Differential Impact of Serotonin Signaling Methylphenidate on Young versus Adult: Insights from Behavioral and Dorsal Raphe Nucleus Neuronal Recordings from Freely Behaving Rats.

Methylphenidate (MPD) remains a cornerstone pharmacological intervention for managing ADHD, yet its increasing usage among ordinary youth and adults outside clinical contexts necessitates a thorough investigation into its developmental effects. This study seeks to simultaneously investigate the behavioral and neuronal changes within the dorsal raphe (DR) nucleus, a center of serotonergic neurons in the mammalian brain, before and after the administration of varying doses of acute and chronic MPD in freely behaving young and adult rats implanted with DR recording electrodes. Wireless neuronal and behavioral recording systems were used over 10 consecutive experimental days. Eight groups were examined: saline, 0.6, 2.5, and 10.0 mg/kg MPD for both young and adult rats. Six daily MPD injections were administered on experimental days 1 to 6, followed by a three-day washout period and MPD re-administration on experimental day 10 (ED10). The analysis of neuronal activity recorded from 504 DR neurons (DRNs) in young rats and 356 DRNs in adult rats reveals significant age-dependent differences in acute and chronic MPD responses. This study emphasizes the importance of aligning electrophysiological evaluations with behavioral outcomes following extended MPD exposure, elucidating the critical role of DRNs and serotonin signaling in modulating MPD responses and delineating age-specific variations in young versus adult rat models.

Approach/Avoidance Behavior to Novel Objects is Correlated with the Serotonergic and Dopaminergic Systems in the Brown Rat (Rattus norvegicus).

The brown rat (Rattus norvegicus) is known to show three types of behavioral responses to novel objects. Whereas some rats are indifferent to novel objects, neophobic and neophilic rats show avoidance and approach behavior, respectively. Here, we compared the dopaminergic, serotonergic, and noradrenergic systems immunohistochemically among these rats. Trapped wild rats and laboratory rats were first individually exposed to the novel objects in their home cage. Wild rats were divided into neophobic and indifferent rats depending on their behavioral responses. Similarly, laboratory rats were divided into neophilic and indifferent rats. Consistent with the behavioral differences, in the paraventricular nucleus of the hypothalamus, Fos expression in corticotropin-releasing hormone-containing neurons was higher in the neophobic rats than in the indifferent rats. In the anterior basal amygdala, the neophobic rats showed higher Fos expression than the indifferent rats. In the posterior basal amygdala, the neophobic and neophilic rats showed lower and higher Fos expressions than the indifferent rats, respectively. When we compared the neuromodulatory systems, in the dorsal raphe, the number of serotonergic neurons and Fos expression in serotonergic neurons increased linearly from neophobic to indifferent to neophilic rats. In the ventral tegmental area, Fos expression in dopaminergic neurons was higher in the neophilic rats than in the indifferent rats. These results demonstrate that approach/avoidance behavior to novel objects is correlated with the serotonergic and dopaminergic systems in the brown rat. We propose that the serotonergic system suppresses avoidance behavior while the dopaminergic system enhances approach behavior to novel objects.

Raphe glucose-sensing serotonergic neurons stimulate KNDy neurons to enhance LH pulses via 5HT2CR: rat and goat studies.

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals.

Tracing Superoxide Anion in Serotonergic Neurons of Living Mouse Brains with Depression by Small-Molecule Fluorescence Probes.

In brains, the serotonergic neurons are the unique resource of the neurotransmitter serotonin, which plays a pivotal role in the physiology of the brain. The dysfunction of serotonergic neurons caused by oxidative stress in the brain is closely related to the occurrence and development of various mental diseases, such as depression. As the biomarker of oxidative stress, the superoxide anion radical (O2•-) can cause oxidative damage to proteins, nucleic acids and lipids, disturbing the function of neurons and brains. A serotonin transporter (SERT) specifically expresses in serotonergic neurons, which is the biomarker of serotonergic neurons. Thus, we created two novel small molecular fluorescent probes (PA-CA and HT-CA) for imaging O2•- in serotonergic neurons of living brains of mice based on specific targeting groups of SERT. Both PA-CA and HT-CA exert excellent SERT-targetable and glorious selectivity for O2•-. Those two probes could monitor the boost of O2•- in living hsert-HEK293 cells that specifically express SERT under oxidative stress. With two-photon fluorescence imaging, we revealed for the first time that O2•- is significantly increased in serotonergic neurons in living brains of mice with depression. More importantly, proteomic analyses suggested that O2•- could oxidize cysteine and histidine in the active site of SERT, which is involved in the development of depression. This work provides new materials for living brain imaging and offers new strategy for unraveling the pathophysiology of depression.

5-hydroxytryptamine produced by enteric serotonergic neurons initiates colorectal cancer stem cell self-renewal and tumorigenesis.

Colorectal cancer stem cells (CSCs) contribute to colorectal tumorigenesis and metastasis. Colorectal CSCs reside within specialized niches and harbor self-renewal and differentiation capacities. However, the niche regulations of CSCs remain unclear. Here, we show that intestinal nerve cells are required for CSC self-renewal and colorectal tumorigenesis. Enteric serotonergic neurons produce 5-hydroxytryptamine (5-HT) to function as a modulator of CSC self-renewal. 5-HT receptors HTR1B/1D/1F are highly expressed in colorectal CSCs and engage with 5-HT to initiate Wnt/ß-catenin signaling. Mechanistically, colorectal cancer (CRC)-enriched microbiota metabolite isovalerate suppresses the enrichment of the NuRD complex onto Tph2 promoter to initiate Tph2 expression, leading to 5-HT production. 5-HT signaling is correlated with CRC severity. Blocking 5-HT signaling in mice not only inhibits the self-renewal of colorectal CSCs but also displays therapeutic efficacy against CRC tumors. Our findings reveal a cross talk between intestinal neurons and tumor cells that serves as an additional layer for CSC regulation.

Adenosine Monophosphate-activated Protein Kinase (AMPK) in serotonin neurons mediates select behaviors during protracted withdrawal from morphine in mice.

Serotonin neurotransmission has been implicated in behavior deficits that occur during protracted withdrawal from opioids. In addition, studies have highlighted multiple pathways whereby serotonin (5-HT) modulates energy homeostasis, however the underlying metabolic effects of opioid withdrawal have not been investigated. A key metabolic regulator that senses the energy status of the cell and regulates fuel availability is Adenosine Monophosphate-activated Protein Kinase (AMPK). To investigate the interaction between cellular metabolism and serotonin in modulating protracted abstinence from morphine, we depleted AMPK in serotonin neurons. Morphine exposure via drinking water generates dependence in these mice, and both wildtype and serotonergic AMPK knockout mice consume similar amounts of morphine with no changes in body weight. Serotonergic AMPK contributes to baseline differences in open field and social interaction behaviors and blocks abstinence induced reductions in immobility following morphine withdrawal in the tail suspension test. Lastly, morphine locomotor sensitization is blunted in mice lacking AMPK in serotonin neurons. Taken together, our results suggest serotonergic AMPK mediates both baseline and protracted morphine withdrawal-induced behaviors.

Cardamonin Modulates Neuropathic Pain through the Possible Involvement of Serotonergic 5-HT1A Receptor Pathway in CCI-Induced Neuropathic Pain Mice Model.

Cardamonin, a naturally occurring chalcone isolated from Alpinia species has shown to possess strong anti-inflammatory and anti-nociceptive activities. Previous studies have demonstrated that cardamonin exerts antihyperalgesic and antiallodynic properties in chronic constriction injury (CCI)-induced neuropathic pain animal model. However, the mechanisms underlying cardamonin's effect have yet to be fully understood. The present study aims to investigate the involvement of the serotonergic system in cardamonin induced antihyperalgesic and antiallodynic effects in CCI-induced neuropathic pain mice model. The neuropathic pain symptoms in the CCI mice model were assessed using Hargreaves Plantar test and von-Frey filament test on day 14 post-surgery. Central depletion of serotonin along the descending serotonergic pathway was done using ρ-chlorophenylalanine (PCPA, 100 mg/kg, i.p.), an inhibitor of serotonin synthesis for four consecutive days before cardamonin treatment, and was found to reverse the antihyperalgesic and antiallodynic effect produced by cardamonin. Pretreatment of the mice with several 5-HT receptor subtypes antagonists: methiothepin (5-HT1/6/77 receptor antagonist, 0.1 mg/kg), WAY 100635 (5-HT1A receptor antagonist, 1 mg/kg), isamoltane (5-HT1B receptor antagonist, 2.5 mg/kg), ketanserin (5-HT2A receptor antagonist, 0.3 mg/kg), and ondansetron (5-HT3 receptor antagonist, 0.5 mg/kg) were shown to abolish the effect of cardamonin induced antihyperalgesic and antiallodynic effects. Further evaluation of the 5-HT1A receptor subtype protein expressions reveals that cardamonin significantly upregulated its expression in the brainstem and spinal cord. Our results suggest that the serotonergic pathway is essential for cardamonin to exert its antineuropathic effect in CCI mice through the involvement of the 5-HT1A receptor subtype in the central nervous system.

Nicotine withdrawal induces hyperalgesia via downregulation of descending serotonergic pathway in the nucleus raphe magnus.

Patients deprived of cigarettes exhibit increased pain sensitivity during perioperative periods, yet the underlying neuroanatomical and molecular bases of this hypersensitivity are unclear. The present study showed that both the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were significantly decreased in a rat model of nicotine withdrawal. These rats showed less tryptophan hydroxylase 2 (TPH2) positive neurons and reduced TPH2 expression in the nucleus raphe magnus (NRM), and thus resulted in decreased 5-hydroxytryptamine (5-HT) levels in cerebrospinal fluid. Intrathecal injection of 5-HT or NRM microinjection of TPH-overexpression adeno-associated virus alleviated nicotine withdrawal-induced hyperalgesia, whereas 5-HT receptor pharmacological blockade by methysergide (a 5-HT receptor antagonist) exacerbated hypersensitivity and diminished the difference between the two groups. Together, these data indicate that hyperalgesia after nicotine withdrawal is mediated by declined descending serotonergic pathways in the NRM. This provides a new perspective to improve the postoperative pain management of patients, especially the smokers.

AAV-mediated inhibition of ULK1 promotes axonal regeneration in the central nervous system in vitro and in vivo.

Axonal damage is an early step in traumatic and neurodegenerative disorders of the central nervous system (CNS). Damaged axons are not able to regenerate sufficiently in the adult mammalian CNS, leading to permanent neurological deficits. Recently, we showed that inhibition of the autophagic protein ULK1 promotes neuroprotection in different models of neurodegeneration. Moreover, we demonstrated previously that axonal protection improves regeneration of lesioned axons. However, whether axonal protection mediated by ULK1 inhibition could also improve axonal regeneration is unknown. Here, we used an adeno-associated viral (AAV) vector to express a dominant-negative form of ULK1 (AAV.ULK1.DN) and investigated its effects on axonal regeneration in the CNS. We show that AAV.ULK1.DN fosters axonal regeneration and enhances neurite outgrowth in vitro. In addition, AAV.ULK1.DN increases neuronal survival and enhances axonal regeneration after optic nerve lesion, and promotes long-term axonal protection after spinal cord injury (SCI) in vivo. Interestingly, AAV.ULK1.DN also increases serotonergic and dopaminergic axon sprouting after SCI. Mechanistically, AAV.ULK1.DN leads to increased ERK1 activation and reduced expression of RhoA and ROCK2. Our findings outline ULK1 as a key regulator of axonal degeneration and regeneration, and define ULK1 as a promising target to promote neuroprotection and regeneration in the CNS.

Activation of PPG neurons following acute stressors differentially involves hindbrain serotonin in male rats.

Within the hindbrain, serotonin (5-HT) functions as a modulator of the central glucagon-like peptide-1 (GLP-1) system. This interaction between 5-HT and GLP-1 is achieved via 5-HT2C and 5-HT3 receptors and is relevant for GLP-1-mediated feeding behavior. The central GLP-1 system is activated by various stressors, activates the hypothalamic pituitary adrenocortical (HPA) axis, and contributes to stress-related behaviors. Whether 5-HT modulates GLP-1's role in the stress response in unknown. We hypothesized that the serotonergic modulation of GLP-1-producing neurons (i.e., PPG neurons) is stimuli-specific and that stressed-induced PPG activity is one of the modalities in which 5-HT plays a role. In this study, we investigated the roles of 5-HT2C and 5-HT3 receptors in mediating the activation of PPG neurons in the nucleus tractus solitarius (NTS) following exposure to three different acute stressors: lithium chloride (LiCl), noncontingent cocaine (Coc), and novel restraint stress (RES). Results showed that increased c-Fos expression in PPG neurons following LiCl and RES-but not Coc-is dependent on hindbrain 5-HT2C and 5-HT3 receptor signaling. Additionally, stressors that depend on 5-HT signaling to activate PPG neurons (i.e., LiCl and RES) increased c-Fos expression in 5-HT-expressing neurons within the caudal raphe (CR), specifically in the raphe magnus (RMg). Finally, we showed that RMg neurons innervate NTS PPG neurons and that some of these PPG neurons lie in close proximity to 5-HT axons, suggesting RMg 5-HT-expressing neurons are the source of 5-HT input responsible for engaging NTS PPG neurons. Together, these findings identify a direct RMg to NTS pathway responsible for the modulatory effect of 5-HT on the central GLP-1 system-specifically via activation of 5-HT2C and 5-HT3 receptors-in the facilitation of acute stress responses.

First Evidence of Kv3.1b Potassium Channel Subtype Expression during Neuronal Serotonergic 1C11 Cell Line Development.

Kv3.1 channel is abundantly expressed in neurons and its dysfunction causes sleep loss, neurodegenerative diseases and depression. Fluoxetine, a serotonin selective reuptake inhibitor commonly used to treat depression, acts also on Kv3.1. To define the relationship between Kv3.1 and serotonin receptors (SR) pharmacological modulation, we showed that 1C11, a serotonergic cell line, expresses different voltage gated potassium (VGK) channels subtypes in the presence (differentiated cells (1C11D)) or absence (not differentiated cells (1C11ND)) of induction. Only Kv1.2 and Kv3.1 transcripts increase even if the level of Kv3.1b transcripts is highest in 1C11D and, after fluoxetine, in 1C11ND but decreases in 1C11D. The Kv3.1 channel protein is expressed in 1C11ND and 1C11D but is enhanced by fluoxetine only in 1C11D. Whole cell measurements confirm that 1C11 cells express (VGK) currents, increasing sequentially as a function of cell development. Moreover, SR 5HT1b is highly expressed in 1C11D but fluoxetine increases the level of transcript in 1C11ND and significantly decreases it in 1C11D. Serotonin dosage shows that fluoxetine at 10 nM blocks serotonin reuptake in 1C11ND but slows down its release when cells are differentiated through a decrease of 5HT1b receptors density. We provide the first experimental evidence that 1C11 expresses Kv3.1b, which confirms its major role during differentiation. Cells respond to the fluoxetine effect by upregulating Kv3.1b expression. On the other hand, the possible relationship between the fluoxetine effect on the kinetics of 5HT1b differentiation and Kv3.1bexpression, would suggest the Kv3.1b channel as a target of an antidepressant drug as well as it was suggested for 5HT1b.

Effects of repeated voluntary or forced exercise on brainstem serotonergic systems in rats.

Voluntary exercise increases stress resistance by modulating stress-responsive neurocircuitry, including brainstem serotonergic systems. However, it remains unknown how exercise produces adaptations to serotonergic systems. Recruitment of serotonergic systems during repeated, daily exercise could contribute to the adaptations in serotonergic systems following exercise, but whether repeated voluntary exercise recruits serotonergic systems is unknown. In this study, we investigated the effects of six weeks of voluntary or forced exercise on rat brain serotonergic systems. Specifically, we analyzed c-Fos and FosB/ΔFosB as markers of acute and chronic cellular activation, respectively, in combination with tryptophan hydroxylase, a marker of serotonergic neurons, within subregions of the dorsal raphe nucleus using immunohistochemical staining. Compared to sedentary controls, rats exposed to repeated forced exercise, but not repeated voluntary exercise, displayed decreased c-Fos expression in serotonergic neurons in the rostral dorsal portion of the dorsal raphe nucleus (DRD) and increased c-Fos expression in serotonergic neurons in the caudal DR (DRC), and interfascicular part of the dorsal raphe nucleus (DRI) during the active phase of the diurnal activity rhythm. Similarly, increases in c-Fos expression in serotonergic neurons in the DRC, DRI, and ventral portion of the dorsal raphe nucleus (DRV) were observed in rats exposed to repeated forced exercise, compared to rats exposed to repeated voluntary exercise. Six weeks of forced exercise, relative to the sedentary control condition, also increased FosB/ΔFosB expression in DRD, DRI, and DRV serotonergic neurons. While both voluntary and forced exercise increase stress resistance, these results suggest that repeated forced exercise, but not repeated voluntary exercise, increases activation of DRI serotonergic neurons, an effect that may contribute to the stress resistance effects of forced exercise. These results also suggest that mechanisms of exercise-induced stress resistance may differ depending on the controllability of the exercise.

Daily administration of Sake Lees (Sake Kasu) reduced psychophysical stress-induced hyperalgesia and Fos responses in the lumbar spinal dorsal horn evoked by noxious stimulation to the hindpaw in the rats.

We tested whether Sake Lees (SL) had inhibitory effects on hyperalgesia in the hindpaw under psychophysical stress conditions. Male rats were subjected to repeated forced swim stress treatments (FST) from Day -3 to Day -1. Intraperiotoneal administration of SL which contained low concentration of ethanol (SLX) was conducted after each FST. On Day 0, formalin-evoked licking behaviors and Fos responses in the lumbar spinal cord (DH) and several areas within the rostral ventromedial medulla (RVM) were quantified as nociceptive responses. FST-induced hyperalgesia in the hindpaw was prevented by repeated SL and SLX treatments. Fos expression was significantly increased in DH and some areas within the RVM under FST, which was prevented by repeated SL or SLX. These findings indicated that daily administration of SL had the potential to alleviate stress-induced hyperalgesia.

A sperm peptide enhances long-term memory in female Drosophila.

Can mating influence cognitive functions such as learning and memory in a permanent way? We have addressed this question using a combined behavioral and in vivo imaging approach, finding that aversive long-term memory performance strongly increases in Drosophila females in response to sperm transfer following mating. A peptide in the male sperm, the sex peptide, is known to cause marked changes in female reproductive behavior, as well as other behaviors such as dietary preference. Here, we demonstrate that this sex peptide enhances memory by acting on a single pair of serotonergic brain neurons, in which activation of the sex peptide receptor stimulates the cyclic adenosine monophosphate/protein kinase A pathway. We thus reveal a strong effect of mating on memory via the neuromodulatory action of a sperm peptide on the female brain.

Serotonin 5-HT4 Receptor Agonists Improve Facilitation of Contextual Fear Extinction in an MPTP-Induced Mouse Model of Parkinson's Disease.

Previously, we found that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease (PD) model mice (PD mice) showed facilitation of hippocampal memory extinction via reduced cyclic adenosine monophosphate (cAMP)/cAMP-dependent response element-binding protein (CREB) signaling, which may cause cognitive impairment in PD. Serotonergic neurons in the median raphe nucleus (MnRN) project to the hippocampus, and functional abnormalities have been reported. In the present study, we investigated the effects of the serotonin 5-HT4 receptor (5-HT4R) agonists prucalopride and velusetrag on the facilitation of memory extinction observed in PD mice. Both 5-HT4R agonists restored facilitation of contextual fear extinction in PD mice by stimulating the cAMP/CREB pathway in the dentate gyrus of the hippocampus. A retrograde fluorogold-tracer study showed that γ-aminobutyric acid-ergic (GABAergic) neurons in the reticular part of the substantia nigra (SNr), but not dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc), projected to serotonergic neurons in the MnRN, which are known to project their nerve terminals to the hippocampus. It is possible that the degeneration of the SNpc DAergic neurons in PD mice affects the SNr GABAergic neurons, and thereafter, the serotonergic neurons in the MnRN, resulting in hippocampal dysfunction. These findings suggest that 5HT4R agonists could be potentially useful as therapeutic drugs for treating cognitive deficits in PD.

RGS2 drives male aggression in mice via the serotonergic system.

Aggressive behavior in our modern, civilized society is often counterproductive and destructive. Identifying specific proteins involved in the disease can serve as therapeutic targets for treating aggression. Here, we found that overexpression of RGS2 in explicitly serotonergic neurons augments male aggression in control mice and rescues male aggression in Rgs2-/- mice, while anxiety is not affected. The aggressive behavior is directly correlated to the immediate early gene c-fos induction in the dorsal raphe nuclei and ventrolateral part of the ventromedial nucleus hypothalamus, to an increase in spontaneous firing in serotonergic neurons and to a reduction in the modulatory action of Gi/o and Gq/11 coupled 5HT and adrenergic receptors in serotonergic neurons of Rgs2-expressing mice. Collectively, these findings specifically identify that RGS2 expression in serotonergic neurons is sufficient to drive male aggression in mice and as a potential therapeutic target for treating aggression.

Anxiolytic and panicolytic-like effects of environmental enrichment seem to be modulated by serotonin neurons located in the dorsal subnucleus of the dorsal raphe.

In a previous study, we showed that exposure of rats to a one-week environmental enrichment (EE) protocol decreases elevated T-maze (ETM) avoidance responses, an anxiolytic-like effect, without altering escape reactions, in clinical terms related to panic disorder. These anxiolytic-like effects were followed by decreased delta FosB-immunoreactivity (delta FosB-ir) in the cingulate cortex, dorsolateral and intermediate lateral septum, hippocampus (cornus of Ammon), anterior and dorsomedial hypothalamus, medial and basolateral amygdala and ventral region of the dorsal raphe nucleus. The purpose of the present study was to further investigate behavioral and neurophysiological alterations induced by EE exposure. For that, in a first experiment we verified if increasing the time of exposure to the same EE protocol used in our previous study (from one to two weeks) altered male Wistar rats' ETM escape responses. All animals were tested in an open field, immediately after the ETM, for locomotor activity assessment. Since anxiety and panic-related reactions have been associated to the functioning of specific subnuclei of the dorsal raphe nucleus (DR), we also evaluated delta FosB-ir in serotonergic cells of DR regions. At last, we analyzed plasma corticosterone levels in animals submitted to EE and to standard housing. Results showed that a two-week exposure to EE decreases both ETM avoidance and escape reactions, inducing anxiolytic and panicolytic-like effects, respectively. There was also a significant decrease in the number of double staining neurons in the midrostral region of the dorsal subnucleus of the dorsal raphe. No changes in corticosterone levels, however, were observed. These results contribute to a better understanding of the effects of EE on anxiety and panic-related responses.

A Shift in the Activation of Serotonergic and Non-serotonergic Neurons in the Dorsal Raphe Lateral Wings Subnucleus Underlies the Panicolytic-Like Effect of Fluoxetine in Rats.

A wealth of evidence indicates that the lateral wings subnucleus of the dorsal raphe nucleus (lwDR) is implicated in the processing of panic-associated stimuli. Escape expression in the elevated T-maze, considered a panic-related defensive behavior, markedly and selectively recruits non-serotonergic cells within this DR subregion and in the dorsal periaqueductal gray (dPAG), another key panic-associated area. However, whether anti-panic drugs may interfere with this pattern of neuronal activation is still unknown. In the present study, the effects of acute (10 mg/kg) or chronic fluoxetine (10 mg/kg/daily/21 days) treatment on the number of serotonergic and non-serotonergic cells induced by escape expression within the rat DR and PAG subnuclei were investigated by immunochemistry. The results showed that chronic, but not acute, treatment with fluoxetine impaired escape expression, indicating a panicolytic-like effect, and markedly decreased the number of non-serotonergic cells that were recruited in the lwDR and dPAG. The same treatment selectively increased the number of serotonergic neurons within the lwDR. Our immunochemistry analyses also revealed that the non-serotonergic cells recruited in the lwDR and dPAG by the escape expression were not nitrergic. Overall, our findings suggest that the anti-panic effect of chronic treatment with fluoxetine is mediated by stimulation of the lwDR-dPAG pathway that controls the expression of panic-associated escape behaviors.

Cav1.2 L-type calcium channels regulate stress coping behavior via serotonin neurons.

Human genetic variation in the gene CACNA1C, which codes for the alpha-1c subunit of Cav1.2 L-type calcium channels (LTCCs), has been broadly associated with enhanced risk for neuropsychiatric disorders including major depression, bipolar and schizophrenia. Little is known about the specific neural circuits through which CACNA1C and Cav1.2 LTCCs impact disease etiology. However, serotonin (5-HT) neurotransmission has been consistently implicated in these neuropsychiatric disorders and Cav1.2 LTCCs may influence 5-HT neuron activity during relevant behavioral states such as stress. We utilized a temporally controlled and 5-HT neuron specific Cacna1c knockout mouse model to assess stress-coping behavior using the forced swim test and dorsal raphe (DR) 5-HT neuron Fos activation. Furthermore, we assessed 5-HT1A receptor function and feedback inhibition of the DR following administration of the 5-HT1A antagonist WAY-100635. We find that 5-HT neuron Cacna1c knockout disrupts active-coping behavior in the forced swim test and that this behavioral effect is rescued by blocking 5-HT1A receptors. Moreover, Cacna1c knockout mice display enhanced Fos expression in caudal DR 5-HT neurons and an enhanced response to a 5-HT1A receptor antagonist in rostral DR 5-HT neurons, indicating that loss of Cacna1c disrupts both 5-HT neuron activation and 5-HT1A dependent feedback inhibition across the caudal to rostral DR. Collectively, these results reveal an important role for 5-HT neuron Cav1.2 LTCCs in stress-coping behavior and 5-HT1A receptor function. This suggests that alterations in CACNA1C function or expression could influence the development or treatment of neuropsychiatric disorder through serotonergic mechanisms.

Role of Estradiol in the Expression of Genes Involved in Serotonin Neurotransmission: Implications for Female Depression.

BACKGROUND: In women, changes in estrogen levels may increase the incidence and/or symptomatology of depression and affect the response to antidepressant treatments. Estrogen therapy in females may provide some mood benefits as a single treatment or might augment clinical response to antidepressants that inhibit serotonin reuptake. OBJECTIVE: We analyzed the mechanisms of estradiol action involved in the regulation of gene expression that modulates serotonin neurotransmission implicated in depression. METHOD: Publications were identified by a literature search on PubMed. RESULTS: The participation of estradiol in depression may include regulation of the expression of tryptophan hydroxylase-2, monoamine oxidase A and B, serotonin transporter and serotonin-1A receptor. This effect is mediated by estradiol binding to intracellular estrogen receptor that interacts with estrogen response elements in the promoter sequences of tryptophan hydroxylase-2, serotonin transporter and monoamine oxidase-B. In addition to directly binding deoxyribonucleic acid, estrogen receptor can tether to other transcription factors, including activator protein 1, specificity protein 1, CCAAT/enhancer binding protein ß and nuclear factor kappa B to regulate gene promoters that lack estrogen response elements, such as monoamine oxidase-A and serotonin 1A receptor. CONCLUSION: Estradiol increases tryptophan hydroxylase-2 and serotonin transporter expression and decreases the expression of serotonin 1A receptor and monoamine oxidase A and B through the interaction with its intracellular receptors. The understanding of molecular mechanisms of estradiol regulation on the protein expression that modulates serotonin neurotransmission will be helpful for the development of new and more effective treatment for women with depression.