Summary 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.

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.

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 increase anxiety. KeywordsStress; Amygdala; Raphe; Fear; Patch Clamp; 5-HT Anxiety disorders affect more than 40 million Americans annually (DuPont et al., 1996), and changes in serotonin (5-HT) functioning have been linked to both their etiology and treatment. Although there is substantial evidence that changes in 5-HT functioning can modulate fear and anxiety-like states in humans and animals alike, the literature is unclear regarding the valence

Ehrlich DE, Ryan SJ, Hazra R, Guo JD, Rainnie DG. Postnatal maturation of GABAergic transmission in the rat basolateral amygdala.

BACKGROUND STEP is a brain-specific protein tyrosine phosphatase that opposes the development of synaptic strengthening and the consolidation of fear memories. In contrast, stress facilitates fear memory formation, potentially by activating corticotrophin releasing factor (CRF) neurons in the anterolateral cell group of the bed nucleus of the stria terminalis (BNSTALG). METHODS Here, using dual-immunofluorescence, single-cell RT-PCR, quantitative RT-PCR, Western blot, and whole cell patch-clamp electrophysiology, we examined the expression and role of STEP in regulating synaptic plasticity in rat BNSTALG neurons, and its modulation by stress. RESULTS STEP was selectively expressed in CRF neurons in the oval nucleus of the BNSTALG. Following repeated restraint stress (RRS), animals displayed a significant increase in anxiety-like behavior, which was associated with a down-regulation of STEP mRNA and protein expression in the BNSTALG as well as selectively enhanced magnitude of long-term potentiation (LTP) induced in Type III, putative CRF neurons. To determine if the changes in STEP expression following RRS were mechanistically related to the facilitation of synaptic strengthening, we examined the effects of intracellular application of STEP on the induction of LTP. STEP completely blocked the RRS-induced facilitation of LTP in BNSTALG neurons. CONCLUSIONS Hence, STEP acts to buffer CRF neurons against excessive activation, while down-regulation of STEP after chronic stress may result in pathological activation of CRF neurons in the BNSTALG and contribute to prolonged states of anxiety. Thus, targeted manipulations of STEP activity might represent a novel treatment strategy for stress-induced anxiety disorders.

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.central amygdala | plasticity | HPA axis | short term synaptic depression | knockout T he neuropeptide corticotrophin-releasing factor (CRF) is involved in initiation of the endocrine, autonomic, and behavioral responses to stressors and its dysfunction is implicated in a variety of mood and anxiety disorders (1). Large populations of CRF-containing neurons are located within brain areas crucial for fear and anxiety, including the central amygdala (CeA), bed nucleus of the stria terminalis (BNST), and paraventricular nucleus of the hypothalamus (PVN). Although site-specific and conditional knockout approaches are increasingly being used (2, 3), the broad expression of CRF receptors and the varied cell types in which the CRF peptide is produced (4, 5) make it challenging to identify region and cell-type specific influences of CRF. One way to manipulate specific subpopulations of CRF neurons is to use mice with the Cre recombinase gene driven by the CRF promoter to allow different subgroups of CRFergic cells to be manipulated.The interaction between CRF and GABA activity in brain structures important for fear and anxiety has been identified as a potential mechanism underlying anxiety disorders (6). CRF administration increases GABA release within the amygdala (7, 8) and causes deficits in GABA(A) receptor-mediated inhibitory transmission (9). Lifelong CRF overexpression leads to changes in GABA receptor subtype expression and sensitivity (1...

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 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2010 December 29. Hence, modulation of 5-HT 7 receptor activity in the BNST ALG may offer a novel avenue for the design of anxiolytic medications.

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.