR gene expression were additive. Increased hippocampal GR gene expression per neuron may increase hippocampal neuronal vulnerability with age or disease.