Serotonergic Afferents Research Articles

Elevated Serotonin in Mouse Spinal Dorsal Horn Is Pronociceptive.

Serotonergic neurons in the rostral ventral medulla (RVM) contribute to bidirectional control of pain through modulation of spinal and trigeminal nociceptive networks. Deficits in this pathway are believed to contribute to pathologic pain states, but whether changes in serotonergic mechanisms are pro- or antinociceptive is debated. We used a combination of optogenetics and fiber photometry to examine these mechanisms more closely. We find that optogenetic activation of RVM serotonergic afferents in the spinal cord of naive mice produces mechanical hypersensitivity and conditioned place aversion (CPA). Neuropathic pain, produced by chronic constriction injury of the infraorbital nerve (CCI-ION), evoked a tonic increase in serotonin (5HT) concentrations within the spinal trigeminal nucleus caudalis (SpVc), measured with liquid chromatography-tandem mass spectroscopy (LC-MS/MS). By contract, CCI-ION had no effect on the phasic serotonin transients in SpVc, evoked by noxious pinch, and measured with fiber photometry of a serotonin sensor. These findings suggest that serotonin release in the spinal cord is pronociceptive and that an increase in sustained serotonin signaling, rather than phasic or event driven increases, potentiate nociception in models of chronic pain.

Reciprocal control of obesity and anxiety\u2013depressive disorder via a GABA and serotonin neural circuit

The high comorbidity between obesity and mental disorders, such as depression and anxiety, often exacerbates metabolic and neurological symptoms significantly. However, neural mechanisms that underlie reciprocal control of feeding and mental states are largely elusive. Here we report that melanocortin 4 receptor (MC4R) neurons located in the dorsal bed nucleus of the stria terminus (dBNST) engage in the regulation of mentally associated weight gain by receiving GABAergic projections from hypothalamic AgRP neurons onto α5-containing GABAA receptors and serotonergic afferents onto 5-HT3 receptors. Chronic treatment with a high-fat diet (HFD) significantly blunts the hyperexcitability of AgRP neurons in response to not only hunger but also anxiety and depression-like stimuli. Such HFD-mediated desensitization reduces GABAergic outputs from AgRP neurons to downstream MC4RdBNST neurons, resulting in severe mental dysregulation. Genetic enhancement of the GABAAR-α5 or suppression of the 5-HT3R within the MC4RdBNST neurons not only abolishes HFD-induced anxiety and depression but also robustly reduces body weight by suppression of food intake. To gain further translational insights, we revealed that combined treatment of zonisamide (enhancing the GABAAR-α5 signaling) and granisetron (a selective 5-HT3R antagonist) alleviates mental dysfunction and yields a robust reversal of diet-induced obesity by reducing total calorie intake and altering food preference towards a healthy low-fat diet. Our results unveil a neural mechanism for reciprocal control of appetite and mental states, which culminates in a novel zonisamide-granisetron cocktail therapy for potential tackling the psychosis-obesity comorbidity.

Lesion of Serotonergic Afferents to the Retrotrapezoid Nucleus Impairs the Tachypneic Response to Hypercapnia in Unanesthetized Animals

Hypercapnia promotes an increase in pulmonary ventilation due to the stimulation of brainstem chemosensory cells that are connected to the respiratory network. Among these cells are the raphe serotonergic neurons which widely send projections to distinct central respiratory compartments. Nevertheless, the physiological role of specific raphe serotonergic projections to other chemosensitive sites on the emergence of hypercapnia ventilatory response in vivo still remains to be elucidated. Here we investigated whether the ventilatory response to hypercapnia requires serotonergic inputs to the chemosensitive cells of the retrotrapezoid nucleus (RTN) in the ventrolateral medulla. To test this, pulmonary ventilation was evaluated under baseline conditions and during hypercapnia (7% CO2) in unanesthetized juvenile Holtzman rats (60–90 g) that received bilateral microinjections of either vehicle (control) or anti-SERT-SAP (0.1 mM, 10 pmol/100 nl) toxin in the RTN to retrogradely destroy serotonergic afferents to this region. Fifteen days after microinjections, baseline ventilation was not different between anti-SERT-SAP (n = 8) and control animals (n = 9). In contrast, the ablation of RTN-projecting serotonergic neurons markedly attenuated the hypercapnia-induced increase in respiratory frequency which was correlated with reduced numbers of serotonergic neurons in the raphe obscurus and magnus, but not in the raphe pallidus. The increase in tidal volume during hypercapnia was not significantly affected by anti-SERT-SAP microinjections in the RTN. Our data indicate that serotoninergic neurons that send projections to the RTN region are required for the processing of ventilatory reflex response during exposure to high CO2 in unanesthetized conditions.

Cross-validated Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Quantitation Protocol for a Pharmaceutical Drug and Its Drug-Target Effects in the Brain Using Time-of-Flight and Fourier Transform Ion Cyclotron Resonance Analyzers.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an established tool in drug development, which enables visualization of drugs and drug metabolites at spatial localizations in tissue sections from different organs. However, robust and accurate quantitation by MALDI-MSI still remains a challenge. We present a quantitative MALDI-MSI method using two instruments with different types of mass analyzers, i.e., time-of-flight (TOF) and Fourier transform ion cyclotron resonance (FTICR) MS, for mapping levels of the in vivo-administered drug citalopram, a selective serotonin reuptake inhibitor, in mouse brain tissue sections. Six different methods for applying calibration standards and an internal standard were evaluated. The optimized method was validated according to authorities’ guidelines and requirements, including selectivity, accuracy, precision, recovery, calibration curve, sensitivity, reproducibility, and stability parameters. We showed that applying a dilution series of calibration standards followed by a homogeneously applied, stable, isotopically labeled standard for normalization and a matrix on top of the tissue section yielded similar results to those from the reference method using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The validation results were within specified limits and the brain concentrations for TOF MS (51.1 ± 4.4 pmol/mg) and FTICR MS (56.9 ± 6.0 pmol/mg) did not significantly differ from those of the cross-validated LC–MS/MS method (55.0 ± 4.9 pmol/mg). The effect of in vivo citalopram administration on the serotonin neurotransmitter system was studied in the hippocampus, a brain region that is the principal target of the serotonergic afferents along with the limbic system, and it was shown that serotonin was significantly increased (2-fold), but its metabolite 5-hydroxyindoleacetic acid was not. This study makes a substantial step toward establishing MALDI-MSI as a fully quantitative validated method.

Compensatory neuritogenesis of serotonergic afferents within the striatum of a transgenic rat model of Parkinson’s disease

The majority of patients with Parkinson’s disease (PD) suffer from L-DOPA-induced dyskinesia (LID). Besides a dysfunctional dopaminergic system, changes of the serotonergic network may be linked to this severe and adverse symptom. Particularly, serotonergic neurons have the potential to synthesize dopamine, likely associated with a disproportional dopamine release within the striatum. We hypothesized that the serotonergic system is adaptively altered in the striatum due to the reduced dopaminergic input. To answer this question, we analyzed a transgenic rat PD model ubiquitously expressing human α-synuclein using a bacterial artificial chromosome. Neurite analysis showed a profound loss of dopaminergic fibers by ~30–40% within the dorsal striatum paralleled by a ~50% reduction of dopaminergic neurons in the substantia nigra pars compacta. In contrast, serotonergic fibers showed an increased fiber density in the dorsal striatum by ~100%, while the number of serotonergic neurons within the raphe nuclei (RN) and its proximal neuritic processes were unaffected. Furthermore, both the dopaminergic and serotonergic fiber density remained unchanged in the neighboring motor cortex M1/M2. Interestingly, essential enzymes required for L-DOPA turnover and dopamine release were expressed in serotonergic neurons of the RN. In parallel, the serotonergic autoreceptor levels involved in a serotonergic negative feedback loop were reduced within the striatum, suggesting a dysfunctional neurotransmitter release. Overall, the increased serotonergic fiber density with its capacity for dopamine release within the striatum suggests a compensatory, site-specific serotonergic neuritogenesis. This maladaptive serotonergic plasticity may be linked to adverse symptoms such as LIDs in PD.

Anxious Behavior of Adult CD1 Mice Perinatally Exposed to Low Concentrations of Ethanol Correlates With Morphological Changes in Cingulate Cortex and Amygdala.

Perinatal ethanol (EtOH) exposure is associated with high incidence of behavioral disorders such as depression and anxiety. The cerebral areas related with these consequences involve the corticolimbic system, in particular the prefrontal cortex, hippocampus, amygdala, and cingulate cortex, although the latter has not been thoroughly studied yet. Different animal models of prenatal or perinatal EtOH exposure have reported morphofunctional alterations in the central nervous system, which could explain behavioral disorders along life; these results focus on youth and adolescents and are still controversial. In the light of these inconclusive results, the aim of this work was to analyze adult behavior in CD1 mice perinatally exposed to low concentrations of EtOH (PEE) during gestation and lactation, and describe the morphology of the cingulate cortex and amygdala with a view to establishing structure/function/behavior correlations. Primiparous CD1 female mice were exposed to EtOH 6% v/v for 20 days prior to mating and continued drinking EtOH 6% v/v during pregnancy and lactation. After weaning, male pups were fed food and water ad libitum until 77 days of age, when behavioral and morphological studies were performed. Mouse behavior was analyzed through light–dark box and open field tests. Parameters related to anxious behavior and locomotor activity revealed anxiogenic behavior in PEE mice. After behavioral studies, mice were perfused and neurons, axons, serotonin transporter, 5HT, CB1 receptor (CB1R) and 5HT1A receptor (5HT1AR) were studied by immunofluorescence and immunohistochemistry in brain sections containing cingulate cortex and amygdala. Cingulate cortex and amygdala cytoarchitecture were preserved in adult PEE mice, although a smaller number of neurons was detected in the amygdala. Cingulate cortex axons demonstrated disorganized radial distribution and reduced area. Serotonergic and endocannabinoid systems, both involved in anxious behavior, showed differential expression. Serotonergic afferents were lower in both brain areas of PEE animals, while 5HT1AR expression was lower in the cingulate cortex and higher in the amygdala. The expression of CB1R was lower only in the amygdala. In sum, EtOH exposure during early brain development induces morphological changes in structures of the limbic system and its neuromodulation, which persist into adulthood and may be responsible for anxious behavior.

Reduced serotonin impairs long-term depression in basolateral amygdala complex and causes anxiety-like behaviors in a mouse model of perimenopause

Perimenopause is characterized by a gradual depletion of ovarian follicles with increased vulnerability to anxiety. However, the underlying mechanisms remain poorly understood. Herein, we show that chronic exposure to 4-vinylcycloxene diepoxide (VCD) in adult female mice (VCD-mice) caused follicles depletion and decline of serum estradiol (E2) and progesterone levels. Serotonin (5-HT) synthesis in dorsal raphe nucleus (DRN) and serotonergic afferents to basolateral amygdala complex (BLA) were reduced in VCD-mice, which were recovered by the supplement E2. VCD-mice appeared anxiety-like behaviors, which was relieved by the treatment with E2 or the co-administration of 5-HT1Ar agonist 8-OH-DPAT and 5-HT2A/Cr agonist DOI. The bath-application of 8-OH-DPAT in the slices obtained from VCD-mice (VCD-slices) corrected the increase in presynaptic glutamate release at external capsule-BLA synaptic transmission. Threshold to induce NMDA receptor (NMDAr)-dependent long-term potentiation (LTP) was declined in VCD-mice with elevation of CaMKII phosphorylation, which was sensitive to 8-OH-DPAT. Notably, the bath-application of 8-OH-DPAT in VCD-slices caused a decrease in the GABAergic feedback inhibition, which was restored by adding DOI. In addition, NMDAr-independent long-term depression (LTD) could not be induced in VCD-mice, which was rescued by the co-application of 8-OH-DPAT with DOI or the GABAA receptor agonist muscimol. Furthermore, the treatment of VCD-mice with E2 could prevent the facilitation of LTP and recover the LTD induction. Thus, the results indicate that the 5-HT deficiency in the BLA of VCD-mice causes the facilitation of LTP via enhanced glutamate release and impairs the LTD induction through diminished GABAergic inhibition, leading to anxiety-like behaviors.

Opposing Cholinergic and Serotonergic Modulation of Layer 6 in Prefrontal Cortex.

Prefrontal cortex is a hub for attention processing and receives abundant innervation from cholinergic and serotonergic afferents. A growing body of evidence suggests that acetylcholine (ACh) and serotonin (5-HT) have opposing influences on tasks requiring attention, but the underlying neurophysiology of their opposition is unclear. One candidate target population is medial prefrontal layer 6 pyramidal neurons, which provide feedback modulation of the thalamus, as well as feed-forward excitation of cortical interneurons. Here, we assess the response of these neurons to ACh and 5-HT using whole cell recordings in acute brain slices from mouse cortex. With application of exogenous agonists, we show that individual layer 6 pyramidal neurons are bidirectionally-modulated, with ACh and 5-HT exerting opposite effects on excitability across a number of concentrations. Next, we tested the responses of layer 6 pyramidal neurons to optogenetic release of endogenous ACh or 5-HT. These experiments were performed in brain slices from transgenic mice expressing channelrhodopsin in either ChAT-expressing cholinergic neurons or Pet1-expressing serotonergic neurons. Light-evoked endogenous neuromodulation recapitulated the effects of exogenous neurotransmitters, showing opposing modulation of layer 6 pyramidal neurons by ACh and 5-HT. Lastly, the addition of 5-HT to either endogenous or exogenous ACh significantly suppressed the excitation of pyramidal neurons in prefrontal layer 6. Taken together, this work suggests that the major corticothalamic layer of prefrontal cortex is a substrate for opposing modulatory influences on neuronal activity that could have implications for regulation of attention.

Differential serotonergic modulation across the main and accessory olfactory bulbs.

There are serotonergic projections to both the main (MOB) and the accessory olfactory bulb (AOB). Current-clamp experiments demonstrate that serotonergic afferents are largely excitatory for mitral cells (MCs) in the MOB where 5-HT2A receptors mediate a direct excitatory action. Serotonergic afferents are predominately inhibitory for MCs in the AOB. There are two types of inhibition: indirect inhibition mediated through the 5-HT2 receptors on GABAergic interneurons and direct inhibition via the 5-HT1 receptors on MCs. Differential 5-HT neuromodulation of MCs across the MOB and AOB could contribute to select behaviours such as olfactory learning or aggression. Mitral cells (MCs) contained in the main (MOB) and accessory (AOB) olfactory bulb have distinct intrinsic membrane properties but the extent of neuromodulation across the two systems has not been widely explored. Herein, we investigated a widely distributed CNS modulator, serotonin (5-HT), for its ability to modulate the biophysical properties of MCs across the MOB and AOB, using an in vitro, brain slice approach in postnatal 15-30day mice. In the MOB, 5-HT elicited three types of responses in 93% of 180 cells tested. Cells were either directly excited (70%), inhibited (10%) or showed a mixed response (13%)- first inhibition followed by excitation. In the AOB, 82% of 148 cells were inhibited with 18% of cells showing no response. Albeit located in parallel partitions of the olfactory system, 5-HT largely elicited MC excitation in the MOB while it evoked two different kinetic rates of MC inhibition in the AOB. Using a combination of pharmacological agents, we found that the MC excitatory responses in the MOB were mediated by 5-HT2A receptors through a direct activation. In comparison, 5-HT-evoked inhibitory responses in the AOB arose due to a polysynaptic, slow-onset inhibition attributed to 5-HT2 receptor activation exciting GABAergic interneurons. The second type of inhibition had a rapid onset as a result of direct inhibition mediated by the 5-HT1 class of receptors. The distinct serotonergic modulation of MCs between the MOB and AOB could provide a molecular basis for differential chemosensory behaviours driven by the brainstem raphe nuclei into these parallel systems.

Cell-Type-Specific Modulation of Sensory Responses in Olfactory Bulb Circuits by Serotonergic Projections from the Raphe Nuclei.

Serotonergic neurons in the brainstem raphe nuclei densely innervate the olfactory bulb (OB), where they can modulate the initial representation and processing of olfactory information. Serotonergic modulation of sensory responses among defined OB cell types is poorly characterized in vivo Here, we used cell-type-specific expression of optical reporters to visualize how raphe stimulation alters sensory responses in two classes of GABAergic neurons of the mouse OB glomerular layer, periglomerular (PG) and short axon (SA) cells, as well as mitral/tufted (MT) cells carrying OB output to piriform cortex. In PG and SA cells, brief (1-4 s) raphe stimulation elicited a large increase in the magnitude of responses linked to inhalation of ambient air, as well as modest increases in the magnitude of odorant-evoked responses. Near-identical effects were observed when the optical reporter of glutamatergic transmission iGluSnFR was expressed in PG and SA cells, suggesting enhanced excitatory input to these neurons. In contrast, in MT cells imaged from the dorsal OB, raphe stimulation elicited a strong increase in resting GCaMP fluorescence with only a slight enhancement of inhalation-linked responses to odorant. Finally, optogenetically stimulating raphe serotonergic afferents in the OB had heterogeneous effects on presumptive MT cells recorded extracellularly, with an overall modest increase in resting and odorant-evoked responses during serotonergic afferent stimulation. These results suggest that serotonergic afferents from raphe dynamically modulate olfactory processing through distinct effects on multiple OB targets, and may alter the degree to which OB output is shaped by inhibition during behavior. Modulation of the circuits that process sensory information can profoundly impact how information about the external world is represented and perceived. This study investigates how the serotonergic system modulates the initial processing of olfactory information by the olfactory bulb, an obligatory relay between sensory neurons and cortex. We find that serotonergic projections from the raphe nuclei to the olfactory bulb dramatically enhance the responses of two classes of inhibitory interneurons to sensory input, that this effect is mediated by increased glutamatergic drive onto these neurons, and that serotonergic afferent activation alters the responses of olfactory bulb output neurons in vivo These results elucidate pathways by which neuromodulatory systems can dynamically regulate brain circuits during behavior.

Brain serotonin determines maternal behavior and offspring survival.

Maternal care is an indispensable component of offspring survival and development in all mammals and necessary for reproductive success. Although brain areas regulating maternal behaviors are innervated by serotonergic afferents, very little is known about the role of this neurotransmitter in these behaviors. To evaluate the contribution of serotonin to maternal care, we used mice with a null mutation in the gene for tryptophan hydroxylase-2 (TPH2), which results in a genetic depletion of brain serotonin, and tested them in a wide range of maternal behavior paradigms. We found that litters born to and reared by TPH2(-/-) mothers showed decreased survival, lower weaning weights and increased cannibalization. In addition, TPH2(-/-) mothers performed poorly in pup retrieval, huddling, nest construction and high-arched back nursing. Aggression in TPH2(-/-) dams was not triggered by lactation and was steadily high. Survival and weaning weight deficits of TPH2(-/-) pups were rescued by cross-fostering and in litters of mixed genotype (TPH2(-/-) and TPH2(-/+) ). However, the maternal behaviors of TPH2(-/-) dams did not improve when rearing either TPH2(+/+) pups or mixed-genotype litters. In addition, TPH2(-/-) pups significantly worsened the behavior of TPH2(+/+) dams with respect to cannibalism, weaning weight and latency to attack. Olfactory and auditory functions of TPH2(-/-) females or anxiety-like behaviors did not account for these maternal alterations as they were equal to their TPH2(+/+) counterparts. These findings illustrate a profound influence of brain serotonin on virtually all elements of maternal behavior and establish that TPH2(-/-) pups can engender maladaptive mothering in dams of both genotypes.

Presynaptic α4β2 Nicotinic Acetylcholine Receptors Increase Glutamate Release and Serotonin Neuron Excitability in the Dorsal Raphe Nucleus

Several behavioral effects of nicotine are mediated by changes in serotonin (5-HT) release in brain areas that receive serotonergic afferents from the dorsal raphe nucleus (DRN). In vitro experiments have demonstrated that nicotine increases the firing activity in the majority of DRN 5-HT neurons and that DRN contains nicotinic acetylcholine receptors (nAChRs) located at both somata and presynaptic elements. One of the most common presynaptic effects of nicotine is to increase glutamate release. Although DRN receives profuse glutamatergic afferents, the effect of nicotine on glutamate release in the DRN has not been studied in detail. Using whole-cell recording techniques, we investigated the effects of nicotine on the glutamatergic input to 5-HT DRN neurons in rat midbrain slices. Low nicotine concentrations, in the presence of bicuculline and tetrodotoxin (TTX), increased the frequency but did not change the amplitude of glutamate-induced EPSCs, recorded from identified 5-HT neurons. Nicotine-induced increase of glutamatergic EPSC frequency persisted 10-20 min after drug withdrawal. This nicotinic effect was mimicked by exogenous administration of acetylcholine (ACh) or inhibition of ACh metabolism. In addition, the nicotine-induced increase in EPSC frequency was abolished by blockade of α4β2 nAChRs, voltage-gated calcium channels, or intracellular calcium signaling but not by α7 nAChR antagonists. These data suggest that both nicotine and endogenous ACh can increase glutamate release through activation of presynaptic α4β2 but not α7 nAChRs in the DRN. The effect involves long-term changes in synaptic function, and it is dependent on voltage-gated calcium channels and presynaptic calcium stores.

Serotonergic innervation and serotonin receptor expression of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei

Pharmacobehavioral studies in experimental animals, and imaging studies in humans, indicate that serotonergic transmission in the amygdala plays a key role in emotional processing, especially for anxiety-related stimuli. The lateral and basolateral amygdaloid nuclei receive a dense serotonergic innervation in all species studied to date. We investigated interrelations between serotonergic afferents and neuropeptide Y (NPY)-producing neurons, which are a subpopulation of inhibitory interneurons in the rat lateral and basolateral nuclei with particularly strong anxiolytic properties. Dual light microscopic immunolabeling showed numerous appositions of serotonergic afferents on NPY-immunoreactive somata. Using electron microscopy, direct membrane appositions and synaptic contacts between serotonin-containing axon terminals and NPY-immunoreactive cellular profiles were unequivocally established. Double in situ hybridization documented that more than 50 %, and about 30–40 % of NPY mRNA-producing neurons, co-expressed inhibitory 5-HT1A and excitatory 5-HT2C mRNA receptor subtype mRNA, respectively, in both nuclei with no gender differences. Triple in situ hybridization showed that individual NPY mRNA-producing interneurons co-express both 5-HT1A and 5-HT2C mRNAs. Co-expression of NPY and 5-HT3 mRNA was not observed. The results demonstrate that serotonergic afferents provide substantial innervation of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei. Studies of serotonin receptor subtype co-expression indicate a differential impact of the serotonergic innervation on this small, but important, population of anxiolytic interneurons, and provide the basis for future studies of the circuitry underlying serotonergic modulation of emotional stimulus processing in the amygdala.

Effects of Repeated 3,4-Methylenedioxymethamphetamine Administration on Neurotransmitter Efflux and Sensory-Evoked Discharge in the Ventral Posterior Medial Thalamus

3,4-Methylenedioxymethamphetamine (MDMA) is known to enhance tactile sensory perception, an effect that contributes to its popularity as a recreational drug. The neurophysiological basis for the effects of MDMA on somatosensation are unknown. However, MDMA interactions with the serotonin transporter (SERT) and subsequent enhancement of serotonin neurotransmission are well known. The rat trigeminal somatosensory system receives serotonergic afferents from the dorsal raphe nucleus. Because these fibers express SERT, they should be vulnerable to MDMA-induced effects. We found that administration of a challenge injection of MDMA (3 mg/kg i.p.) after repeated MDMA treatment (3 mg/kg per day for 4 days) elicits both serotonin and norepinephrine efflux in the ventral posterior medial (VPM) thalamus of Long-Evans hooded rats, the main relay along the lemniscal portion of the rodent trigeminal somatosensory pathway. We evaluated the potential for repeated MDMA administration to modulate whisker-evoked discharge of individual neurons in this region. After surgically implanting stainless steel eight-wire multichannel electrode bundles, we recorded spike train activity of single cells while activating the whisker pathway using a piezoelectric mechanical stimulator. We found that repeated MDMA administration increased the spontaneous firing rate but reduced both the magnitude and duration of whisker-evoked discharge in individual VPM thalamic neurons. The time course of drug action on neuronal firing patterns was generally consistent with fluctuations in neurotransmitter efflux as shown from our microdialysis studies. On the basis of these results, we propose that single use and repeated administration of MDMA may "distort," rather than enhance, tactile experiences in humans, in part, by disrupting normal spike firing patterns through somatosensory thalamic relay circuits.

Serotonin and circadian rhythms.

All mammal behaviors and functions exhibit synchronization with environmental rhythms. This is accomplished through an internal mechanism that generates and modulates biological rhythms. The circadian timing system, responsible for this process, is formed by connected neural structures. Pathways receive and transmit environmental cues to the central oscillator, the hypothalamic suprachiasmatic nucleus, which mediates physiological and behavioral alterations. The suprachiasmatic nucleus has three major inputs: the retinohypothalamic tract (a direct projection from the retina), the geniculohypothalamic tract (an indirect photic projection originating in the intergeniculate leaflet), and a dense serotonergic plexus from the raphe nuclei. The serotonergic pathway, a source of non-photic cues to the suprachiasmatic nucleus, modulates its activity. The importance of raphe nuclei in circadian rhythms, especially in photic responses, has been demonstrated in many studies. Serotonin is the raphe neurotransmitter that triggers phase shifts, inhibits light-induced phase-shifts, and plays a role in controlling the sleep-wake cycle. All data to date have demonstrated the importance of the raphe, through serotonergic afferents, in adjusting circadian rhythms and must therefore be considered a component of the circadian timing system. The aim of this paper is to review the literature addressing the involvement of serotonin in the modulation of circadian rhythm.

MDMA (3,4-Methylenedioxymethamphetamine)-Mediated Distortion of Somatosensory Signal Transmission and Neurotransmitter Efflux in the Ventral Posterior Medial Thalamus

MDMA (3,4-methylenedioxymethamphetamine, Ecstasy) is reported to enhance tactile sensory perception, an effect that is believed to contribute to its popularity as a recreational drug. To date, no literature exists that addresses the neurophysiological mechanisms underlying the effects of MDMA on somatosensation. However, MDMA interactions with the serotonin transporter protein (SERT) are well known. The rat trigeminal somatosensory system has been studied extensively and receives serotonergic afferents from the dorsal raphe nucleus. Given that these fibers express SERT, they should be vulnerable to MDMA-induced effects. We found that short-term low-dose MDMA administration (3 mg/kg i.p.) led to a significant increase in 5-hydroxytryptamine (5-HT) efflux in the ventral posterior medial (VPM) thalamus, the main relay along the lemniscal portion of the rodent trigeminal somatosensory pathway. We further evaluated the potential for MDMA to modulate whisker-evoked discharge (WED) of individual neurons in this region. After surgically implanting stainless steel 8-wire multichannel electrode bundles, we recorded spike train activity from single cells of halothane-anesthetized rats while mechanically activating the whisker pathway. We found that short-term low-dose MDMA (3 mg/kg i.p.) increased the spontaneous firing rate but reduced the magnitude and duration of WED in individual VPM thalamic neurons. It is noteworthy that the time course of drug action on neuronal firing patterns was generally consistent with increased 5-HT efflux as shown from our microdialysis studies. Based on these results, we propose the working hypothesis that MDMA may "distort" rather than enhance tactile experiences in humans, in part, by disrupting normal spike firing patterns through somatosensory thalamic relay circuits.

CB1-cannabinoid receptors are involved in the modulation of non-synaptic [3H]serotonin release from the rat hippocampus

In the present study we investigated whether serotonin release in the hippocampus is subject to regulation via cannabinoid receptors. Both rat and mouse hippocampal slices were preincubated with [3H]serotonin ([3H]5-HT) and superfused with medium containing serotonin reuptake inhibitor citalopram hydrobromide (300nM). The cannabinoid receptor agonist R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN55,212-2, 1μM) did not affect either the resting or the electrically evoked [3H]5-HT release. In the presence of the ionotropic glutamate receptor antagonists d(−)-2-amino-5-phosphonopentanoic acid (AP-5, 50μM) and 6-cyano-7-nitroquinoxaline-2,3-dione-disodium (CNQX, 10μM) the evoked [3H]5-HT release was decreased significantly. Similar findings were obtained when CNQX (10μM) was applied alone with WIN55,212-2. This effect was abolished by the selective cannabinoid receptor subtype 1 (CB1) antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716, 1μM) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide trifluoroacetate salt (AM251, 1μM). Similarly to that observed in rats, WIN55,212-2 (1μM) decreased the evoked [3H]5-HT efflux in wild-type mice (CB1+/+). The inhibitory effect of WIN55,212-2 (1μM) was completely absent in hippocampal slices derived from mice genetically deficient in CB1 cannabinoid receptors (CB1−/−).Relatively selective degeneration of fine serotonergic axons by the neurotoxin parachloramphetamine (PCA) reduced significantly the tritium uptake and the evoked [3H]5-HT release. In addition, PCA, eliminated the effect of WIN55,212-2 (1μM) on the stimulation-evoked [3H]5-HT efflux. In contrast to the PCA-treated animals, WIN55,212-2 (1μM) reduced the [3H]5-HT efflux in the saline-treated group.Our data suggest that a subpopulation of non-synaptic serotonergic afferents express CB1 receptors and activation of these CB1 receptors leads to a decrease in 5-HT release.

A comparative study of cytoarchitecture and serotonergic afferents in the suprachiasmatic nucleus of primates ( Cebus apella and Callithrix jacchus) and rats (Wistar and Long Evans strains)

The suprachiasmatic nucleus, an essential diencephalic component of the circadian timing system, plays a role in the generation and modulation of behavioral and neuroendocrine rhythms in mammals. Its cytoarchitecture, neurochemical and hodological characteristics have been investigated in various mammalian species, particularly in rodents. In most species, two subdivisions, based on these aspects and considered to reflect functional specialization within the nucleus, can be recognized. Many studies reveal a typical dense innervation by serotonergic fibers in this nucleus, mainly in the ventromedial area, overlapping the retinal afferents. However, a different pattern occurs in certain animals, which lead us to investigate the distribution of serotonergic afferents in the suprachiasmatic nucleus of the Capuchin monkey, Cebus apella, compared to the marmoset, Callithrix jacchus, and two Rattus norvegicus lines (Long Evans and Wistar), and to reported findings for other mammalian species. Our morphometric data show the volume and length of the suprachiasmatic nucleus along the rostrocaudal axis to be greatest in C. apella > C. jacchus > Long Evans ≥ Wistar rats, in agreement with their body sizes. In C. apella, however, the serotonergic terminals occupy only some 10% of the nucleus' area, less than the 25% seen in the marmoset and rats. The distribution of the serotonergic fibers in C. apella does not follow the characteristic ventral organization pattern seen in the rodents. These findings raise questions concerning the intrinsic organization of the nucleus, as well as regarding the functional relationship between serotonergic input and retinal afferents in this diurnal species.

Endogenous Serotonin Excites Striatal Cholinergic Interneurons via the Activation of 5-HT 2C, 5-HT6, and 5-HT7 Serotonin Receptors: Implications for Extrapyramidal Side Effects of Serotonin Reuptake Inhibitors

The striatum is richly innervated by serotonergic afferents from the raphe nucleus. We explored the effects of this input on striatal cholinergic interneurons from rat brain slices, by means of both conventional intracellular and whole-cell patch-clamp recordings. Bath-applied serotonin (5-HT, 3-300 microM), induced a dose-dependent membrane depolarization and increased the rate of spiking. This effect was mimicked by the 5-HT reuptake blockers citalopram and fluvoxamine. In voltage-clamped neurons, 5-HT induced an inward current, whose reversal potential was close to the K(+) equilibrium potential. Accordingly, the involvement of K(+) channels was confirmed either by increasing extracellular K(+) concentration and by blockade of K(+) channels with barium. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) profiling demonstrated the presence of 5-HT2C, 5-HT6, and 5-HT7 receptor mRNAs in identified cholinergic interneurons. The depolarization/inward current induced by 5-HT was partially mimicked by the 5-HT2 receptor agonist 2,5-dimethoxy-4-iodoamphetamine and antagonized by both ketanserin and the selective 5-HT2C antagonist RS102221, whereas the selective 5-HT3 and 5-HT4 receptor antagonists tropisetron and RS23597-190 had no effect. The depolarizing response to 5-HT was also reduced by the selective 5-HT6 and 5-HT7 receptor antagonists SB258585 and SB269970, respectively, and mimicked by the 5-HT7 agonist, 5-CT. Accordingly, activation of either 5-HT6 or 5-HT7 receptor induced an inward current. The 5-HT response was attenuated by U73122, blocker of phospholipase C, and by SQ22,536, an inhibitor of adenylyl cyclase. These results suggest that 5-HT released by serotonergic fibers originating in the raphe nuclei has a potent excitatory effect on striatal cholinergic interneurons.

Effects of serotonin on perifornical‐lateral hypothalamic area neurons in rat

The hypocretin (HCRT) system of the perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the facilitation of behavioral arousal. HCRT neurons receive serotonergic afferents from the dorsal raphe nucleus. Although in-vitro pharmacological studies suggest that serotonin (5-HT) inhibits HCRT neurons, the in-vivo effects of 5-HT on HCRT neurons in the PF-LHA and associated behavioral changes have not been described. We examined the effects of 5-HT delivered locally into the PF-LHA using reverse microdialysis on its neuronal activity and the consequent sleep-wake changes in rats. First, we quantified Fos expression (Fos-IR) in HCRT and other PF-LHA neurons following unilateral 5-HT perfusion in awake rats. Second, we determined the transient effects of 5-HT perfusion on the extracellular activity of the PF-LHA neurons recorded via microwires placed adjacent to the microdialysis probe. Third, we examined the effects of 5-HT perfusion into the PF-LHA on the sleep-wake profiles of the rats during the lights-off period. Unilateral perfusion of 5-HT into the PF-LHA in awake rats dose-dependently decreased the number of HCRT neurons exhibiting Fos-IR. 5-HT also inhibited the discharge activity of four of five responsive wake-related, putative HCRT neurons. However, unilateral perfusion of 5-HT into the PF-LHA did not produce significant behavioral changes during the 2-h recording period. These results confirm the in-vitro findings that 5-HT exerts inhibitory influences on HCRT neurons but further suggest that the inactivation of a limited number of HCRT neurons by unilateral 5-HT microdialysis may not be sufficient to induce behavioral changes.