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.

The serotonin (5-hydroxytryptamine; 5-HT) system has a well-characterized role in depression. Recent reports describe comorbidities of mood-immune disorders, suggesting an immunological component may contribute to the pathogenesis of depression as well. Chemokines, immune proteins which mediate leukocyte trafficking, and their receptors are widely distributed in the brain, mediate neuronal patterning, and modulate various neuropathologies. The purpose of this study was to investigate the neuroanatomical relationship and functional impact of the chemokine stromal cell-derived factor-1alpha/CXCL12 and its receptor, CXCR4, on the serotonin dorsal raphe nucleus (DRN) system in the rat using anatomical and electrophysiological techniques. Immunohistochemical analysis indicates that over 70% of 5-HT neurons colocalize with CXCL12 and CXCR4. At a subcellular level, CXCL12 localizes throughout the cytoplasm whereas CXCR4 concentrates to the outer membrane and processes of 5-HT neurons. CXCL12 and CXCR4 also colocalize on individual DRN cells. Furthermore, electrophysiological studies demonstrate CXCL12 depolarization of 5-HT neurons indirectly via glutamate synaptic inputs. CXCL12 also enhances the frequency of spontaneous inhibitory and excitatory postsynaptic currents (sIPSC and sEPSC). CXCL12 concentration-dependently increases evoked IPSC amplitude and decreases evoked IPSC paired-pulse ratio selectively in 5-HT neurons, effects blocked by the CXCR4 antagonist AMD3100. These data indicate presynaptic enhancement of GABA and glutamate release at 5-HT DRN neurons by CXCL12. Immunohistochemical analysis further shows CXCR4 localization to DRN GABA neurons, providing an anatomical basis for CXCL12 effects on GABA release. Thus, CXCL12 indirectly modulates 5-HT neurotransmission via GABA and glutamate synaptic afferents. Future therapies targeting CXCL12 and other chemokines may treat serotonin related mood disorders, particularly depression experienced by immune-compromised individuals.

The dorsal (DR) and median (MR) raphe nuclei contain 5-hydroxytryptamine (5-HT) cell bodies that give rise to the majority of the ascending 5-HT projections to the forebrain. The DR and MR have differential roles in mediating stress, anxiety and depression. Glutamate and GABA activity sculpt putative 5-HT neuronal firing and 5-HT release in a seemingly differential manner in the MR and DR, yet isolated glutamate and GABA activity within the DR and MR has not been systematically characterized. Visualized whole-cell voltage-clamp techniques were used to record excitatory and inhibitory postsynaptic currents (EPSC and IPSC) in 5-HT-containing neurons. There was a regional variation in action potential-dependent (spontaneous) and basal [miniature (m)] glutamate and GABAergic activity. mEPSC activity was greater than mIPSC activity in the DR, whereas in the MR the mIPSC activity was greater. These differences in EPSC and IPSC frequency indicate that glutamatergic and GABAergic input have distinct cytoarchitectures in the DR and MR. 5-HT(1B) receptor activation decreased mEPSC frequency in the DR and the MR, but selectively inhibited mIPSC activity only in the MR. This finding, in concert with its previously described function as an autoreceptor, suggests that 5-HT(1B) receptors influence the ascending 5-HT system through multiple mechanisms. The disparity in organization and integration of glutamatergic and GABAergic input to DR and MR neurons and their regulation by 5-HT(1B) receptors may contribute to the distinction in MR and DR regulation of forebrain regions and their differential function in the aetiology and pharmacological treatment of psychiatric disease states.

Drug craving triggered by cues that were once associated with drug intoxication is a major contributor to continued drug-seeking behaviors. Addictive drugs engage molecular pathways of associative learning and memory. Reactivated memories are vulnerable to disruption by interference with the process of reconsolidation, hence targeting reconsolidation could be a strategy to reduce cue-induced drug craving and relapse. Here we examined the circuitry of cocaine contextual memory reconsolidation and explored neuroplasticity following memory reactivation. Mice underwent chemogenetic inhibition of either nucleus accumbens (NA) neurons or the glutamatergic projection neurons from the ventral hippocampus (vHPC) to NA using inhibitory designer receptors exclusively activated by designer drugs (iDREADD). Mice underwent cocaine conditioned place preference followed by reactivation of the cocaine contextual memory. Clozapine-N-oxide (CNO) was administered after memory reactivation to inhibit either NA neurons or the accumbens-projecting vHPC neurons during the reconsolidation period. When retested 3 days later, a significant reduction in the previously established preference for the cocaine context was found in both conditions. FosTRAP2-Ai14 mice were used to identify neurons activated by cocaine memory recall and to evaluate plasticity in NA medium spiny neurons (MSNs) and vHPC pyramidal neurons upon recall of cocaine memories. Results indicate a significant increase in dendritic spine density in NA MSNs activated by cocaine memory recall, particularly of the thin spine type. Sholl analysis indicated longer dendritic length and more branching of NA MSNs after cocaine memory recall than without memory reactivation. vHPC neurons showed increased spine density, with the most robust change in stubby spines. These results implicate a circuit involving glutamatergic projections from the vHPC onto NA neurons which is necessary for the reconsolidation of cocaine memories. Interruption of cocaine memory reconsolidation reduced drug-seeking behavior.

The serotonin system is intimately linked to both the mediation of anxiety and long-term effects of cocaine, potentially through interaction of inhibitory 5-HT2C receptor and gamma-aminobutyric acid (GABA) networks. This study characterized the function of the dorsal raphe (DR) 5-HT2C receptor and GABA network in anxiety produced by chronic cocaine withdrawal. C57BL/6 mice were injected with saline or cocaine (15 mg/kg) 3 times daily for 10 days, and tested on the elevated plus maze 30 min, 25 h, or 7 days after the last injection. Cocaine-withdrawn mice showed heightened anxiety-like behavior at 25 h of withdrawal, as compared to saline controls. Anxiety-like behavior was not different when mice were tested 30 min or 7 days after the last cocaine injection. Electrophysiology data revealed that serotonin cells from cocaine-withdrawn mice exhibited increased GABA inhibitory postsynaptic currents (IPSCs) in specific DR subregions dependent on withdrawal time (25 h or 7 d), an effect that was absent in cells from non-withdrawn mice (30 min after the last cocaine injection). Increased IPSC activity was restored to baseline levels following bath application of the 5-HT2C receptor antagonist, SB 242084. In a separate cohort of cocaine-injected mice at 25 h of withdrawal, both global and intra-DR blockade of 5-HT2C receptors prior to elevated plus maze testing attenuated anxiety-like behavior. This study demonstrates that DR 5-HT2C receptor blockade prevents anxiety-like behavior produced by cocaine withdrawal, potentially through attenuation of heightened GABA activity, supporting a role for the 5-HT2C receptor in mediating anxiety produced by cocaine withdrawal.

Within glomeruli, the initial sites of synaptic integration in the olfactory pathway, olfactory sensory axons terminate on dendrites of projection and juxtaglomerular (JG) neurons. JG cells form at least two major circuits: the classic intraglomerular circuit consisting of external tufted (ET) and periglomerular (PG) cells and an interglomerular circuit comprised of the long-range connections of short axon (SA) cells. We examined the projections and the synaptic inputs of identified JG cell chemotypes using mice expressing green fluorescent protein (GFP) driven by the promoter for glutamic acid decarboxylase (GAD) 65 kDa, 67 kDa, or tyrosine hydroxylase (TH). Virtually all (97%) TH+ cells are also GAD67+ and are thus DAergic-GABAergic neurons. Using a combination of retrograde tracing, whole-cell patch-clamp recording, and single-cell three-dimensional reconstruction, we show that different JG cell chemotypes contribute to distinct microcircuits within or between glomeruli. GAD65+ GABAergic PG cells ramify principally within one glomerulus and participate in uniglomerular circuits. DAergic-GABAergic cells have extensive interglomerular projections. DAergic-GABAergic SA cells comprise two subgroups. One subpopulation contacts 5-12 glomeruli and is referred to as "oligoglomerular." Approximately one-third of these oligoglomerular DAergic SA cells receive direct olfactory nerve (ON) synaptic input, and the remaining two-thirds receive input via a disynaptic ON-->ET-->SA circuit. The second population of DAergic-GABAergic SA cells also disynaptic ON input and connect tens to hundreds of glomeruli in an extensive "polyglomerular" network. Although DAergic JG cells have traditionally been considered PG cells, their interglomerular connections argue that they are more appropriately classified as SA cells.

Opioids have immunomodulatory functions and may alter susceptibility to immune disorders. Behavioral studies also indicate that chemokines, molecules expressed by immune cells, block opioid-induced analgesia in the periaqueductal grey (PAG). Bi-directional heterologous desensitization of opioid and chemokine receptors has been described in cell systems. We report the anatomical and functional interactions of chemokine receptors with the mu-opioid receptor (MOR) in the rat brain. The chemokine receptors, CXCR4 and CX3CR1, as well as their chemokine substrates, CXCL12 and CX3CL1, are widely expressed in the central nervous system (CNS). Immunohistochemical techniques were utilized to investigate MOR-CXCR4 and MOR-CX3CR1 receptor colocalization in multiple brain areas. Our results demonstrate co-expression of these receptors on individual neurons in several regions including cingulate cortex, hippocampus, and PAG, suggesting functional receptor interactions. Whole-cell patch-clamp recordings of PAG neurons in a rat brain slice preparation were used to examine morphine or chemokine (CXCL12, CX3CL1) effects alone, or in combination on neuronal membrane properties. Morphine (10 μM) hyperpolarized and reduced input resistance of PAG neurons. CXCL12 and CX3CL1 (10 nM) had no impact on either parameter. In the presence of CXCL12, morphine's electrophysiological effects were blocked in all neurons examined, whereas with CX3CL1, morphine's effects were blocked in 57% of neurons studied. The data provide electrophysiological evidence for MOR-CXCR4 and MOR-CX3CR1 heterologous desensitization in the PAG at the single-cell level. These interactions may contribute to the limited utility of opioid analgesics for inflammatory pain treatment and supports chemokines as neuromodulators.

Swim stress regulates forebrain 5-hydroxytryptamine (5-HT) release in a complex manner and its effects are initiated in the serotonergic dorsal raphe nucleus (DRN). The purpose of this study was to examine the effects of swim stress on the physiology of DRN neurons in conjunction with 5-HT immunohistochemistry. Basic membrane properties, 5-HT(1A) and 5-HT(1B) receptor-mediated responses and glutamatergic excitatory postsynaptic currents (EPSCs) were measured using whole-cell patch clamp techniques. Rats were forced to swim for 15min and 24h later DRN brain slices were prepared for electrophysiology. Swim stress altered the resting membrane potential, input resistance and action potential duration of DRN neurons in a neurochemical-specific manner. Swim stress selectively elevated glutamate EPSC frequency in 5-HT DRN neurons. Swim stress non-selectively reduced EPSC amplitude in all DRN cells. Swim stress elevated the 5-HT(1B) receptor-mediated inhibition of glutamatergic synaptic activity that selectively targeted 5-HT cells. Non-5-HT DRN neurons appeared to be particularly responsive to the effects of a milder handling stress. Handling elevated EPSC frequency, reduced EPSC decay time and enhanced a 5-HT(1B) receptor-mediated inhibition of mEPSC frequency selectively in non-5-HT DRN cells. These results indicate that swim stress has both direct, i.e., changes in membrane characteristics, and indirect effects, i.e., via glutamatergic afferents, on DRN neurons. These results also indicate that there are distinct local glutamatergic afferents to neurochemically specific populations of DRN neurons, and furthermore that these distinct afferents are differentially regulated by swim stress. These cellular changes may contribute to the complex effects of swim stress on 5-HT neurotransmission and/or the behavioral changes underlying the forced swimming test model of depression.

Alcoholism afflicts 1 in 13 US adults, and comorbidity with depression is common. Levels of serotonin (5-HT) metabolites in alcoholic or depressed humans and rat strains are lower compared to healthy counterparts. Rats bred for ethanol (EtOH) preference are common in EtOH studies, however out-bred strains better model the range of EtOH consumption in humans. We examined voluntary EtOH consumption in out-bred Sprague-Dawley (SD) rats placed in the 20% EtOH intermittent access drinking paradigm (IA). Acquisition of 20% EtOH consumption (g EtOH/kg/24h) was assessed during the first 6-8 weeks of IA. Rats naturally separated into two groups (Drinkers or Non-drinkers) based on EtOH intake above or below 0.5 g/kg/24h prior to treatment intervention. We examined the effect of central 5-HT depletion on EtOH consumption by infusing 5,7-dihyroxytryptamine (5,7-DHT; i.c.v., 200-300 μg) or vehicle and measured EtOH consumption for 4 weeks post-operatively in IA. Compared to baseline, there was no effect of vehicle or 5,7-DHT on EtOH consumption during the post-operative period. Quantification of 5-HT depletion in the dorsal raphe nucleus (DRN) using tryptophan hydroxylase-2 (TPH2) immunohistochemistry resulted in a 76% decrease in staining with 5,7-DHT treatment. Interestingly, preservation of the ventromedial (VM) sub-regions was evident in all animals treated with 5,7-DHT, regardless of drinking behavior. In addition, Drinkers treated with 5,7-DHT had significantly more TPH2 depletion in the DRN compared to Non-drinkers. Our findings indicate that out-bred SD rats exhibit a natural EtOH consumption behavior (Drinker or Non-drinker) that is stable across time and independent of 5-HT depletion in the CNS. In addition, rats that regularly consumed >0.5 g EtOH/kg had greater sensitivity to 5,7-DHT in the DRN, indicating an interaction between EtOH and sensitivity of DRN 5-HT cells to neurotoxic substances. This may contribute to the dysfunctionality of the 5-HT system in alcoholic humans and lead to a better understanding of current pharmacological treatments for this addiction.

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.