Acute engagement of Gq-mediated signaling in the bed nucleus of the stria terminalis induces anxiety-like behavior.

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of Gq-mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that Gq-mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple Gq-coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the Gq-coupled receptor 5-HT2CR in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

Pituitary adenylate cyclase-activating polypeptide (PACAP) in the bed nucleus of the stria terminalis (BNST) increases corticosterone in male and female rats.

Single nucleotide polymorphisms (SNP) in the genes for pituitary adenylate cyclase-activating polypeptide (PACAP) and the PAC1 receptor have been associated with several psychiatric disorders whose etiology has been associated with stressor exposure and/or dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. In rats, exposure to repeated variate stress has been shown to increase PACAP and its cognate PAC1 receptor expression in the bed nucleus of the stria terminalis (BNST), a brain region implicated in anxiety and depression-related behaviors as well as the regulation of HPA axis activity. We have argued that changes in BNST PACAP signaling may mediate the changes in emotional behavior and dysregulation of the HPA axis associated with anxiety and mood disorders. The current set of studies was designed to determine whether BNST PACAP infusion leads to activation of the HPA axis as determined by increases in plasma corticosterone. We observed an increase in plasma corticosterone levels 30min following BNST PACAP38 infusion in male and female rats, which was independent of estradiol (E2) treatment in females, and we found that plasma corticosterone levels were increased at both 30min and 60min, but returned to baseline levels 4h following the highest dose. PACAP38 infusion into the lateral ventricles immediately above the BNST did not alter plasma corticosterone level, and the increased plasma corticosterone following BNST PACAP was not blocked by BNST corticotropin releasing hormone (CRH) receptor antagonism. These results support others suggesting that BNST PACAP plays a key role in regulating stress responses.

Bi-directional modulation of bed nucleus of stria terminalis neurons by 5-HT: molecular expression and functional properties of excitatory 5-HT receptor subtypes.

Activation of neurons in the anterolateral bed nucleus of the stria terminalis (BNST(ALG)) plays an important role in mediating the behavioral response to stressful and anxiogenic stimuli. Application of 5-HT elicits complex postsynaptic responses in BNST(ALG) neurons, which includes (1) membrane hyperpolarization (5-HT(Hyp)), (2) hyperpolarization followed by depolarization (5-HT(Hyp-Dep)), (3) depolarization (5-HT(Dep)) or (4) no response (5-HT(NR)). We have shown that the inhibitory response is mediated by activation of postsynaptic 5-HT(1A) receptors. Here, we used a combination of in vitro whole-cell patch-clamp recording and single cell reverse transcriptase polymerase chain reaction (RT-PCR) to determine the pharmacological properties and molecular profile of 5-HT receptor subtypes mediating the excitatory response to 5-HT in BNST(ALG) neurons. We show that the depolarizing component of both the 5-HT(Hyp/Dep) and the 5-HT(Dep) response was mediated by activation of 5-HT(2A), 5-HT(2C) and/or 5-HT(7) receptors. Single cell RT-PCR data revealed that 5-HT(7) receptors (46%) and 5-HT(1A) receptors (41%) are the most prevalent receptor subtypes expressed in BNST(ALG) neurons. Moreover, 5-HT receptor subtypes are differentially expressed in type I-III BNST(ALG) neurons. Hence, 5-HT(2C) receptors are almost exclusively expressed by type III neurons, whereas 5-HT(7) receptors are expressed by type I and II neurons, but not type III neurons. Conversely, 5-HT(2A) receptors are found predominantly in type II neurons. Finally, bi-directional modulation of individual neurons occurs only in type I and II neurons. Significantly the distribution of 5-HT receptor subtypes in BNST(ALG) neurons predicted the observed expression pattern of 5-HT responses determined pharmacologically. Together, these results suggest that 5-HT can differentially modulate the excitability of type I-III neurons, and further suggest that bi-directional modulation of BNST(ALG) neurons occurs primarily through an interplay between 5-HT(1A) and 5-HT(7) receptors. Hence, modulation of 5-HT(7) receptor activity in the BNST(ALG) may offer a novel avenue for the design of anxiolytic medications.

Identification of cell-type-specific promoters within the brain using lentiviral vectors.

The development of cell-type-specific mini-promoters for genetic studies is complicated by a number of issues. Here, we describe a general method for the relatively rapid screening of specific promoter activity in cell culture, in acute brain slice preparations and in vivo. Specifically, we examine the activity of an approximately 3 kb promoter region from the neuroactive peptide cholecystokinin (CCK) compared to the commonly used cytomegalovirus promoter. We find a high degree of cell-type selectivity in vivo using lentiviral approaches in rats and traditional transgenic approaches in mice. Appropriate colocalization of Cre-recombinase and CCK gene expression is found within the hippocampus, when the CCK promoter is driving either the expression of Cre-recombinase or green fluorescent protein. We also demonstrate fluorescent identification of CCK-positive interneurons that allows for cell-type-specific electrophysiologic studies in rats and mice. In conclusion, these studies identify a functional mini-promoter for the CCK gene and outline a novel and sensitive general method to test activity of selective promoters in vitro and in vivo. This approach may allow for the more rapid identification of specific promoters for use with transgenic animals, in genetically modified viruses, and in the design of targeted, therapeutic gene-delivery systems.

5-hydroxytryptamine1A-like receptor activation in the bed nucleus of the stria terminalis: electrophysiological and behavioral studies.

The anteriorlateral bed nucleus of the stria terminalis (BNST AL) and the serotonergic system are believed to modulate behavioral responses to stressful and/or anxiogenic stimuli. However, although the BNST AL receives heavy serotonergic innervation, the functional significance of this input is not known. Data obtained from in vitro whole-cell patch clamp recording in the rat BNST slice show that exogenous application of 5-hydroxytryptamine (5-HT) evoked a heterogeneous response in BNST AL neurons. The principal action of 5-HT in this region was inhibitory, evoking a membrane hyperpolarization (5-HTHyp) and a concomitant reduction in input resistance in the majority of neurons tested. The broad-spectrum 5-HT1 agonist, 5-carboxamindotryptamine (5-CT), but not R(+/-)8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT), mimicked the 5-HTHyp response in the BNST. Moreover, the outward current mediating 5-HTHyp was inwardly rectifying and sensitive to the G protein activated inwardly rectifying K+ (G IRK) channel blocker, tertiapin-Q. In the CNS 5-HT1A receptors are thought to couple to GIRK channels, suggesting that 5-HTHyp in BNST AL neurons was mediated by activation of 5-HT1A-like receptors. This was confirmed by the blockade of both 5-HTHyp and 5-CTHyp by the specific 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt (WAY100635 200nM). Furthermore, an in vivo examination of the functional consequences of 5-HT1A-like induced inhibition of BNST neurons revealed that infusion of 5-CT into the BNST significantly reduced the acoustic startle response, without affecting the general motor activity of the animals. These data point to the possibility that 5-HT1A mediated inhibition of the BNST AL could contribute to an anxiolytic action. Hence, we propose that in response to stressful stimuli, enhanced levels of 5-HT in the BNST AL plays a critical homeostatic role in feedback inhibition of the anxiogenic response to these stimuli.

Inescapable shock induces resistance to the effects of dexamethasone.

Administration of bacterial endotoxin (lipopolysachharide; LPS) elevates proinflammatory cytokines, such as interleukin-1beta (IL-1beta) and IL-6, and activates the hypothalamic-pituitary-adrenal (HPA) axis. Corticosterone (CORT), the glucocorticoid (GC) effector hormone of the HPA axis in rats, inhibits both proinflammatory cytokine production/release and activity of the HPA axis itself. Exposure to chronic or repeated stressors often induces resistance to the effects of GCs. The following experiments were conducted to test the hypothesis that an acute stressor, inescapable tailshock (IS), alters responsivity of the HPA axis and proinflammatory cytokine system to dexamethasone (DEX), a synthetic GC. First, we examined the ability of various doses of DEX to suppress proinflammatory cytokine and HPA activity in response to LPS challenge 24 h after either home cage (HCC) or IS treatment. Upon finding resistance to DEX in IS animals, we examined the duration of the altered response to DEX by testing animals 1, 4 and 21 days after IS. To test whether IS animals were selectively resistant to the suppressive effects of DEX on the response to LPS, the ability of DEX to suppress HPA activity in response to a non-inflammatory stressor, exposure to an elevated "pedestal", was assessed. Again, DEX resistance was observed in IS animals. Finally, we examined whether changes in the responsivity to DEX were dependent upon the controllability of the stressor. The induction of DEX resistance was independent of the degree of behavioral control that the animal had over the stressor. Thus, a single session of IS induces DEX resistance of both HPA axis and cytokine responses measured in vivo.

Blockade of alpha1 adrenoreceptors in the dorsal raphe nucleus prevents enhanced conditioned fear and impaired escape performance following uncontrollable stressor exposure in rats.

Previous research has shown that the effect of exposure to uncontrollable stressors on conditioned fear responding and escape behavior in rats is dependent on serotonergic neural activity in the dorsal raphe nucleus (DRN). The role that norepinephrine released in the DRN plays in producing the behavioral consequences of exposure to inescapable tail shock in rats was investigated in the present study. The selective alpha1 adrenoreceptor antagonist benoxathian was injected into the DRN before exposure to inescapable tail shock or before behavioral testing conducted 24 h later. Benoxathian prevented the impairment of escape responding produced by inescapable shock, but did not reverse this effect when given before testing. The enhancement of conditioned fear produced by prior inescapable shock was attenuated by benoxathian administered before inescapable shock or before behavioral testing. These results support the view that noradrenergic input to the DRN is necessary to produce the behavioral effects of inescapable tail shock.

Inescapable shock-induced potentiation of morphine analgesia in rats: sites of action.

Inescapable shock (IS) enhances analgesia to systemic morphine (MOR) 24 hr later. IS activates serotonin neurons in the dorsal raphe nucleus (DRN), rendering them hyperexcitable. These studies tested whether IS potentiates the analgesic effect of MOR microinjected in the DRN, as predicted by this hypothesis. To test site specificity, the effect of previous IS was examined on MOR microinjected lateral to the DRN and into 2 other sites that support MOR analgesia, the nucleus raphe magnus (NRM) and spinal cord. Twenty-four hours after IS, potentiated analgesia was observed after 0.5 microg MOR microinjected into, but not lateral to, the DRN. Potentiated analgesia was also observed after NRM (1.0 microg) and spinal cord (3.0 microg) MOR microinjections. These data suggest that IS-induced excitability changes within the DRN synergize with opiates microinjected in other analgesia areas and that this potentiates the responses to opiates 24 hr after IS.

Activation of serotonin-immunoreactive cells in the dorsal raphe nucleus in rats exposed to an uncontrollable stressor.

The dorsal raphe nucleus (DRN) and its serotonergic terminal regions have been suggested to be part of the neural substrate by which exposure to uncontrollable stressors produces poor escape responding and enhanced conditioned fear expression. Such stressor exposure is thought to selectively activate DRN serotonergic neurons in such a way as to render them transiently sensitized to further input. As a result of this sensitized state, behavioral testing procedures are thought to cause excess serotonergic activity in brain regions that control these behaviors. The present studies were conducted to investigate activity in the DRN following exposure to escapable and yoked, inescapable tailshock. Neural activity was characterized using immunohistochemistry to detect the immediate early gene product Fos in serotonin-immunoreactive cells in the DRN. Inescapable tailshock led to greater serotonergic neural activity than did escapable tailshock, supporting the hypothesis that uncontrollable stressors preferentially activate serotonergic neurons in the DRN.

Interleukin-1 induces c-Fos immunoreactivity in primary afferent neurons of the vagus nerve.

Peripheral administration of bacterial endotoxin, an immune stimulant, induces evidence of activation in vagal primary afferent neurons. To determine whether interleukin-1beta (IL-1beta) is part of the molecular pathway leading to this activation, we assessed the expression of the neuronal activation marker c-Fos in vagal primary afferent neurons after intraperitoneal injections of IL-1beta (2 microg/kg). IL-1beta, but not vehicle, induced c-Fos expression, demonstrating that IL-1beta is likely an important signal from the immune system to the vagus nerve, and thus the brain.

Amiloride inhibits taste nerve responses to NaCl and KCl in Sprague-Dawley and Fischer 344 rats.

In a two-bottle test, Sprague-Dawley rats preferentially consume a greater amount of hypotonic and isotonic NaCl solutions relative to water, whereas inbred Fischer 344 (F344) rats fail to prefer NaCl solutions at any concentration relative to water. To determine whether taste contributes to this strain difference, we measured the integrated neural responses of the chorda tympani nerve to a concentration range of NaCl and KCl solutions. The amiloride-sensitive component of the taste nerve response was assessed by adding amiloride during salt stimulation in Experiment 1, and by pretreating the taste receptors with amiloride prior to salt stimulation in Experiment 2. Adding amiloride to NaCl during sustained neural activity suppressed chorda tympani nerve responses more than pretreating the tongue with amiloride. Adding amiloride during salt stimulation also partially suppressed chorda tympani neuron responses to KCl, a presumed control stimulus. The neural responses of the chorda tympani nerve to NaCl and KCl were similar for salt-avoiding F344 and salt-preferring Sprague-Dawley rats. However, amiloride pretreatment suppressed the taste nerve responses to NaCl significantly less in F344 rats than in Sprague-Dawley rats. The strain difference in the amiloride-sensitive component of the taste response may contribute to the difference in NaCl preference.

Calfhood weights, body measurements and measures of fatness versus criteria of overall size and shape for predicting yearling performance in beef cattle.

Data were collected over a period of 4 yr from bulls and heifers of two location-breed groups (East Tennessee-Polled Hereford, Cumberland Plateau-Angus). Variables measured and recorded at the time all calves were weaned (at an average age of approximately 230 d) were age (in d at time of data collection; AGE), weight (WT), visual condition score (COND), ultrasonically measured subcutaneous fat thickness (FAT), body length (LT), heart girth (HG) and hip width (HIP). These variables were included in principal-components (PC) analyses. The first principal component (PC1) was a measure of overall size, accounting for 66 to 69% of the total variation. Remaining principal components contrasted body shapes. Stepwise multiple regression analyses (separate for each location-sex subgroup) were conducted using various combinations of the weaning variables listed above, or principal components of these variables, to predict birth to yearling average daily gain (BYADG), weaning to yearling average daily gain (WYADG) and yearling condition score (YCOND). Condition score (COND) appeared to be the most valuable complement to AGE and WT, with LT somewhat useful also. In equations including principal components, after AGE, PC1 most frequently entered first, generally followed by PC2. Coefficients of multiple determination (R2) were largest for BYADG (greater than .50), followed by YCOND (.15 to .50) and WYADG (less than .15). Longer-bodied, thinner-fleshed animals of a given age and weight tended to have larger gains and smaller YCOND.