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Swim Stress differentially blocks CRF receptor mediated responses in dorsal raphe nucleus
Abstract
Modulation of the serotonergic (5-HT) neurotransmitter system arising from the dorsal raphe nucleus (DR) is thought to support the behavioral effects of swim stress, i.e., immobility. In vivo pharmacological and anatomical studies suggest that corticotropin-releasing factor (CRF) and γ-aminobutyric acid (GABA) synaptic transmission closely interact to set the response of the DR to swim stress. To investigate the cellular basis of these physiological mechanisms the effects of ovine CRF (oCRF) on GABA(A)-dependent miniature inhibitory postsynaptic currents (mIPSCs) in 5-HT and non-5-HT DR neurons in acute mesencephalic slices obtained from rats either naïve or 24h after a 15 min swim stress session were tested. In this study, the effect of swim stress alone was to decrease the holding current, i.e., hyperpolarize the neuron, and to increase the amplitude and charge of mIPSCs recorded from non-5-HT neurons. Ovine CRF (10 nM) induced an increase in mIPSC frequency in 5-HT neurons recorded from naïve rats, an effect that was suppressed by swim stress. The inward current elicited by oCRF in both 5-HT and non-5-HT neurons was also blocked by swim stress. Ovine CRF increased mIPSCs amplitude and charge in both 5-HT and non-5-HT neurons, but this effect was not modified by swim stress. In concert with our previous findings that swim stress decreased input resistance, action potential threshold and action potential duration and increased glutamatergic synaptic activity the overall primary effect of swim stress is to increase the excitability of 5-HT neurons. These data provide a mechanism at the cellular level for the immobility induced by swim stress and identifies critical components of the raphe circuitry responsible for the altered output of 5-HT neurons induced by swim stress.
... Low doses of CRF in the DRN inhibit 5-HT neurons via CRF receptor type1 (CRF-R1), whereas high doses of CRF activate 5-HT neurons via CRF receptor type 2 (CRF-R2) (Kirby et al. 2000;Lukkes et al. 2008;Price et al. 1998;Price and Lucki 2001). Although there is evidence that 24 h after exposure to swim stress, DRN-5-HT neurons exhibited increased excitability (Lamy and Beck 2010), acute stress generally inhibits DRN-5-HT activity (Kirby et al. 2000;Price et al. 1998;Price et al. 2002). Anatomical studies showed that DRN GABAergic interneurons receive strong CRF-containing projections (Waselus et al. 2005) and have dense expression of CRF-R1 receptors (Howerton et al. 2014;Lowry et al. 2000;Roche et al. 2003;Valentino et al. 2001). ...
Rationale
The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Our previous data show that stressors can inhibit 5-HT neuronal activity and release by stimulating the release of the stress neurohormone corticotropin-releasing factor (CRF) within the serotonergic dorsal raphe nucleus (DRN). The inhibitory effects of CRF on 5-HT DRN neurons are indirect, mediated by CRF-R1 receptors located on GABAergic afferents.
Objectives
We tested the hypothesis that DRN CRF-R1 receptors contribute to stress-induced reinstatement of morphine-conditioned place preference (CPP). We also examined the role of this circuitry in stress-induced negative affective state with 22-kHz distress ultrasonic vocalizations (USVs), which are naturally emitted by rats in response to environmental challenges such as pain, stress, and drug withdrawal.
Methods
First, we tested if activation of CRF-R1 receptors in the DRN with the CRF-R1-preferring agonist ovine CRF (oCRF) would reinstate morphine CPP and then if blockade of CRF-R1 receptors in the DRN with the CRF-R1 antagonist NBI 35965 would attenuate swim stress–induced reinstatement of morphine CPP. Second, we tested if intra-DRN pretreatment with NBI 35965 would attenuate foot shock stress–induced 22-kHz USVs.
Results
Intra-DRN injection of oCRF reinstated morphine CPP, while intra-DRN injection of NBI 35965 attenuated swim stress–induced reinstatement. Moreover, intra-DRN pretreatment with NBI 35965 significantly reduced 22-kHz distress calls induced by foot shock.
Conclusions
These data provide evidence that stress-induced negative affective state is mediated by DRN CRF-R1 receptors and may contribute to reinstatement of morphine CPP.
... Prior stress history differentially affects the function of CRF at these different cellular substrates. For example, both stressor exposure as well as basal stress hyperresponsivity ablate the ability of CRF-R1 to increase GABAergic transmission onto serotonergic dorsal raphe neurons (Lamy and Beck, 2010;Lemos et al., 2011), thereby reducing CRF mediated-inhibition of serotonin release. Likewise, different types of stressor were shown to disrupt CRF's ability to enhance dopamine transmission both in the VTA and the NAc (Beckstead et al., 2009;Lemos et al., 2012), an effect that our findings here confirm and extend upon. ...
Acute stressors can stimulate appetitive and exploratory behaviors, not just produce negative affect that impair performance. Corticotropin releasing factor (CRF), which is released in the brain in response to stress, acts on different targets and circuits to mediate both the negative and positive effects of stress. In the nucleus accumbens, CRF facilitates appetitive behavior through mechanisms not fully understood. Here we report that cholinergic interneurons (CINs) are a novel target for CRF actions in the striatum. CRF enhances the spontaneous firing via activation of CRF-type 1 receptors expressed on CINs. This causes the activation muscarinic acetylcholine receptors type 5, which mediate CRF potentiation of dopamine transmission in the striatum. Repeated stress selectively dampens some CRF functions but spare effect on CINs and changes CRF-R1 expression in a cell-specific manner. These data highlight the existence of diverse CRF targets within the striatum, which vary in their resilience to stress.
... Stressor exposure induces a series of neuroendocrine, physiological, and behavioral adaptations that promote an appropriate response to the stressor. Central to these diverse functions is CRF signaling which in a number of brain regions promotes a number of immediate [48,49,51,[177][178][179][180][181][182][183][184] and persistent [50,52,60,121,[185][186][187] cellular changes. These effects are of particular interest in LC, where the interaction of CRF with its receptor CRFR1 activates cAMP-dependent intracellular signaling cascades, increasing tonic discharge and promoting anxiety-like behavior [64,77,188,189]. ...
Neural plasticity plays a critical role in mediating short- and long-term brain responses to environmental stimuli. A major effector of plasticity throughout many regions of the brain is stress. Activation of the locus coeruleus (LC) is a critical step in mediating the neuroendocrine and behavioral limbs of the stress response. During stressor exposure, activation of the hypothalamic-pituitary-adrenal axis promotes release of corticotropin-releasing factor in LC, where its signaling promotes a number of physiological and cellular changes. While the acute effects of stress on LC physiology have been described, its long-term effects are less clear. This review will describe how stress changes LC neuronal physiology, function, and morphology from a genetic, cellular, and neuronal circuitry/transmission perspective. Specifically, we describe morphological changes of LC neurons in response to stressful stimuli and signal transduction pathways underlying them. Also, we will review changes in excitatory glutamatergic synaptic transmission in LC neurons and possible stress-induced modifications of AMPA receptors. This review will also address stress-related behavioral adaptations and specific noradrenergic receptors responsible for them. Finally, we summarize the results of several human studies which suggest a link between stress, altered LC function, and pathogenesis of posttraumatic stress disorder.
... * P < 0.05. GABA A Rs within the CNS, in a receptor subtype and brain region-specific manner (Caldji et al., 2003;Lamy and Beck, 2010;Martisova et al., 2012;Crawford et al., 2013;Liu et al., 2014;Corteen et al., 2015). However, the effect of stress on peripheral GABA A Rs is poorly understood. ...
Gamma aminobutyric acid (GABA) subtype A receptors (GABAARs) are integral membrane ion channels composed of five individual proteins or subunits. Up to 19 different GABAAR subunits (α1-6, β1-3, γ1-3, δ, ε, θ, π, and ρ1-3) have been identified, resulting in anatomically, physiologically, and pharmacologically distinct multiple receptor subtypes, and therefore GABA-mediated inhibition, across the central nervous system (CNS). Additionally, GABAAR-modulating drugs are important tools in clinical medicine, although their use is limited by adverse effects. While significant advances have been made in terms of characterizing the GABAAR system within the brain, relatively less is known about the molecular phenotypes within the peripheral nervous system of major organ systems. This represents a potentially missed therapeutic opportunity in terms of utilizing or repurposing clinically available GABAAR drugs, as well as promising research compounds discarded due to their poor CNS penetrance, for the treatment of peripheral disorders. In addition, a broader understanding of the peripheral GABAAR subtype repertoires will contribute to the design of therapies which minimize peripheral side-effects when treating CNS disorders. We have recently provided a high resolution molecular and function characterization of the GABAARs within the enteric nervous system of the mouse colon. In this study, the aim was to determine the constituent GABAAR subunit expression profiles of the mouse bladder, heart, liver, kidney, lung, and stomach, using reverse transcription polymerase chain reaction and western blotting with brain as control. The data indicate that while some subunits are expressed widely across various organs (α3-5), others are restricted to individual organs (γ2, only stomach). Furthermore, we demonstrate complex organ-specific developmental expression plasticity of the transporters which determine the chloride gradient within cells, and therefore whether GABAAR activation has a depolarizing or hyperpolarizing effect. Finally, we demonstrate that prior exposure to early life psychosocial stress induces significant changes in peripheral GABAAR subunit expression and chloride transporters, in an organ- and subunit-specific manner. Collectively, the data demonstrate the molecular diversity of the peripheral GABAAR system and how this changes dynamically in response to life experience. This provides a molecular platform for functional analyses of the GABA-GABAAR system in health, and in diseases affecting various peripheral organs.
... CRF regulates DRN-5-HT neurons in a bidirectional and CRF receptor subtype-specific manner (Kirby et al., 2000;Valentino et al., 2010). Although there is evidence that 24 h after exposure to swim stress, DRN-5-HT neurons exhibited increased excitability (Lamy and Beck, 2010), acute stress as well as intra-DRN injection of low doses of CRF primarily inhibit DRN-5-HT activity (Kirby et al., 2000;Price et al., 2002). Additional support for these findings come from the observation that the neuronal excitability of DRN-5-HT neurons is decreased in Wistar-Kyoto rats, a stress hyperresponsive strain (Lemos et al., 2011). ...
The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Stressors and stress hormones can inhibit the dorsal raphe nucleus (DRN)-5-HT system, which composes the majority of forebrain-projecting 5-HT. This inhibition is mediated via stimulation of GABA synaptic activity at DRN-5-HT neurons. Using swim stress-induced reinstatement of morphine conditioned place-preference, recent data from our laboratory indicate that morphine history sensitizes DRN-5-HT neurons to GABAergic inhibitory effects of stress. Moreover, GABAA receptor-mediated inhibition of the serotonergic DRN is required for this reinstatement. In our current experiment, we tested the hypothesis that GABAergic sensitization of DRN-5-HT neurons is a neuroadaptation elicited by multiple classes of abused drugs across multiple models of stress-induced relapse by applying a chemical stressor (yohimbine) to induce reinstatement of previously extinguished cocaine self-administration in Sprague-Dawley rats. Whole-cell patch-clamp recordings of GABA synaptic activity in DRN-5-HT neurons were conducted after the reinstatement. Behavioral data indicate that yohimbine triggered reinstatement of cocaine self-administration. Electrophysiology data indicate that 5-HT neurons in the cocaine group exposed to yohimbine had increased amplitude of inhibitory postsynaptic currents compared to yoked-saline controls exposed to yohimbine or unstressed animals in both drug groups. These data, together with previous findings, indicate that interaction between psychostimulant or opioid history and chemical or physical stressors may increase postsynaptic GABA receptor density and/or sensitivity in DRN-5-HT neurons. Such mechanisms may result in serotonergic hypofunction and consequent dysphoric mood states which confer vulnerability to stress-induced drug reinstatement.
... DR-5-HT neurons are strongly regulated by CRF in a bimodal manner, in which activation of difference CRF receptor subtypes can have opposing effects on DR-5-HT neurons (Valentino et al. 2010). Although there is evidence that 24 hours after exposure to swim stress DR-5-HT neurons exhibited increased excitability (Lamy and Beck 2010), acute stress as well as intraDR injection of low doses of CRF inhibits DR-5-HT activity (Kirby et al. 2000;Price et al. 1998;Price et al. 2002) resulting in changes of 5-HT release in targeted brain regions, e.g. decreased 5-HT levels in the lateral septum, amygdala and thalamus-hypothalamus but increased 5-HT in the striatum (Briones-Aranda et al. 2005;Kirby et al. 1995;Kirby and Lucki 1998). ...
The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Our data indicate that stress inhibits the dorsal raphe nucleus (DRN)-5-HT system via stimulation of GABA synaptic activity by the stress neurohormone corticotropin-releasing factor and, more recently, that morphine history sensitizes DRN-5-HT neurons to GABAergic inhibitory effects of stress.
We tested the hypothesis that DRN GABAA receptors contribute to stress-induced reinstatement of morphine-conditioned place preference (CPP).
First, we tested if activation of GABAA receptors in the DRN would reinstate morphine CPP. Second, we tested if blockade of GABAA receptors in the DRN would attenuate swim stress-induced reinstatement of morphine CPP. CPP was induced by morphine (5 mg/kg) in a 4-day conditioning phase followed by a conditioning test. Upon acquiring conditioning criteria, subjects underwent 4 days of extinction training followed by an extinction test. Upon acquiring extinction criteria, animals underwent a reinstatement test. For the first experiment, the GABAA receptor agonist muscimol (50 ng) or vehicle was injected into the DRN prior to the reinstatement test. For the second experiment, the GABAA receptor antagonist bicuculline (75 ng) or vehicle was injected into the DRN prior to a forced swim stress, and then, animals were tested for reinstatement of CPP.
Intraraphe injection of muscimol reinstated morphine CPP, while intraraphe injection of bicuculline attenuated swim stress-induced reinstatement.
These data provide evidence that GABAA receptor-mediated inhibition of the serotonergic DRN contributes to stress-induced reinstatement of morphine CPP.
... To determine whether recorded cells were 5-HT-containing or not, slices were immunostained for tryptophan hydroxylase (TPH), an enzyme in the biosynthetic pathway for 5-HT production as previously described (Beck et al., 2004;Crawford et al., 2010;Kirby et al., 2008;Kirby et al., 2007;Lamy and Beck, 2010;Lemos et al., 2006;Lemos et al., 2010). Neurons that were positively stained with TPH were classified as 5-HT whereas those without TPH were identified as non-5-HT. ...
The median (MR) and dorsal raphe (DR) nuclei contain the majority of the 5-hydroxytryptamine (5-HT, serotonin) neurons that project to limbic forebrain regions, are important in regulating homeostatic functions and are implicated in the etiology and treatment of mood disorders and schizophrenia. The primary synaptic inputs within and to the raphe are glutamatergic and GABAergic. The DR is divided into three subfields, i.e., ventromedial (vmDR), lateral wings (lwDR) and dorsomedial (dmDR). Our previous work shows that cell characteristics of 5-HT neurons and the magnitude of the 5-HT(1A) and 5-HT(1B) receptor-mediated responses in the vmDR and MR are not the same. We extend these observations to examine the electrophysiological properties across all four raphe subfields in both 5-HT and non-5-HT neurons. The neurochemical topography of glutamatergic and GABAergic cell bodies and nerve terminals were identified using immunohistochemistry and the morphology of the 5-HT neurons was measured. Although 5-HT neurons possessed similar physiological properties, important differences existed between subfields. Non-5-HT neurons were indistinguishable from 5-HT neurons. GABA neurons were distributed throughout the raphe, usually in areas devoid of 5-HT neurons. Although GABAergic synaptic innervation was dense throughout the raphe (immunohistochemical analysis of the GABA transporters GAT1 and GAT3), their distributions differed. Glutamate neurons, as defined by vGlut3 anti-bodies, were intermixed and co-localized with 5-HT neurons within all raphe subfields. Finally, the dendritic arbor of the 5-HT neurons was distinct between subfields. Previous studies regard 5-HT neurons as a homogenous population. Our data support a model of the raphe as an area composed of functionally distinct subpopulations of 5-HT and non-5-HT neurons, in part delineated by subfield. Understanding the interaction of the cell properties of the neurons in concert with their morphology, local distribution of GABA and glutamate neurons and their synaptic input, reveals a more complicated and heterogeneous raphe. These results provide an important foundation for understanding how specific subfields modulate behavior and for defining which aspects of the circuitry are altered during the etiology of psychological disorders.
The dorsal raphe nucleus (DRN) provides the major source of serotonin to the central nervous system (CNS) and modulates diverse neural functions including mood. Furthermore, DRN cellular networks are engaged in the stress-response at the CNS level allowing for adaptive behavioural responses, whilst stress-induced dysregulation of DRN and serotonin release is implicated in psychiatric disorders. Therefore, identifying the molecules regulating DRN activity is fundamental to understand DRN function in health and disease. GABAA receptors (GABAARs) allow for brain region, cell type and subcellular domain-specific GABA-mediated inhibitory currents and are thus key regulators of neuronal activity. Yet, the GABAAR subtypes expressed within the neurochemically diverse cell types of the mouse DRN are poorly described. In this study, immunohistochemistry and confocal microscopy revealed that all serotonergic neurons expressed immunoreactivity for the GABAAR alpha2 and 3 subunits, although the respective signals were co-localised to varying degrees with inhibitory synaptic marker proteins. Only a topographically located sub-population of serotonergic neurons exhibited GABAAR alpha1 subunit immunoreactivity. However, all GABAergic as well as non-GABAergic, non-serotonergic neurons within the DRN expressed GABAAR alpha1 subunit immunoreactivity. Intriguingly, immunoreactivity for the GABAAR gamma2 subunit was enriched on GABAergic rather than serotonergic neurons. Finally, repeated restraint stress increased the expression of the GABAAR alpha3 subunit at the mRNA and protein level. The study demonstrates the identity and location of distinct GABAAR subunits within the cellular networks of the mouse DRN and that stress impacts on the expression levels of particular subunits at the gene and protein level.
Extracellular electrophysiological recordings in freely moving cats have shown that serotonergic neurons from the dorsal raphe nucleus (DRN) fire tonically during wakefulness, decrease their activity during slow wave sleep (SWS), and are nearly quiescent during paradoxical sleep (PS). The mechanisms at the origin of the modulation of activity of these neurons are still unknown. Here, we show in the unanesthetized rat that the iontophoretic application of the GABA A antagonist bicuculline on dorsal raphe serotonergic neurons induces a tonic discharge during SWS and PS and an increase of discharge rate during quiet waking. These data strongly suggest that an increase of a GABAergic inhibitory tone present during wakefulness is responsible for the decrease of activity of the dorsal raphe serotonergic cells during slow wave and paradoxical sleep. In addition, by combining retrograde tracing with cholera toxin B subunit and glutamic acid decarboxylase immunohistochemistry, we demonstrate that the GABAergic innervation of the dorsal raphe nucleus arises from multiple distant sources and not only from interneurons as classically accepted. Among these afferents, GABAergic neurons located in the lateral preoptic area and the pontine ventral periaqueductal gray including the DRN itself could be responsible for the reduction of activity of the serotonergic neurons of the dorsal raphe nucleus during slow wave and paradoxical sleep, respectively.
Corticotropin-releasing factor (CRF) is the primary factor involved in controlling the release of ACTH from the anterior pituitary and also acts as a neurotransmitter in a variety of brain systems. The actions of CRF are mediated by G-protein coupled membrane bound receptors and a high affinity CRF receptor, CRF1, has been previously cloned and functionally characterized. We have recently isolated a cDNA encoding a second member of the CRF receptor family, designated CRF2, which displays approximately 70% homology at the nucleotide level to the CRF1 receptor and exhibits a distinctive pharmacological profile. The present study utilized in situ hybridization histochemistry to localize the distribution of CRF2 receptor mRNA in rat brain and pituitary gland and compared this with the distribution of CRF1, receptor expression. While CRF1 receptor expression was very high in neocortical, cerebellar, and sensory relay structures, CRF2 receptor expression was generally confined to subcortical structures. The highest levels of CRF2 receptor mRNA in brain were evident within the lateral septal nucleus, the ventromedial hypothalamic nucleus and the choroid plexus. Moderate levels of CRF2 receptor expression were evident in the olfactory bulb, amygdaloid nuclei, the paraventricular and suraoptic nuclei of the hypothalamus, the inferior colliculus and 5-HT-associated raphe nuclei of the midbrain. CRF2-expressing cells were also evident in the bed nucleus of the stria terminalis, the hippocampal formation and anterior and lateral hypothalmic areas. In addition, CRF2 receptor mRNA was also found in cerebral arterioles throughout the brain. Within the pituitary gland, CRF2 receptor mRNA was detectable only at very low levels in scattered cells while CRF1 receptor mRNA was readily detectable in anterior and intermediate lobes. This heterogeneous distribution of CRF1 and CRF2 receptor mRNA suggests distinctive functional roles for each receptor in CRF-related systems. The CRF1 receptor may be regarded as the primary neuroendocrine pituitary CRF receptor and important in cortical, cerebellar and sensory roles of CRF. The anatomical distribution of CRF2 receptor mRNA indicates a role for this novel receptor in hypothalamic neuroendocrine, autonomic and general behavioral actions of central CRF.
It has been argued that exposure to inescapable shock produces later behavioral changes such as poor shuttle box escape learning because it leads to the conditioning of intense fear, which later transfers to the shuttle box test situation and interferes with escape. Both fear, as assessed by freezing, and escape were measured in Sprague-Dawley rats 24 hrs after exposure to inescapable shock. Lesions of the basolateral region and central nucleus of the amygdala eliminated the fear that transfers to the shuttle box after inescapable shock, as well as the fear conditioned in the shuttle box by the shuttle box shocks. However, the amygdala lesions did not reduce the learning deficit produced by inescapable shock. In contrast, dorsal raphe nucleus lesions did not reduce the fear that transfers to the shuttle box after inescapable shock, but eliminated the enhanced fear conditioning in the shuttle box as well as the escape deficit. The implications of these results for the role of fear and anxiety in mediating inescapable shock effects are discussed. (PsycINFO Database Record (c) 2012 APA, all rights reserved)
Two G protein-coupled receptors have been identified that bind corticotropin-releasing factor (CRF) and urocortin (UCN) with high affinity. Hybridization histochemical methods were used to shed light on controversies concerning their localization in rat brain, and to provide normative distributional data in mouse, the standard model for genetic manipulation in mammals. The distribution of CRF-R1 mRNA in mouse was found to be fundamentally similar to that in rat, with expression predominating in the cerebral cortex, sensory relay nuclei, and in the cerebellum and its major afferents. Pronounced species differences in distribution were few, although more subtle variations in the relative strength of R1 expression were seen in several forebrain regions. CRF-R2 mRNA displayed comparable expression in rat and mouse brain, distinct from, and more restricted than that of CRF-R1. Major neuronal sites of CRF-R2 expression included aspects of the olfactory bulb, lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, medial and posterior cortical nuclei of the amygdala, ventral hippocampus, mesencephalic raphe nuclei, and novel localizations in the nucleus of the solitary tract and area postrema. Several sites of expression in the limbic forebrain were found to overlap partially with ones of androgen receptor expression. In pituitary, rat and mouse displayed CRF-R1 mRNA signal continuously over the intermediate lobe and over a subset of cells in the anterior lobe, whereas CRF-R2 transcripts were expressed mainly in the posterior lobe. The distinctive expression pattern of CRF-R2 mRNA identifies additional putative central sites of action for CRF and/or UCN. Constitutive expression of CRF-R2 mRNA in the nucleus of the solitary tract, and stress-inducible expression of CRF-R1 transcripts in the paraventricular nucleus may provide a basis for understanding documented effects of CRF-related peptides at a loci shown previously to lack a capacity for CRF-R expression or CRF binding. Other such “mismatches” remain to be reconciled. J. Comp. Neurol. 428:191–212, 2000. © 2000 Wiley-Liss, Inc.
Stress-related psychiatric disorders such as anxiety and depression involve dysfunction of the serotonin [5-hydroxytryptamine (5-HT)] system. Previous studies have found that the stress neurohormone corticotropin-releasing factor (CRF) inhibits 5-HT neurons in the dorsal raphe nucleus (DRN) in vivo. The goals of the present study were to characterize the CRF receptor subtypes (CRF-R1 and -R2) and cellular mechanisms underlying CRF-5-HT interactions. Visualized whole-cell patch-clamp recording techniques in brain slices were used to measure spontaneous or evoked GABA synaptic activity in DRN neurons of rats and CRF effects on these measures. CRF-R1 and -R2-selective agonists were bath applied alone or in combination with receptor-selective antagonists. CRF increased presynaptic GABA release selectively onto 5-HT neurons, an effect mediated by the CRF-R1 receptor. CRF increased postsynaptic GABA receptor sensitivity selectively in 5-HT neurons, an effect to which both receptor subtypes contributed. CRF also had direct effects on DRN neurons, eliciting an inward current in 5-HT neurons mediated by the CRF-R2 receptor and in non-5-HT neurons mediated by the CRF-R1 receptor. These results indicate that CRF has direct membrane effects on 5-HT DRN neurons as well as indirect effects on GABAergic synaptic transmission that are mediated by distinct receptor subtypes. The inhibition of 5-HT DRN neurons by CRF in vivo may therefore be primarily an indirect effect via stimulation of inhibitory GABA synaptic transmission. These results regarding the cellular mechanisms underlying the complex interaction between CRF, 5-HT, and GABA systems could contribute to the development of novel treatments for stress-related psychiatric disorders.
Stress is a common trigger in affective disorder onset, yet the mechanism and predisposing factors of vulnerability remain unknown. Effective disease prevention requires a critical balance of responses within the serotonergic raphe nucleus, including a coordination of corticotropin-releasing factor (CRF) actions at both of its receptors, CRF receptor-1 and CRF receptor-2. Mice deficient in CRF receptor-2 (R2KO) were used as a model of maladaptive stress responsivity to examine the physiological and molecular markers of stress dysregulation within the raphe in the absence of this receptor. After chronic stress, R2KO mice failed to display the robust stress-mediated adaptations characteristic of control mice, including elevations in tryptophan hydroxylase-2 and CRF receptor-1 expression and concordant increases in behavioral arousal. As a further indication of failed homeostatic mechanisms, R2KO mice displayed indices of cell death in the raphe after stress exposure, with elevations in proapoptotic factors but a failure to mount adaptive increases in antiapoptotic factors found in control mice. In vitro electrophysiological characterization of the specific influence of CRF on the raphe revealed both basal differences and a failure to respond to CRF administration in R2KO mice. These results support a requirement for homeostatic maintenance in response to stress in the raphe, where dysregulation may be a critical predictor of affective disorder onset.
Serotonergic (5-HT) neurons of the brainstem dorsal raphe nucleus (DRN) have been implicated in a diversity of physiological and behavioral processes in vertebrates. However, despite extensive information about the intrinsic properties and the efferent projections of this neurochemical system, little information is available regarding the afferents that control its activity. This study investigated the neurotransmitters that regulate the activity of DRN-5-HT neurons under physiologically relevant conditions, by utilizing microiontophoresis in combination with single-unit recordings in the awake, head-restrained cat. This made it possible to examine the direct effects of neurotransmitters on DRN-5-HT neuronal activity, and, through the use of specific antagonists, to study the roles of these neurotransmitter inputs during physiological conditions that influence DRN-5-HT neuronal activity. The results indicate that (1) iontophoretic application of the GABA antagonist bicuculline reversed the typical suppression of neuronal activity seen during slow wave sleep, but had no effect on maintained activity during wakefulness. The suppression of neuronal activity during REM sleep was generally unaffected by application of bicuculline. This suggests a role for a GABAergic input to DRN-5-HT neurons in controlling some aspects of their state-dependent activity. (2) Iontophoretic application of the excitatory amino acid (EAA) antagonist kynurenic acid reduced the magnitude of the neuronal response evoked by phasic auditory stimuli, but had no effect on the spontaneous activity of these neurons, suggesting a role for an EAA input to the DRN in mediating the response to phasic sensory stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)
Extracellular electrophysiological recordings in freely moving cats have shown that serotonergic neurons from the dorsal raphe nucleus (DRN) fire tonically during wakefulness, decrease their activity during slow wave sleep (SWS), and are nearly quiescent during paradoxical sleep (PS). The mechanisms at the origin of the modulation of activity of these neurons are still unknown. Here, we show in the unanesthetized rat that the iontophoretic application of the GABA(A) antagonist bicuculline on dorsal raphe serotonergic neurons induces a tonic discharge during SWS and PS and an increase of discharge rate during quiet waking. These data strongly suggest that an increase of a GABAergic inhibitory tone present during wakefulness is responsible for the decrease of activity of the dorsal raphe serotonergic cells during slow wave and paradoxical sleep. In addition, by combining retrograde tracing with cholera toxin B subunit and glutamic acid decarboxylase immunohistochemistry, we demonstrate that the GABAergic innervation of the dorsal raphe nucleus arises from multiple distant sources and not only from interneurons as classically accepted. Among these afferents, GABAergic neurons located in the lateral preoptic area and the pontine ventral periaqueductal gray including the DRN itself could be responsible for the reduction of activity of the serotonergic neurons of the dorsal raphe nucleus during slow wave and paradoxical sleep, respectively.
In vivo studies suggest that the stress-related neuropeptide corticotropin-releasing factor (CRF) modulates serotonergic neurotransmission. To investigate the underlying mechanisms for this interaction, the present study examined the effects of CRF in vitro on dorsal raphe neurons that displayed electrophysiological and pharmacological properties consistent with a serotonergic phenotype. In the presence of either 1 or 2 mm Ca(2+), perfusion of ovine CRF or rat/human CRF rapidly and reversibly increased firing rates of a subpopulation (19 of 70, 27%) of serotonergic neurons predominantly located in the ventral portion of the dorsal raphe nucleus. For a given responsive neuron, the excitatory effects of CRF were reproducible, and there was no tachyphylaxis. Excitatory effects were dose-dependent (over the range of 0.1-1.6 micrometer) and were completely absent after exposure to the competitive CRF receptor antagonists alpha-helical CRF(9-41) or rat/human [d-Phe(12), Nle(21, 38), alpha-Me-Leu(37)]-CRF(12-41). Both the proportion of responsive neurons and the magnitude of excitatory responses to CRF in the ventral portion of the caudal dorsal raphe nucleus were markedly potentiated in slices prepared from animals previously exposed to isolation and daily restraint stress for 5 d. Immunohistochemical staining of the recorded slices revealed close associations between CRF-immunoreactive varicose axons and tryptophan hydroxylase-immunoreactive neurons in the area of the recordings, providing anatomical evidence for potential direct actions of CRF on serotonergic neurons. The electrophysiological properties and the distribution of responsive neurons within the dorsal raphe nucleus are consistent with the hypothesis that endogenous CRF activates a topographically organized mesolimbocortical serotonergic system.
Two G protein-coupled receptors have been identified that bind corticotropin-releasing factor (CRF) and urocortin (UCN) with high affinity. Hybridization histochemical methods were used to shed light on controversies concerning their localization in rat brain, and to provide normative distributional data in mouse, the standard model for genetic manipulation in mammals. The distribution of CRF-R1 mRNA in mouse was found to be fundamentally similar to that in rat, with expression predominating in the cerebral cortex, sensory relay nuclei, and in the cerebellum and its major afferents. Pronounced species differences in distribution were few, although more subtle variations in the relative strength of R1 expression were seen in several forebrain regions. CRF-R2 mRNA displayed comparable expression in rat and mouse brain, distinct from, and more restricted than that of CRF-R1. Major neuronal sites of CRF-R2 expression included aspects of the olfactory bulb, lateral septal nucleus, bed nucleus of the stria terminalis, ventromedial hypothalamic nucleus, medial and posterior cortical nuclei of the amygdala, ventral hippocampus, mesencephalic raphe nuclei, and novel localizations in the nucleus of the solitary tract and area postrema. Several sites of expression in the limbic forebrain were found to overlap partially with ones of androgen receptor expression. In pituitary, rat and mouse displayed CRF-R1 mRNA signal continuously over the intermediate lobe and over a subset of cells in the anterior lobe, whereas CRF-R2 transcripts were expressed mainly in the posterior lobe. The distinctive expression pattern of CRF-R2 mRNA identifies additional putative central sites of action for CRF and/or UCN. Constitutive expression of CRF-R2 mRNA in the nucleus of the solitary tract, and stress-inducible expression of CRF-R1 transcripts in the paraventricular nucleus may provide a basis for understanding documented effects of CRF-related peptides at a loci shown previously to lack a capacity for CRF-R expression or CRF binding. Other such "mismatches" remain to be reconciled.
The serotonergic dorsal raphé nucleus (DRN) is innervated by corticotropin-releasing factor (CRF)-immunoreactive fibers and contains CRF receptor-binding sites, suggesting that endogenous CRF regulates this system. The present study examined the possibility that CRF in the DRN regulates the release of serotonin (5-HT) in forebrain terminal regions. Intracerebroventricular administration of CRF produced a bimodal effect on extracellular levels of 5-HT in the lateral septum. Doses of 0.3 and 1.0 microg decreased extracellular 5-HT levels, whereas both a higher (3.0 microg) and a lower (0.1 microg) dose had no effect. The reduction of extracellular 5-HT in the lateral septum by CRF (0.3 microg, i.c.v.) was blocked by pretreatment with the CRF receptor antagonist d-PheCRF(12-41) (3.0 microg, i.c.v.). Direct administration of CRF (30 ng) into the DRN reduced extracellular 5-HT levels in the lateral septum and the striatum. Furthermore, injection of d-PheCRF(12-41) (10 ng) into the DRN before ventricular administration of CRF (0.3 microg, i.c.v.) blocked the decrease in extracellular 5-HT in both the lateral septum and striatum. Taken together, these data support the hypothesis that CRF may modulate 5-HT release in terminal regions via its effects at the level of the DRN. This modulation supports a potential interaction between CRF and 5-HT in stress-related psychiatric disorders in which both systems have been implicated.
Anatomical evidence indicates that medial prefrontal cortex (mPFC) neurons project to the dorsal raphe nucleus (DR). In this study, we functionally characterized this descending pathway in rat brain. Projection neurons in the mPFC were identified by antidromic stimulation from the DR. Electrical stimulation of the mPFC mainly inhibited the activity of DR 5-HT neurons (55 of 66). Peristimulus time histograms showed a silence of 150 +/- 9 msec poststimulus (latency, 36 +/- 1 msec). The administration of WAY-100635 and picrotoxinin partly reversed this inhibition, indicating the involvement of 5-HT(1A) and GABA(A) receptors. In rats depleted of 5-HT with p-chlorophenylalanine, the electrical stimulation of mPFC mainly activated 5-HT neurons (31 of 40). The excitations (latency, 17 +/- 1 msec) were antagonized by MK-801 and NBQX. Likewise, MK-801 prevented the rise in DR 5-HT release induced by electrical stimulation of mPFC. The application of 8-OH-DPAT in mPFC significantly inhibited the firing rate of DR 5-HT neurons and, in dual-probe microdialysis experiments, reduced the 5-HT output in mPFC and DR. Furthermore, the application of WAY-100635 in mPFC significantly antagonized the reduction of 5-HT release produced by systemic 8-OH-DPAT administration in both areas. These results indicate the existence of a complex regulation of DR 5-HT neurons by mPFC afferents. The stimulus-induced excitation of some 5-HT neurons by descending excitatory fibers releases 5-HT, which inhibits the same or other DR neurons by acting on 5-HT(1A) autoreceptors. Afferents from the mPFC also inhibit 5-HT neurons through the activation of GABAergic interneurons. Ascending serotonergic pathways may control the activity of this descending pathway by acting on postsynaptic 5-HT(1A) receptors.
The dorsal raphe nucleus (DR)-serotonin (5-HT) system has been implicated in depression and is dramatically affected by swim stress, an animal model with predictive value for antidepressants. Accumulating evidence implicates the stress-related neuropeptide corticotropin-releasing factor (CRF) in the effect of swim stress on this system. This study investigated neural circuits within the DR that are activated by swim stress as revealed by neuronal expression of the immediate early gene, c-fos. Swim stress increased c-fos expression in the dorsolateral subregion of the DR. The majority of c-fos-expressing neurons were doubly labeled for GABA (85 +/- 5%), whereas relatively few were immunolabeled for 5-HT (4 +/- 1%), glutamate (0.5 +/- 0.3%) or calbindin (1.5 +/- 0.3%). Dual immunohistochemical labeling revealed that c-fos-expressing neurons in the dorsolateral DR were enveloped by dense clusters of CRF-immunoreactive fibers and also contained immunolabeling for CRF receptor, suggesting that c-fos-expressing neurons in the DR were specifically targeted by CRF. Consistent with this, the CRF receptor 1 antagonist, antalarmin, prevented swim-stress-elicited c-fos expression in the dorsolateral DR. Together with previous findings that both swim stress and CRF decrease 5-HT release in certain forebrain regions, these results suggest that swim stress engages CRF inputs to GABA neurons in the dorsolateral DR that function to inhibit 5-HT neurons and 5-HT release in the forebrain. This circuitry may underlie some of the acute behavioral responses to swim stress as well as the neuronal plasticity involved in long-term behavioral changes produced by this stress.
A number of clinical investigations and postmortem brain studies have provided evidence that excessive corticotropin-releasing hormone (CRH) secretion and neurotransmission is involved in the pathophysiology of depressive illness, and several studies have suggested that the hyperactivity in CRH neurotransmission extends beyond the hypothalamus involving several extra-hypothalamic brain regions. The present study was designed to test the hypothesis that CRH levels are increased in specific brainstem regions of suicide victims with a diagnosis of major depression. Frozen tissue sections of the pons containing the locus coeruleus and caudal raphe nuclei from 11 matched pairs of depressed suicide and control male subjects were processed for radioimmunocytochemistry using a primary antiserum to CRH and a ([125])I-IgG secondary antibody. The optical density corresponding to the level of CRH-immunoreactivity (IR) was quantified in specific pontine regions from the film autoradiographic images. The level of CRH-IR was increased by 30% in the locus coeruleus, 39% in the median raphe and 45% in the caudal dorsal raphe in the depressed suicide subjects compared to controls. No difference in CRH-IR was found in the dorsal tegmentum or medial parabrachial nucleus between the subject groups. These findings reveal that CRH-IR levels are specifically increased in norepinephrine- and serotonin-containing pontine nuclei of depressed suicide men, and thus they are consistent with the hypothesis that CRH neurotransmission is elevated in extra-hypothalamic brain regions of depressed subjects.
Increasing evidence suggests that postnatal events, such as handling or maternal separation, can produce long-term changes in brain function. These are often expressed as changes in the profile of endocrine or behavioral responses to stress. Changes in gamma-aminobutyric acid type A receptors (GABARs), which mediate the majority of fast synaptic inhibition in adult brain, have been proposed as one potential mediator of these behavioral effects. In the current article, we use a combination of single-cell electrophysiology and antisense mRNA amplification to demonstrate permanent molecular and functional differences in GABARs within hippocampal dentate granule neurons after as few as two episodes of neonatal handling with brief maternal separation. Adult animals that as pups experienced handling with maternal separation maintained a more immature GABAR phenotype and exhibited increased activity in response to swim stress. These findings demonstrate the exquisite sensitivity of the developing GABAergic system to even subtle environmental manipulations and provide an unique molecular mechanism by which postnatal handling with maternal separation may alter stress-related behavior.
The dorsal raphe (DR)-serotonin (5-HT) system has been implicated in stress-related psychiatric disorders. Stress may impact on this system through corticotropin-releasing factor (CRF), which densely innervates the DR. CRF binds to CRF-R1 and CRF-R2 receptors in the DR and has complex and opposing effects depending on the dose used and the endpoint examined. To clarify the impact of CRF on the DR-5-HT system, the effects of selectively activating CRF-R2 receptors (the predominant subtype) on extracellular DR neuronal activity were examined in halothane-anesthetized rats. Because the DR is neurochemically heterogeneous, when possible, neurons were labeled with neurobiotin for subsequent neurochemical classification as 5-HT or non-5-HT. Relatively low doses of urocortin II (UII) (0.1-10 ng) injected into the DR inhibited most (79%; n = 34) neurons, whereas a higher dose (30 ng) inhibited 28% and activated 41% (n = 29). An analysis of effects on neurochemically identified neurons revealed that 5-HT neurons were inhibited by 0.1-10 ng of UII and activated by 30 ng of UII. Activation of 5-HT neurons by 30 ng of UII likely resulted from disinhibition because the majority of non-5-HT neurons were inhibited by this dose. Antisauvagine-30, but not antalarmin, antagonized UII, implicating CRF-R2 receptors in the effects. The results suggest that activation of CRF-R2 on DR-5-HT neurons inhibits neuronal activity, whereas activation of CRF-R2 receptors on non-5-HT neurons may indirectly excite DR-5-HT neurons through disinhibition. Importantly, the tone of the DR-5-HT system can be regulated in a dynamic manner through CRF-R2 activation, being either decreased or increased depending on the level of endogenous or exogenous ligand.
The dorsal raphe nucleus (DR) has a topographic neuroanatomy consistent with the idea that different parts of this nucleus subserve different functions. Here we use dual in situ hybridization to describe the rostral-caudal neurochemical distribution of three major cell groups, serotonin (5-hydroxytryptamine; 5-HT), gamma-aminobutyric acid (GABA), and catecholamine, and their relative colocalization with each other and mRNA encoding four different receptor subtypes that have been described to influence DR responses, namely, 5HT-1A, alpha(1b) adrenergic (alpha(1b) ADR), and corticotropin-releasing factor type 1 (CRF-R1) and 2 (CRF-R2) receptors. Serotonergic and GABAergic neurons were distributed throughout the rostral-caudal extent of the DR, whereas catecholaminergic neurons were generally restricted to the rostral half of the nucleus. These phenotypes essentially represent distinct cell populations, because the neurochemical markers were rarely colocalized. Both 5HT-1A and alpha(1b) ADR mRNA were highly expressed throughout the DR, and the vast majority of serotonergic neurons expressed both receptors. A smaller percentage of GABAergic neurons also expressed 5HT-1A or alpha(1b) ADR mRNA. Very few catecholaminergic cells expressed either 5HT-1A or alpha(1b) ADR mRNA. CRF-R1 mRNA was detected only at very low levels within the DR, and quantitative colocalization studies were not technically feasible. CRF-R2 mRNA was mainly expressed at the middle and caudal levels of the DR. At midlevels, CRF-R2 mRNA was expressed exclusively in serotonin neurons, whereas, at caudal levels, approximately half the CRF-R2 mRNA was expressed in GABAergic neurons. The differential distribution of distinct neurochemical phenotypes lends support to the idea of functional differentiation of the DR.
The dorsal raphe nucleus (DR)-serotonin (5-HT) system has been implicated in depression and is dramatically affected by swim stress, an animal model with predictive value for antidepressants. Accumulating evidence implicates the stress-related neuropeptide corticotropin-releasing factor (CRF) in the effect of swim stress on this system. This study investigated neural circuits within the DR that are activated by swim stress as revealed by neuronal expression of the immediate early gene, c-fos. Swim stress increased c-fos expression in the dorsolateral subregion of the DR. The majority of c-fos-expressing neurons were doubly labeled for GABA (85 +/- 5%), whereas relatively few were immunolabeled for 5-HT (4 +/- 1%), glutamate (0.5 +/- 0.3%) or calbindin (1.5 +/- 0.3%). Dual immunohistochemical labeling revealed that c-fos-expressing neurons in the dorsolateral DR were enveloped by dense clusters of CRF-immunoreactive fibers and also contained immunolabeling for CRF receptor, suggesting that c-fos-expressing neurons in the DR were specifically targeted by CRF. Consistent with this, the CRF receptor 1 antagonist, antalarmin, prevented swim-stress-elicited c-fos expression in the dorsolateral DR. Together with previous findings that both swim stress and CRF decrease 5-HT release in certain forebrain regions, these results suggest that swim stress engages CRF inputs to GABA neurons in the dorsolateral DR that function to inhibit 5-HT neurons and 5-HT release in the forebrain. This circuitry may underlie some of the acute behavioral responses to swim stress as well as the neuronal plasticity involved in long-term behavioral changes produced by this stress.
The dorsal raphe nucleus (DRN) serotonin (5-HT) system has been implicated in acute responses to stress and in stress-related psychiatric disorders such as anxiety and depression. Substantial findings suggest that the neuropeptide corticotropin-releasing factor (CRF) is instrumental in modulating the activity of this system during stress. Because the DRN is neurochemically heterogeneous, dual immunoelectron microscopy was used to examine cellular substrates for interactions between CRF and either 5-HT or gamma-aminobutyric acid (GABA) in the dorsolateral and ventromedial DRN. CRF immunoreactivity was identified primarily within axon terminals, where immunolabeling was particularly enriched in dense-core vesicles. Although CRF terminals targeted 5-HT-containing dendrites in the dorsolateral DRN (16%; n = 251 terminals), synaptic contacts with dendrites that lacked detectable 5-HT immunolabeling were more numerous (48%). In contrast, dual labeling for CRF and GABA (n = 240 terminals) in the dorsolateral DRN revealed that substantially more CRF terminals contacted GABA dendrites (42%) as opposed to unlabeled dendrites (29%). In the ventromedial DRN, contacts between CRF axon terminals and either 5-HT-labeled dendrites or GABA-containing dendrites were fewer than in the dorsolateral DRN. As in the dorsolateral DRN, CRF terminals more frequently contacted GABA dendrites than 5-HT dendrites (30% vs. 8%, respectively). The findings support physiological studies suggesting that CRF has both direct and indirect effects on DRN-5-HT neurons and further implicate GABA as a primary mediator by which CRF and stressors alter the activity of the DRN-5-HT system. (C) 2004 Wiley-Liss, Inc.
A depressed state can be induced in mice by forcing them to swim in a narrow cylinder from which they cannot escape. After a brief period of vigorous activity the mice adopt a characteristic immobile posture which is readily identifiable. Immobility was reduced by tricyclic antidepressants, monoamine oxidase inhibitors and atypical antidepressants, as well as by electroconvulsive shock. Psychostimulants also reduced immobility but in contrast to antidepressants caused marked motor stimulation. Immobility was not affected by minor or major tranquilisers. These findings, closely parallel to those we have previously reported in rats, suggest that the procedure is selectively sensitive to antidepressant treatments. The mouse procedure is, however, more rapid and less costly than that with rats and is thus more suitable for the primary screening of antidepressant drugs.
The serotonergic dorsal raphe nucleus is innervated by corticotropin-releasing factor (CRF) and expresses CRF receptors, suggesting that endogenous CRF impacts on this system. The present study characterized interactions between CRF and the dorsal raphe serotonin (5-HT) system. The effects of intracerebroventricularly (i.c.v.) administered CRF on microdialysate concentrations of 5-HT in the lateral striatum of freely moving rats were determined. CRF had biphasic effects, with 0.1 and 0.3 mu g decreasing and 3.0 mu g increasing 5-HT dialysate concentrations. I.C.V. administration of CRF inhibited neuronal activity of the majority of dorsal raphe neurons at both low (0.3 mu g) and high (3 mu g) doses. Likewise, intraraphe administration of CRF (0.3 and 1.0 ng) had predominantly inhibitory effects on discharge rate. Together, these results suggest that CRF is positioned to regulate the function of the dorsal raphe serotonergic system via actions within the cell body region. This regulation may play a role in stress-related psychiatric disorders in which 5-HT has been implicated. (C) 1998 American College of Neuropsychopharmacology. Published by Elsevier Science Inc.
The differential projections from the dorsal raphe and median raphe nuclei of the midbrain were autoradiographically traced in the rat brain after 3H-proline micro-injections. Six ascending fiber tracts were identified, the dorsal raphe nucleus being the sole source of four tracts and sharing one with the median raphe nucleus. The tracts can be classified as those lying within the medial forebrain bundle (dorsal raphe forebrain tract and the median raphe forebrain tract) and those lying entirely outside (dorsal raphe arcuate tract, dorsal raphe periventricular tract, dorsal raphe cortical tract, and raphe medial tract). The dorsal raphe forebrain tract lies in the ventrolateral aspect of the medial forebrain bundle (MFB) and projects mainly to lateral forebrain areas (e.g., basal ganglion, amygdala, and the pyriform cortex). The median raphe forebrain tract lies in the ventromedial aspect of the MFB and projects to medial forebrain areas (e.g., cingulate cortex, medial septum, and hippocampus). The dorsal raphe cortical tract lies ventrolaterally to the medial longitudinal fasciculus and projects to the caudate-putamen and the parieto-temporal cortex. The dorsal raphe periventricular tract lies immediately below the midbrain aqueduct and projects rostrally to the periventricular region of the thalamus and hypothalamus. The dorsal raphe arcuate tract curves laterally from the dorsal raphe nucleus to reach the ventrolateral edge of the midbrain and projects to ventrolateral geniculate body nuclei and the hypothalamic suprachiasmatic nuclei. Finally, the raphe medial tract receives fibers from both the median and dorsal raphe nuclei and runs ventrally between the fasciculus retroflexus and projects to the interpeduncular nucleus and the midline mammillary body.
Further studies were done to test whether the fiber tracts travelling in the MFB contained 5-HT. Unilateral (left) injections of 5,7-dihydroxytryptamine (5 μgm/400 nl) 18 days before midbrain raphe microinjections of 3H-proline produced a reduction in the grain concentrations in all the ascending fibers within the MFB. Furthermore, pharmacological and behavioural evidence was obtained to show that the 5-HT system had been unilaterally damaged; these animals displayed preferential ipsilateral turning in a rotameter which was strongly reversed to contralateral turning after 5-hydroxytryptophan administration.
The results show that DR and MR nuclei have numerous ascending projections whose axons contain the transmitter 5-HT. The results agree with the neuroanatomical distribution of the 5-HT system previously determined biochemically, histochemically, and neurophysiologically. The midbrain serotonin system seems to be organized by a series of fiber pathways. The fast transport rate in these fibers was found to be about 108 mm/day.
The serotonergic dorsal raphe nucleus is innervated by corticotropin-releasing factor (CRF) and expresses CRF receptors, suggesting that endogenous CRF impacts on this system. The present study characterized interactions between CRF and the dorsal raphe serotonin (5-HT) system. The effects of intracerebroventricularly (i.c.v.) administered CRF on microdialysate concentrations of 5-HT in the lateral striatum of freely moving rats were determined. CRF had biphasic effects, with 0.1 and 0.3 μg decreasing, and 3.0 μg increasing 5-HT dialysate concentrations. I.C.V. administration of CRF inhibited neuronal activity of the majority of dorsal raphe neurons at both low (0.3 μg) and high (3 μg) doses. Likewise, intraraphe administration of CRF (0.3 and 1.0 ng) had predominantly inhibitory effects on discharge rate. Together, these results suggest that CRF is positioned to regulate the function of the dorsal raphe serotonergic system via actions within the cell body region. This regulation may play a role in stress-related psychiatric disorders in which 5-HT has been implicated.
The stress-related neuropeptide corticotropin-releasing factor (CRF) is involved in determining behavioral strategies for responding to stressors, in part through its regulation of the dorsal raphe (DR)-serotonin (5-HT) system. CRF(1) and CRF(2) receptor subtypes have opposing effects on this system that are associated with active versus passive coping strategies, respectively.
Immunoelectron microscopy and in vivo single-unit recordings were used to assess CRF receptor distribution and neuronal responses, respectively, in the DR of stressed and unstressed rats.
Here we show that in unstressed rats CRF(1) and CRF(2) are differentially distributed within DR cells, with CRF(1) being prominent on the plasma membrane and CRF(2) being cytoplasmic. Stress experience reverses this distribution, such that CRF(2) is recruited to the plasma membrane and CRF(1) tends to internalize. As a consequence of this stress-induced cellular redistribution of CRF receptors, neuronal responses to CRF change from inhibition to a CRF(2)-mediated excitation.
Given evidence that CRF(1) and CRF(2) activation are associated with distinct behavioral responses to stress, the stress-triggered reversal in receptor localization provides a cellular mechanism for switching behavioral strategies for coping with stressors.
A MAJOR problem in the search for new antidepressant drugs is the lack
of animal models which both resemble depressive illness and are
selectively sensitive to clinically effective antidepressant treatments.
We have been working on a new behavioural model in the rat which
attempts to meet these two requirements. The method is based on the
observation that a rat, when forced to swim in a situation from which
there is no escape, will, after an initial period of vigorous activity,
eventually cease to move altogether making only those movements
necessary to keep its head above water. We think that this
characteristic and readily identifiable behavioural immobility indicates
a state of despair in which the rat has learned that escape is
impossible and resigns itself to the experimental conditions. This
hypothesis receives support from results presented below which indicate
that immobility is reduced by different treatments known to be
therapeutic in depression including three drugs, iprindole, mianserin
and viloxazine which although clinically active1-3 show
little or no `antidepressant' activity in the usual animal
tests4-6.
A depressed state can be induced in mice by forcing them to swim in a narrow cylinder from which they cannot escape. After a brief period of vigorous activity the mice adopt a characteristic immobile posture which is readily identifiable. Immobility was reduced by tricyclic antidepressants, monoamine oxidase inhibitors and atypical antidepressants, as well as by electroconvulsive shock. Psychostimulants also reduced immobility but in contrast to antidepressants caused marked motor stimulation. Immobility was not affected by minor or major tranquilisers. These findings, closely parallel to those we have previously reported in rats, suggest that the procedure is selectively sensitive to antidepressant treatments. The mouse procedure is, however, more rapid and less costly than that with rats and is thus more suitable for the primary screening of antidepressant drugs.
Retrograde tracer injections of fluorescein- and rhodamine-labelled latex microspheres centered in the parvicellular zone of the hypothalamic paraventricular nucleus and pontine lateral parabrachial nucleus revealed that 36% of the labelled neurons in the dorsal raphe nucleus send collaterals to both structures. These cells were organized in a well-distinguishable cluster within the dorsal raphe nucleus. By combining retrograde tracing with immunocytochemistry, it was found that less than 8% of the double-labelled cells stained positively for serotonin. Of the remaining raphe nuclei that were examined, only the median raphe nucleus contributed a minor nonserotoninergic projection to the paraventricular or lateral parabrachial nuclei. Few of the retrogradely labelled cells in the median raphe nucleus contained both tracers. These results suggest that nonserotoninergic and serotoninergic neurons in the dorsal raphe nucleus, via collateral branching, may simultaneously influence the activity of two central nervous system nuclei involved in autonomic control.
The cerebral cortex of the rat and other mammals is innervated by two morphologically distinct classes of serotoninergic (5-HT) axon terminals: fine axons with minute varicosities and beaded axons characterized by large, spherical varicosities. Fine and beaded 5-HT axons exhibit different regional and laminar distributions in forebrain and arise from separate brainstem nuclei, the dorsal and median raphe nuclei, respectively. Tha present neuroanatomic study, based on immunocytochemical methods to visualize 5-HT axons, demonstrates that the two axon types differ markedly in their vulnerability to the neurotoxic amphetamine derivatives, methylenedioxyamphetamine (MDA), and p-chloroamphetamine (PCA). While both drugs cause extensive degeneration of fine 5-HT axons throughout forebrain, beaded 5-HT axons are consistently spared. Fine 5-HT axons, which richly innervate most regions of dorsal forebrain in control rats, are rarely seen 2 weeks after treatment with MDA or PCA; this loss of fine axons reflects a marked denervation that persists for months after drug administration. The serotoninergic axon terminals remaining after MDA or PCA administration are almost entirely of the beaded type and appear to be unaffected by both drugs. Over a wide range of doses (2.5–40 mg/kg PCA) and survival times (2 weeks to 2 months), these spared 5-HT axons with large, spherical varicosities cannot be distinguished from the normal, beaded 5-HT axons in control rats by morphologic criteria. Moreover, beaded 5-HT axons exhibit a highly characteristic regional distribution which is the same in control as in MDA- and PCA-treated rats: these axons innervate specific zones or layers within parietal and occipital cortex, hippocampus, cingulate cortex, entorhinal cortex, and the olfactory bulb, among other forebrain areas, and they form a dense plexus lining the ventricular system.
Corticotropin-releasing factor (CRF) is the primary factor involved in controlling the release of ACTH from the anterior pituitary and also acts as a neurotransmitter in a variety of brain systems. The actions of CRF are mediated by G-protein coupled membrane bound receptors and a high affinity CRF receptor, CRF1, has been previously cloned and functionally characterized. We have recently isolated a cDNA encoding a second member of the CRF receptor family, designated CRF2, which displays approximately 70% homology at the nucleotide level to the CRF1 receptor and exhibits a distinctive pharmacological profile. The present study utilized in situ hybridization histochemistry to localize the distribution of CRF2 receptor mRNA in rat brain and pituitary gland and compared this with the distribution of CRF1, receptor expression. While CRF1 receptor expression was very high in neocortical, cerebellar, and sensory relay structures, CRF2 receptor expression was generally confined to subcortical structures. The highest levels of CRF2 receptor mRNA in brain were evident within the lateral septal nucleus, the ventromedial hypothalamic nucleus and the choroid plexus. Moderate levels of CRF2 receptor expression were evident in the olfactory bulb, amygdaloid nuclei, the paraventricular and suraoptic nuclei of the hypothalamus, the inferior colliculus and 5-HT-associated raphe nuclei of the midbrain. CRF2-expressing cells were also evident in the bed nucleus of the stria terminalis, the hippocampal formation and anterior and lateral hypothalmic areas. In addition, CRF2 receptor mRNA was also found in cerebral arterioles throughout the brain. Within the pituitary gland, CRF2 receptor mRNA was detectable only at very low levels in scattered cells while CRF1 receptor mRNA was readily detectable in anterior and intermediate lobes. This heterogeneous distribution of CRF1 and CRF2 receptor mRNA suggests distinctive functional roles for each receptor in CRF-related systems. The CRF1 receptor may be regarded as the primary neuroendocrine pituitary CRF receptor and important in cortical, cerebellar and sensory roles of CRF. The anatomical distribution of CRF2 receptor mRNA indicates a role for this novel receptor in hypothalamic neuroendocrine, autonomic and general behavioral actions of central CRF.
The unique distribution of corticotropin-releasing factor (CRF) and its receptors within the central nervous system, its pre-eminent role in mediating the endocrine, behavioural, autonomic and immunological effects of stress and its potent effects after direct administration into the CNS all support the hypothesis that alterations in CRF neuronal systems contribute to the pathophysiology of depression and certain anxiety disorders. This report summarizes a series of preclinical and clinical investigations which have sought to test the hypothesis that CRF-containing neurons show alterations in depression and anxiety, and that drugs used to treat these disorders alter CRF neuronal circuits. Direct injection of CRF into the locus ceruleus or nearby parabrachial nucleus evokes an anxiogenic response. Stress increases CRF concentrations in the locus ceruleus, whereas alprazolam, a benzodiazepine anxiolytic, decreases the concentration of the peptide in the same area. Clinical studies reveal that drug-free depressed patients show: (1) hyperactivity of the hypothalamo-pituitary-adrenal axis; (2) increased CRF concentrations in the cerebrospinal fluid; (3) a blunted release of ACTH in response to CRF; (4) a reduced density of CRF receptors in the frontal cortex; (5) pituitary and adrenal gland hypertrophy. These findings are all concordant with hypersecretion of CRF from hypothalamic and extrahypothalamic CRF neurons in depression.
Recently, local injection of morphine in the dorsal raphe nucleus (DRN) has been shown to increase serotonin release in the forebrain of unanesthetized rats. This study investigated the site of action of opioids in rat brain slices containing the DRN. Postsynaptic currents (PSCs), measured intracellularly under voltage clamp, were induced in serotonergic neurons with bath and microiontophoretic applications of NMDA to activate local neurons. Met-enkephalin (ENK) suppressed spontaneous and NMDA-induced GABAergic inhibitory PSCs. This effect, which was mimicked by the mu agonist DAMGO but not the kappa-agonist U50488 or the delta-agonist DPDPE, was reversed by the mu antagonist CTOP. ENK also suppressed spontaneous and NMDA-induced glutamatergic excitatory PSCs. By searching with focal microiontophoretic NMDA applications, GABAergic and glutamatergic cells projecting on serotonergic neurons were found in the DRN and the adjacent periaqueductal gray. Consistent with the reduction in PSCs, ENK inhibited/hyperpolarized the great majority (81%) of non-serotonergic neurons recorded extra- and intracellularly in the DRN; the ENK effect reversed polarity at -99 +/- 9 mV, close to the potassium reversal potential. In contrast, ENK inhibited/hyperpolarized only 28% of serotonergic neurons; in the affected cells, the ENK effect, blocked by CTOP, had its reversal potential shifted with change of extracellular potassium in agreement with the value predicted by the Nernst equation for a potassium conductance; serotonin occluded the ENK inhibition. Taken together, these results indicate that opioids inhibit both local GABAergic and glutamatergic cells projecting onto DRN serotonergic neurons.
Corticotropin-releasing hormone (CRH) has been implicated in a variety of physiological and behavioral responses to stress, as well as in the pathophysiology of certain psychiatric disorders. Although studies in rodents support a neuromodulatory influence of CRH on monoamine neurotransmission in a number of brain regions, little information in available to support a similar role for CRH in the human brain. The present study used immunocytochemistry to characterize the anatomical organization of CRH-immunoreactive axons in the human brainstem. Substantial regional differences in the density and distribution of CRH-immunoreactive axons were found in the dopamine-, noradrenaline- and serotonin-containing cell body regions of the human brainstem. Dense networks of CRH-immunoreactive axons were found in the medial subnuclei of the ventral mesencephalon and in the dorsolateral region of the locus coeruleus. Moderate densities of CRH-positive fibers were located in the median and dorsal raphe, whereas lower numbers of CRH-labeled axons appeared in the substantia nigra pars compacta. In addition, differences in CRH innervation density were observed within each region. For example, the dorsal tier of the substantia nigra contained a greater density of CRH-labeled axons than the ventral tier. In all monoamine-containing nuclei, CRH-labeled axons exhibited numerous beaded varicosities and fine intervaricose segments. The differential distribution of CRH-containing axons across these human brainstem nuclei suggests that the influence of CRH on monoamine function may be neurotransmitter-specific.
The effects of repeated exposure to forced swimming was examined on extracellular concentrations of 5-hydroxytryptamine (5-HT), behavioral, and endocrine responses in rats. Animals were exposed to a 15-min swimming session on two consecutive days. On the first day, the swimming session increased extracellular 5-HT by 80 % over baseline in the striatum and reduced 5-HT to 40 % below baseline in the lateral septum. On the second day, however, the swimming session produced no effect on 5-HT in either brain region. Ratings of behavior showed that rats demonstrated climbing and swimming behaviors and developed immobility during the initial swimming session and that ratings of immobility increased and swimming decreased during the second swimming session. Immobility was positively correlated and swimming was negatively correlated with changes in extracellular 5-HT in the lateral septum but not in the striatum. Plasma corticosterone was equally elevated by 950 % after either 1 or 2 days of swimming exposure. These results show that there is rapid adaptation to the effects of repeated forced swimming on the regionally-specific, bi-directional response of extracellular 5-HT. In addition, changes in extracellular 5-HT in the lateral septum may be related to the behaviors produced during the forced swimming test that underlie its utility as an animal model of depression.
To further define the operational boundaries on fast inhibition in neocortex, whole cell recordings were made from layer V pyramidal neurons in neocortical slices to evaluate evoked inhibitory postsynaptic currents (IPSCs) and spontaneous miniature IPSCs (mIPSCs). Stimulating electrodes were placed in layers VI and I/II to determine whether simultaneous stimulation of deep and superficial laminae could extend the magnitude of maximal IPSCs evoked by deep-layer stimulation alone. The addition of superficial-layer stimulation did not increase maximal IPSC amplitude, confirming the strict limit on fast inhibition. Spontaneous miniature IPSCs were recorded in the presence of tetrodotoxin. The frequency of spontaneous mIPSCs ranged from 10.0 to 33.1 Hz. mIPSC amplitude varied considerably, with a range of 5. 0-128.2 pA and a mean value of 20.7+/-4.1 pA (n = 12 cells). The decay phase of miniature IPSCs was best fit by a single exponential, similar to evoked IPSCs. The mean time constant of decay was 6.4+/-0.6 ms, with a range of 0.2-20.1 ms. The mean 10-90% rise time was 1.9+/-0.2 ms, ranging from 0.2 to 6.3 ms. Evaluation of mIPSC kinetics revealed no evidence of dendritic filtering. Amplitude histograms of mIPSCs exhibited skewed distributions with several discernable peaks that, when fit with Gaussian curves, appeared to be spaced equidistantly, suggesting that mIPSC amplitudes varied quantally. The mean separation of Gaussian peaks ranged from 6.1 to 7.8 pA. The quantal distributions did not appear to be artifacts of noise. Exposure to saline containing low Ca(2+) and high Mg(2+) concentrations reduced the number of histogram peaks, but did not affect the quantal size. Mean mIPSC amplitude and quantal size varied with cell holding potential in a near-linear manner. Statistical evaluation of amplitude histograms verified the multimodality of mIPSC amplitude distributions and corroborated the equidistant spacing of peaks. Comparison of mIPSC values with published data from single GABA channel recordings suggests that the mean mIPSC conductance corresponds to the activation of 10-20 GABA(A) receptor channels, and that the release of a single inhibitory quantum opens 3-6 channels. Further comparison of mIPSCs with evoked inhibitory events suggests that a single interneuron may form, on average, 4-12 functional synapses with a pyramidal cell, and that 10-12 individual interneurons are engaged during recruitment of maximal population IPSCs. This suggests that inhibitory circuits are much more restricted in both the size of the unit events and effective number of connections when compared with excitatory inputs.
The present study examined the regional localization of corticotropin-releasing factor (CRF)- and 5-hydroxytryptamine (5-HT)-immunoreactive (IR) fibers within the rat dorsal raphe nucleus (DRN) using immunohistochemistry. Additionally, the effects of CRF, administered intracerebroventricularly (0.1-3.0 micrograms) or intraraphe (0.3-30 ng), on discharge rates of putative 5-HT DRN neurons were quantified using in vivo single unit recording in halothane-anesthetized rats. CRF-IR fibers were present at all rostrocaudal levels of the DRN and exhibited a topographical distribution. CRF produced predominantly inhibitory effects on DRN discharge at lower doses and these effects diminished or became excitatory at higher doses. Inhibition of DRN discharge by CRF was attenuated by the nonselective CRF antagonist, DPheCRF12-41 and the CRF-R1-selective antagonist, antalarmin, implicating the CRF-R1 receptor subtype in these electrophysiological effects. The present findings provide anatomical and physiological evidence for an impact of CRF on the DRN-5HT system.
Regulation of serotonin release by gamma-aminobutyric acid (GABA) and glutamate was examined by microdialysis in unanaesthetized rats. The GABA(A) receptor agonist muscimol, or the glutamate receptor agonists kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate or N-methyl-D-aspartate were infused into the dorsal raphe nucleus (DRN) while extracellular serotonin was measured in the DRN and nucleus accumbens. Muscimol produced decreases, and the glutamate receptor agonists produced increases in serotonin. To determine if these receptors have a tonic influence on serotonergic neurons, glutamate or GABA(A) receptor antagonists were infused into the DRN. Kynurenate, a nonselective glutamate receptor blocker, produced a small, 30% decrease in serotonin. A similar decrease was obtained with combined infusion of AP-5 and DNQX into the DRN. The GABAA receptor blocker bicuculline produced an approximately three-fold increase in DRN serotonin. In conclusion, glutamate neurotransmitters have a weak tonic excitatory influence on serotonergic neurons in the rat DRN. However, the predominate influence is mediated by GABA(A) receptors.
The purpose of the present study was to characterize the synaptic currents induced by bath-applied serotonin (5-HT) in 5-HT cells of the dorsal raphe nucleus (DRN) and to determine which 5-HT receptor subtypes mediate these effects. In rat brain slices, 5-HT induced a concentration-dependent increase in the frequency of inhibitory postsynaptic currents (IPSCs) in 5-HT neurons recorded intracellularly in the ventral part of the DRN (EC(50): 86 microM); 5-HT also increased IPSC amplitude. These effects were blocked by the GABA(A) receptor antagonist, bicuculline (10 microM) and by the fast sodium channel blocker, TTX, suggesting that 5-HT had increased impulse flow in local GABAergic neurons. DAMGO (300 nM), a selective mu-agonist, markedly suppressed the increase in IPSC frequency induced by 5-HT (100 microM) in the DRN. A near maximal concentration of the selective 5-HT(2A) antagonist, MDL100,907 (30 nM), produced a large reduction ( approximately 70%) in the increase in IPSC frequency induced by 100 microM 5-HT; SB242,084 (30 nM), a selective 5-HT(2C) antagonist, was less effective ( approximately 24% reduction). Combined drug application suppressed the increase in 5-HT-induced IPSC frequency almost completely, suggesting involvement of both 5-HT(2A) and 5-HT(2C) receptors. Unexpectedly, the phenethylamine hallucinogen, DOI, a partial agonist at 5-HT(2A/2C) receptors, caused a greater increase (+334%) in IPSC frequency than did 5-HT 100 microM (+80%). This result may be explained by an opposing 5-HT(1A) inhibitory effect since the selective 5-HT(1A) antagonist, WAY-100635, enhanced the 5-HT-induced increase in IPSCs. These results indicate that within the DRN-PAG area there may be a negative feedback loop in which 5-HT induces an increase in IPSC frequency in 5-HT cells by exciting GABAergic interneurons in the DRN via 5-HT(2A) and, to a lesser extent, 5-HT(2C) receptors. Increased GABA tone may explain the previous observation of an indirect suppression of firing of a subpopulation of 5-HT cells in the DRN induced by phenethylamine hallucinogens in vivo.
The dorsal raphe nucleus (DR) is innervated by fibers containing the stress-related neurohormone corticotropin-releasing factor (CRF), which alters DR neuronal activity and serotonin release in rats. This study examined the relative distribution of CRF-immunoreactive fibers in the rat DR by using light level densitometry. Additionally, CRF-immunoreactive processes within specific subregions of the DR were examined at the ultrastructural level by using electron microscopy. CRF-immunoreactive fibers were organized within the DR along a caudal-rostral gradient, such that proceeding rostrally, innervation shifted from dorsolateral to ventromedial. Numerous CRF-immunoreactive axon terminals containing dense-core vesicles were found in both the caudal dorsolateral region and the rostral ventromedial/interfascicular region. These formed synaptic specializations with unlabeled dendrites and frequently contacted nonlabeled axon terminals. Semiquantitative analysis revealed certain differences between the two regions with respect to the types of associations made by CRF-immunoreactive terminals. Associations with dendrites were more frequent in the dorsolateral vs. ventromedial region (65% of 171 terminals vs. 39% of 233 terminals, respectively), whereas associations with axon terminals were more frequent in the ventromedial/interfascicular vs. the dorsolateral region (72% of 233 terminals vs. 57% of 171 terminals, respectively). Additionally, synaptic specializations between CRF-immunoreactive terminals and dendrites were more frequently asymmetric in the dorsolateral region (60%) and symmetric (49%) in the ventromedial/interfascicular region. Regional differences in CRF terminal interactions in the DR could account for the reported heterogeneous effects of CRF on DR neuronal activity and forebrain serotonin release. Importantly, the present results provide anatomical substrates for regulation of the DR by endogenous CRF.
Recent electrophysiological studies demonstrate that the ventral medial prefrontal cortex has a powerful inhibitory influence on 5-hydroxytryptamine (5-HT) neurones in the dorsal raphe nucleus. Here we utilised a combination of anatomical and electrophysiological methods to characterise the cellular substrate underlying this effect.Anterograde tracing (Phaseolus vulgaris leucoagglutinin) using electron microscopy demonstrated a pathway from the ventral medial prefrontal cortex that makes neuronal contacts throughout the dorsal raphe nucleus. These contacts were predominantly asymmetrical synapses adjoining GABA immunoreactive dendrites and spines. In vivo extracellular recordings were made in the dorsal raphe nucleus of the anaesthetised rat from a subpopulation of non-5-HT neurones. These neurones were fast-firing, irregular and with short spike width, properties strongly reminiscent of immunochemically identified GABA interneurones in other brain regions. Recordings of classical 5-HT neurones were also included. Electrical stimulation of the ventral medial prefrontal cortex elicited a rapid onset (16 ms latency), orthodromic excitation of the non-5-HT neurones (13/25 neurones). This stimulation also caused a pronounced inhibition of most 5-HT neurones tested, with a longer latency (30 ms), and this was partially blocked by locally applied bicuculline. These data provide the first evidence that the ventral medial prefrontal cortex influences the activity of large numbers of raphe 5-HT neurones by targeting a local network of GABA neurones. This circuitry predicts that physiological and pathological changes in the ventral medial prefrontal cortex will impact on significant parts of the forebrain 5-HT system.
Swim stress decreases extracellular serotonin (5-HT) levels in the rat lateral septum, and adaptation to this effect occurs with repeated swimming. Corticotropin-releasing factor (CRF) administered into the dorsal raphe nucleus (DRN) also decreases 5-HT release in the lateral septum, suggesting that CRF may mediate the effects of swim stress.
The hypothesis that endogenous CRF mediates the reduction of 5-HT levels in the lateral septum evoked by swim stress and is involved in the adaptation that occurs with repeated swim stress was tested.
Extracellular 5-HT levels in rat lateral septum were quantified by means of in vivo microdialysis. Extracellular single unit activity was recorded from the DRN.
Intracerebroventricular (i.c.v.) administration of a CRF receptor antagonist prevented the ability of swim stress to decrease 5-HT release in the lateral septum. Prior exposure to swim stress reduced the ability of both CRF (i.c.v.) and a subsequent swim stress to decrease lateral septum 5-HT release (cross adaptation). Additionally, the effects of CRF, administered into the DRN, on DR neuronal discharge were attenuated in rats with a history of swim stress. Finally, administration of a CRF receptor antagonist (i.c.v.) between two swim stress sessions restored the neurochemical response to swim stress (i.e., 5-HT levels were reduced during the second exposure to swim).
Endogenous CRF modulates 5-HT transmission during acute environmental stress and is also integral to adaptation of the 5-HT response produced by repeated stress. Modulation of the 5-HT system by CRF during acute stress may underlie certain coping behaviors, while stress-induced adaptation of this effect may be involved in psychiatric manifestations of repeated stress.
Serotonergic systems play an important role in the regulation of behavioural, autonomic and endocrine responses to stressful stimuli. This includes modulation of both the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-spinal-adrenal (HSA) axis, which converge at the level of the adrenal cortex to regulate glucocorticoid secretion. Paradoxically, serotonin can either facilitate or inhibit HPA axis activity and stress-related physiological or behavioural responses. A detailed analysis of the brainstem raphé complex and its ascending projections reveals that facilitatory and inhibitory effects of serotonergic systems on glucocorticoid secretion may be due to influences of topographically organized and functionally diverse serotonergic systems. (i) A serotonergic system arising from the middle and caudal dorsal raphé nucleus and projecting to a distributed central autonomic control system and a lateral 'emotional motor system'. Evidence suggests that serotonin can sensitize this subcortical circuit associated with autonomic arousal, anxiety and conditioned fear. (ii) A serotonergic system arising from the median raphé nucleus and projecting extensively and selectively to a ventral subiculum projection system. Evidence suggests that serotonin facilitates this limbic circuit associated with inhibition of ultradian, circadian and stress-induced activity of both the HPA axis and the HSA axis. These new perspectives, based on functional anatomical considerations, provide a hypothetical framework for investigating the role of serotonergic systems in the modulation of ultradian, circadian and stress-induced neuroendocrine function.
The dorsal (DRN) and median raphe nuclei (MRN) are two major sources of serotonergic projections to forebrain that are involved in regulation of behavioral state and motor activity, and implicated in affective disorders such as depression and schizophrenia. To investigate afferent influences on serotonergic neurons, this study compared the role of endogenous GABA and glutamate in the DRN and MRN using microdialysis and measurement of locomotor activity in freely behaving rats. Local infusion of the GABA(A) receptor antagonist bicuculline increased serotonin (5-HT) efflux in the DRN but not the MRN. In contrast, infusion of glutamate receptor antagonists produced larger decreases in 5-HT efflux in the MRN compared with the DRN. Moreover, glutamate receptor antagonists attenuated the increase in 5-HT efflux produced by GABA receptor blockade in the DRN. Thus, the disinhibitory effect of GABA blockers could be ascribed in part to an enhanced influence of glutamate. Measurements of locomotor activity indicate that changes in 5-HT were not simply correlated with behavioral activity induced by drug infusion. In summary, the role of inhibitory and excitatory afferents was strikingly different in the DRN and MRN. GABA afferents were the predominant tonic influence on serotonergic neurons in the DRN. In contrast, glutamatergic but not GABAergic afferents had a strong tonic influence on serotonergic neurons in the MRN.
The dorsal (DR) and median raphe (MR) nuclei contain 5-hydroxytryptamine (serotonin, 5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain limbic areas that control emotional behavior. In the past, the electrophysiological identification of neurochemically identified 5-HT neurons has been limited. Recent technical developments have made it possible to re-examine the electrophysiological characteristics of identified 5-HT- and non-5-HT-containing neurons. Visualized whole cell electrophysiological techniques in combination with fluorescence immunohistochemistry for 5-HT were used. In the DR, both 5-HT- and non-5-HT-containing neurons exhibited similar characteristics that have historically been attributed to putative 5-HT neurons. In contrast, in the MR, the 5-HT-and non-5-HT-containing neurons had very different characteristics. Interestingly, the MR 5-HT-containing neurons had a shorter time constant and larger afterhyperpolarization (AHP) amplitude than DR 5-HT-containing neurons. The 5-HT(1A) receptor-mediated response was also measured. The efficacy of the response elicited by 5-HT(1A) receptor activation was greater in 5-HT-containing neurons in the DR than the MR, whereas the potency was similar, implicating greater autoinhibition in the DR. Non-5-HT-containing neurons in the DR were responsive to 5-HT(1A) receptor activation, whereas the non-5-HT-containing neurons in the MR were not. These differences in the cellular characteristics and 5-HT(1A) receptor-mediated responses between the MR and DR neurons may be extremely important in understanding the role of these two 5-HT circuits in normal physiological processes and in the etiology and treatment of pathophysiological states.
Appropriate regulatory control of the hypothalamo-pituitary-adrenocortical stress axis is essential to health and survival. The following review documents the principle extrinsic and intrinsic mechanisms responsible for regulating stress-responsive CRH neurons of the hypothalamic paraventricular nucleus, which summate excitatory and inhibitory inputs into a net secretory signal at the pituitary gland. Regions that directly innervate these neurons are primed to relay sensory information, including visceral afferents, nociceptors and circumventricular organs, thereby promoting 'reactive' corticosteroid responses to emergent homeostatic challenges. Indirect inputs from the limbic-associated structures are capable of activating these same cells in the absence of frank physiological challenges; such 'anticipatory' signals regulate glucocorticoid release under conditions in which physical challenges may be predicted, either by innate programs or conditioned stimuli. Importantly, 'anticipatory' circuits are integrated with neural pathways subserving 'reactive' responses at multiple levels. The resultant hierarchical organization of stress-responsive neurocircuitries is capable of comparing information from multiple limbic sources with internally generated and peripherally sensed information, thereby tuning the relative activity of the adrenal cortex. Imbalances among these limbic pathways and homeostatic sensors are likely to underlie hypothalamo-pituitary-adrenocortical dysfunction associated with numerous disease processes.
Since corticotropin-releasing factor (CRF) was first characterized, a growing family of ligands and receptors has evolved. The mammalian family members include CRF, urocortinI (UcnI), UcnII, and UcnIII, along with two receptors, CRFR1 and CRFR2, and a CRF binding protein. These family members differ in their tissue distribution and pharmacology. Studies have provided evidence supporting an important role of this family in regulation of the endocrine and behavioral responses to stress. Although CRF appears to play a stimulatory role in stress responsivity through activation of CRFR1, specific actions of UcnII and UcnIII on CRFR2 may be important for dampening stress sensitivity. As the only ligand with high affinity for both receptors, UcnI's role may be promiscuous. Regulation of the relative contribution of the two CRF receptors to brain CRF pathways may be essential in coordinating physiological responses to stress. The development of disorders related to heightened stress sensitivity and dysregulation of stress-coping mechanisms appears to involve regulatory mechanisms of CRF family members.