Ji-Dong Guo

Neuroanatomical evidence for reciprocal regulation of the corticotrophin-releasing factor and oxytocin systems in the hypothalamus and the bed nucleus of the stria terminalis of the rat: Implications for balancing stress and affect.

Activation of corticotrophin releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus (PVN) is necessary for establishing the classic endocrine response to stress, while activation of forebrain CRF neurons mediates affective components of the stress response. Previous studies have reported that mRNA for CRF2 receptor (CRFR2) is expressed in the bed nucleus of the stria terminalis (BNST) as well as hypothalamic nuclei, but little is known about the localization and cellular distribution of CRFR2 in these regions. Using immunofluorescence with confocal microscopy, as well as electron microscopy, we demonstrate that in the BNST CRFR2-immunoreactive fibers represent moderate to strong labeling on axons terminals. Dual-immunofluorescence demonstrated that CRFR2-fibers co-localize oxytocin (OT), but not arginine-vasopressin (AVP), and make perisomatic contacts with CRF neurons. Dual-immunofluorescence and single cell RT-PCR demonstrate that in the hypothalamus, CRFR2 immunoreactivity and mRNA are found in OT, but not in CRF or AVP-neurons. Furthermore, CRF neurons of the PVN and BNST express mRNA for the oxytocin receptor, while the majority of OT/CRFR2 neurons in the hypothalamus do not. Finally, using adenoviral-based anterograde tracing of PVN neurons, we show that OT/CRFR2-immunoreactive fibers observed in the BNST originate in the PVN. Our results strongly suggest that CRFR2 located on oxytocinergic neurons and axon terminals might regulate the release of this neuropeptide and hence might be a crucial part of potential feedback loop between the hypothalamic oxytocin system and the forebrain CRF system that could significantly impact affective and social behaviors, in particular during times of stress.

The response of neurons in the bed nucleus of the stria terminalis to serotonin: Implications for anxiety

Substantial evidence has suggested that the activity of the bed nucleus of the stria terminalis (BNST) mediates many forms of anxiety-like behavior in human and non-human animals. These data have led many investigators to suggest that abnormal processing within this nucleus may underlie anxiety disorders in humans, and effective anxiety treatments may restore normal BNST functioning. Currently some of the most effective treatments for anxiety disorders are drugs that modulate serotonin (5-HT) systems, and several decades of research have suggested that the activation of 5-HT can modulate anxiety-like behavior. Despite these facts, relatively few studies have examined how activity within the BNST is modulated by 5-HT. Here we review our own investigations using in vitro whole-cell patch-clamp electrophysiological methods on brain sections containing the BNST to determine the response of BNST neurons to exogenous 5-HT application. Our data suggest that the response of BNST neurons to 5-HT is complex, displaying both inhibitory and excitatory components, which are mediated by 5-HT(1A), 5-HT(2A), 5-HT(2C) and 5-HT(7) receptors. Moreover, we have shown that the selective activation of the inhibitory response to 5-HT reduces anxiety-like behavior, and we describe data suggesting that the activation of the excitatory response to 5-HT may be anxiogenic. We propose that in the normal state, the function of 5-HT is to dampen activity within the BNST (and consequent anxiety-like behavior) during exposure to threatening stimuli; however, we suggest that changes in the balance of the function of BNST 5-HT receptor subtypes could alter the response of BNST neurons to favor excitation and produce a pathological state of increased anxiety.

Group II metabotropic glutamate receptors in anxiety circuitry: correspondence of physiological response and subcellular distribution.

Activation of group II metabotropic glutamate receptors (mGluR2/3) in the amygdala plays a critical role in the regulation of fear and anxiety states. Previous studies using nonselective agonists have suggested this action can result from activation of either pre‐ or postsynaptic mGluR2/3. Here, we have used a combination of whole‐cell patch clamp recording with highly selective agonists (LY354740 and LY379268) and immunoelectron microscopy to examine structure‐function relationships for mGluR2/3 in the basolateral amygdala (BLA) and bed nucleus of the stria terminalis (BNST). Stimulation of mGluR2/3 evoked a direct, TTX‐insensitive membrane hyperpolarization in all BLA projection neurons tested, but only about half of BNST neurons. The membrane hyperpolarization was mediated by activation of an outward potassium current or blockade of a tonically active inward Ih current in different groups of BLA neurons. In both regions, mGluR2/3 caused a long‐lasting reduction of glutamate release from presynaptic afferent terminals even at concentrations that failed to elicit a direct postsynaptic response. The localization of mGluR2/3 differed regionally, with postsynaptic labeling significantly more common in BLA than BNST, corresponding to the strength of postsynaptic responses recorded there. Our results demonstrate a complex role for mGluR2/3 receptors in modulating anxiety circuitry, including direct inhibition and reduction of excitatory drive. The combination of direct inhibition of projection neurons within the BLA and suppression of excitatory neurotransmission in the BNST may be responsible for the anxiolytic actions of group II mGluR agonists. J. Comp. Neurol. 505:682–700, 2007.

Central CRF neurons are not created equal: phenotypic differences in CRF-containing neurons of the rat paraventricular hypothalamus and the bed nucleus of the stria terminalis

Corticotrophin-releasing factor (CRF) plays a key role in initiating many of the endocrine, autonomic, and behavioral responses to stress. CRF-containing neurons of the paraventricular nucleus of the hypothalamus (PVN) are classically involved in regulating endocrine function through activation of the stress axis. However, CRF is also thought to play a critical role in mediating anxiety-like responses to environmental stressors, and dysfunction of the CRF system in extra-hypothalamic brain regions, like the bed nucleus of stria terminalis (BNST), has been linked to the etiology of many psychiatric disorders including anxiety and depression. Thus, although CRF neurons of the PVN and BNST share a common neuropeptide phenotype, they may represent two functionally diverse neuronal populations. Here, we employed dual-immunofluorescence, single-cell RT-PCR, and electrophysiological techniques to further examine this question and report that CRF neurons of the PVN and BNST are fundamentally different such that PVN CRF neurons are glutamatergic, whereas BNST CRF neurons are GABAergic. Moreover, these two neuronal populations can be further distinguished based on their electrophysiological properties, their co-expression of peptide neurotransmitters such as oxytocin and arginine-vasopressin, and their cognate receptors. Our results suggest that CRF neurons in the PVN and the BNST would not only differ in their response to local neurotransmitter release, but also in their action on downstream target structures.

Cell-type specific deletion of GABA(A)α1 in corticotropin-releasing factor-containing neurons enhances anxiety and disrupts fear extinction

Corticotropin-releasing factor (CRF) is critical for the endocrine, autonomic, and behavioral responses to stressors, and it has been shown to modulate fear and anxiety. The CRF receptor is widely expressed across a variety of cell types, impeding progress toward understanding the contribution of specific CRF-containing neurons to fear dysregulation. We used a unique CRF-Cre driver transgenic mouse line to remove floxed GABA(A)α1 subunits specifically from CRF neurons [CRF-GABA(A)α1 KO]. This process resulted in mice with decreased GABA(A)α1 expression only in CRF neurons and increased CRF mRNA within the amygdala, bed nucleus of the stria terminalis (BNST) and paraventricular nucleus of the hypothalamus. These mice show normal locomotor and pain responses and no difference in depressive-like behavior or Pavlovian fear conditioning. However, CRF-GABA(A)α1 KO increased anxiety-like behavior and impaired extinction of conditioned fear, coincident with an increase in plasma corticosterone concentration. These behavioral impairments were rescued with systemic or BNST infusion of the CRF antagonist R121919. Infusion of Zolpidem, a GABA(A)α1-preferring benzodiazepine-site agonist, into the BNST of the CRF-GABA(A)α1 KO was ineffective at decreasing anxiety. Electrophysiological findings suggest a disruption in inhibitory current may play a role in these changes. These data indicate that disturbance of CRF containing GABA(A)α1 neurons causes increased anxiety and impaired fear extinction, both of which are symptoms diagnostic for anxiety disorders, such as posttraumatic stress disorder.

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.

Presynaptic 5-HT1B receptor-mediated serotonergic inhibition of glutamate transmission in the bed nucleus of the stria terminalis

Activation of neurons in the bed nucleus of the stria terminalis (BNST) plays a critical role in stress and anxiety-related behaviors. Previously, we have shown that serotonin (5-HT) can directly modulate BNST neuronal excitability by an action at postsynaptic receptors. In this study we built upon that work to examine the effects of 5-HT on excitatory neurotransmission in an in vitro rat BNST slice preparation. Bath application of 5-HT reversibly reduced the amplitude of evoked excitatory postsynaptic currents (eEPSCs). These effects were mimicked by the 5-HT(1B/D) receptor agonist, sumatriptan, and by the 5-HT(1B) receptor selective agonist, CP93129. Conversely, the effects of 5-HT and sumatriptan could be blocked by the 5-HT(1B) receptor-selective antagonist, GR55562. In contrast, the 5-HT(1A) receptor agonist 8-OH DPAT or antagonist WAY 100635 could not mimic or block the effect of 5-HT on eEPSCs. Together, these data suggest that the 5-HT-induced attenuation of eEPSCs was mediated by 5-HT(1B) receptor activation. Moreover, sumatriptan had no effect on the amplitude of the postsynaptic current elicited by pressure applied AMPA, suggesting a possible presynaptic locus for the 5-HT(1B) receptor. Furthermore, 5-HT, sumatriptan and CP93129 all increased the paired pulse ratio of eEPSCs while they concomitantly decreased the amplitude of eEPSCs, suggesting that these agonists act to reduce glutamate release probability at presynaptic locus. Consistent with this observation, sumatriptan decreased the frequency of miniature EPSCs, but had no effect on their amplitude. Taken together, these results suggest that 5-HT suppresses glutamatergic neurotransmission in the BNST by activating presynaptic 5-HT(1B) receptors to decrease glutamate release from presynaptic terminals. This study illustrates a new pathway by which the activity of BNST neurons can be indirectly modulated by 5-HT, and suggests a potential new target for the development of novel treatments for depression and anxiety disorders.

Oxytocin in the nucleus accumbens shell reverses CRFR2-evoked passive stress-coping after partner loss in monogamous male prairie voles

Loss of a partner can have severe effects on mental health. Here we explore the neural mechanisms underlying increased passive stress-coping, indicative of depressive-like behavior, following the loss of the female partner in the monogamous male prairie vole. We demonstrate that corticotropin-releasing factor receptor 2 (CRFR2) in the nucleus accumbens shell mediates social loss-induced passive coping. Further, we show that partner loss compromises the oxytocin system through multiple mechanisms. Finally, we provide evidence for an interaction of the CRFR2 and oxytocin systems in mediating the emotional consequences of partner loss. Our results suggest that chronic activation of CRFR2 and suppression of striatal oxytocin signaling following partner loss result in an aversive emotional state that may share underlying mechanisms with bereavement. We propose that the suppression of oxytocin signaling is likely adaptive during short separations to encourage reunion with the partner and may have evolved to maintain long-term partnerships. Additionally, therapeutic strategies targeting these systems should be considered for treatment of social loss-mediated depression.

Postnatal maturation of GABAergic transmission in the rat basolateral amygdala.

Many psychiatric disorders, including anxiety and autism spectrum disorders, have early ages of onset and high incidence in juveniles. To better treat and prevent these disorders, it is important to first understand normal development of brain circuits that process emotion. Healthy and maladaptive emotional processing involve the basolateral amygdala (BLA), dysfunction of which has been implicated in numerous psychiatric disorders. Normal function of the adult BLA relies on a fine balance of glutamatergic excitation and GABAergic inhibition. Elsewhere in the brain GABAergic transmission changes throughout development, but little is known about the maturation of GABAergic transmission in the BLA. Here we used whole cell patch-clamp recording and single-cell RT-PCR to study GABAergic transmission in rat BLA principal neurons at postnatal day (P)7, P14, P21, P28, and P35. GABAA currents exhibited a significant twofold reduction in rise time and nearly 25% reduction in decay time constant between P7 and P28. This corresponded with a shift in expression of GABAA receptor subunit mRNA from the α2- to the α1-subunit. The reversal potential for GABAA receptors transitioned from depolarizing to hyperpolarizing with age, from around -55 mV at P7 to -70 mV by P21. There was a corresponding shift in expression of opposing chloride pumps that influence the reversal, from NKCC1 to KCC2. Finally, we observed short-term depression of GABAA postsynaptic currents in immature neurons that was significantly and gradually abolished by P28. These findings reveal that in the developing BLA GABAergic transmission is highly dynamic, reaching maturity at the end of the first postnatal month.

A transcriptomic analysis of type I-III neurons in the bed nucleus of the stria terminalis

The activity of neurons in the anterolateral cell group of the bed nucleus of the stria terminalis (BNST(ALG)) plays a critical role in anxiety- and stress-related behaviors. Histochemical studies have suggested that multiple distinct neuronal phenotypes exist in the BNST(ALG). Consistent with this observation, the physiological properties of BNST(ALG) neurons are also heterogeneous, and three distinct cell types can be defined (Types I-III) based primarily on their expression of four key membrane currents, namely I(h), I(A), I(T), and I(K(IR)). Significantly, all four channels are multimeric proteins and can comprise of more than one pore-forming α subunit. Hence, differential expression of α subunits may further diversify the neuronal population. However, nothing is known about the relative expression of these ion channel α subunits in BNST(ALG) neurons. We have addressed this lacuna by combining whole-cell patch-clamp recording together with single-cell reverse transcriptase polymerase chain reaction (scRT-PCR) to assess the mRNA transcript expression for each of the subunits for the four key ion channels in Type I-III neurons of the BNST(ALG.) Here, cytosolic mRNA from single neurons was probed for the expression of transcripts for each of the α subunits of I(h) (HCN1-HCN4), I(T) (Ca(v)3.1-Ca(v)3.3), I(A) (K(v)1.4, K(v)3.4, K(v)4.1-K(v) 4.3) and I(K(IR)) (Kir2.1-Kir2.4). An unbiased hierarchical cluster analysis followed by discriminant function analysis revealed that a positive correlation exists between the physiological and genetic phenotype of BNST(ALG) neurons. Thus, the analysis segregated BNST(ALG) neurons into 3 distinct groups, based on their α subunit mRNA expression profile, which positively correlated with our existing electrophysiological classification (Types I-III). Furthermore, analysis of mRNA transcript expression in Type I-Type III neurons suggested that, whereas Type I and III neurons appear to represent genetically homologous cell populations, Type II neurons may be further subdivided into three genetically distinct subgroups. These data not only validate our original classification scheme, but further refine the classification at the molecular level, and thus identifies novel targets for potential disruption and/or pharmacotherapeutic intervention in stress-related anxiety-like behaviors.