A Basal Forebrain Site Coordinates the Modulation of Endocrine and Behavioral Stress Responses via Divergent Neural Pathways

Abstract

The bed nuclei of the stria terminalis (BST) are critically important for integrating stress-related signals between the limbic forebrain and hypothalamo-pituitary-adrenal (HPA) effector neurons in the paraventricular hypothalamus (PVH). Nevertheless, the circuitry underlying BST control over the stress axis and its role in depression-related behaviors has remained obscure. Utilizing optogenetic approaches in rats, we have identified a novel role for the anteroventral subdivision of BST in the coordinated inhibition of both HPA output and passive coping behaviors during acute inescapable (tail suspension, TS) stress. Follow-up experiments probed axonal pathways emanating from the anteroventral BST which accounted for separable endocrine and behavioral functions subserved by this cell group. The PVH and ventrolateral periaqueductal gray were recipients of GABAergic outputs from the anteroventral BST that were necessary to restrain stress-induced HPA activation and passive coping behavior, respectively, during TS and forced swim tests. In contrast to other BST subdivisions implicated in anxiety-like responses, these results direct attention to the anteroventral BST as a nodal point in a stress-modulatory network for coordinating neuroendocrine and behavioral coping responses, wherein impairment could account for core features of stress-related mood disorders.

Significance statement: Dysregulation of the neural pathways modulating stress-adaptive behaviors is implicated in stress-related psychiatric illness. While aversive situations activate a network of limbic forebrain regions thought to mediate such changes, little is known about how this information is integrated to orchestrate complex stress responses. Here we identify novel roles for the anteroventral bed nuclei of the stria terminalis in inhibiting both stress hormone output and passive coping behavior via divergent projections to regions of the hypothalamus and midbrain. Inhibition of these projections produced features observed with rodent models of depression, namely stress hormone hypersecretion and increased passive coping behavior, suggesting that dysfunction in these networks may contribute to expression of pathological changes in stress-related disorders.

Keywords: HPA; bed nuclei of the stria terminalis; behavioral coping; corticosterone; paraventricular hypothalamus; periaqueductal gray area.

Figure 1.

Endocrine and behavioral consequences of avBST somata photoinhibition. a–e, Dark-field image (left) of a coronal section showing YFP-fluorescent neuron soma after AAV microinjection into avBST, with a schematic coronal section (b) illustrating fiber optic placement for the bilateral photoinhibition of neuronal somata therein. Example photomicrographs showing Fos immunoperoxidase labeling in PVH from unstressed YFP control (c), as well as YFP (d) and Arch (e) rats subjected to TS, illustrating a marked increase in Fos immunoreactivity after avBST somata photoinhibition. Scale bar, 200 μm (a, c–e). f, Quantification of Fos-immunoreactive nuclei reveals a significant induction as a result of stress exposure (*p < 0.05) and further enhancement associated with photoinhibition of avBST neuron axons (†p < 0.05). n = 3 YFP + control, n = 7 YFP + stress, n = 6 Arch + stress. g, Analysis of ACTH levels before and after 10 min TS coincident with 561 nm illumination (light green shaded area) of avBST somata. h, Plasma levels of CORT were significantly elevated in Arch animals 30 min after the onset of stress versus YFP controls (*p < 0.05). Arch stimulation resulted in a prolonged elevation of plasma titers of ACTH and CORT (at 30 min) after stress onset compared with YFP control animals (*p < 0.05). i, Photoinhibition of avBST somata was associated with a significant increase in immobility behavior during TS. n = 6 YFP, n = 6 Arch (f–i).

Figure 2.

Neurophysiological confirmation of Arch-mediated photoinhibition. a, Epifluorescent image (left) displaying the microelectrode path (dashed line) along which neurophysiological activity was recorded ventral to the anterior commissure (ac) to confirm suppression of neuronal activity with 561 nm light, with schematic diagram (right) illustrating optrode placement during the recording session. Scale bar, 200 μm. b–d, Example of activity over 15 min recording sessions in the absence (gray raster plot; b) and presence (green raster plot; c) of constant 561 nm illumination in a putative Arch-expressing avBST unit, with summary histogram (d) illustrating suppression of neuronal firing (gray line, no laser session; green line, 561 nm laser session). e–g, In a YFP-microinjected animal, neuronal firing was unchanged between the unilluminated (e) and 561 nm illuminated periods (f), which are summarized in a histogram (g). Green bars indicate continuous laser illumination over 50 ms blocks.

Figure 3.

Changes in Fos induction after unilateral avBST somata photoexcitation. a, Sagittal diagram depicting bilateral AAV-ChR2 microinjection into, and fiber-optic placement above, avBST for unilateral avBST somata photoexcitation. b, Coronal view). c, Dark-field image illustrating fiber-optic (blue outline) placement ventral to the anterior commissure (ac) and immediately dorsal to ChR2:YFP-expressing neurons in avBST. d, Brightfield photomicrograph showing Fos immunoperoxidase staining in PVH contralateral and ipsilateral (contra and ipsi, respectively) to the photoexcited side of avBST after TS. Scale bar, 200 μm (c, d). e, Counts of Fos-positive nuclei revealed significant increases within the photoexcited side of avBST in both stressed and unstressed control animals compared with the contralateral side of unstressed animals (*p < 0.05). In stressed animals, illumination was associated with further increases in Fos induction relative to the contralateral side (†p < 0.05). f, Whereas stressed animals displayed overall increases in Fos induction in PVH relative to controls (*p < 0.05), the side of PVH ipsilateral to avBST photoexcitation showed abrogated responses relative to the contralateral side (†p < 0.05). n = 3 control, n = 5 stress (e, f).

Figure 4.

Endocrine and behavioral consequences of bilateral avBST photoexcitation. Analysis of ACTH levels before (a) and after 10 min of TS concurrent with bilateral photoexcitation (light blue shaded area) of avBST somata failed to reveal any significant differences for any individual time point, whereas CORT levels (b) were significantly reduced in ChR2 animals immediately after TS (at 10 min) and at 90 min after its onset (*p < 0.05). i, Immobility behavior during TS did not differ between ChR2 and YFP groups (p = 0.4). n = 7 per group (a–c).

Figure 5.

Neurophysiological confirmation of ChR2-mediated photoexcitation. a–c, Example of neurophysiological activity in the absence (a; gray raster plot, 15 min recording session) and presence (b; blue raster plot, 15 min recording session) of 20 Hz pulsed 473 nm light in a putative ChR2-expressing avBST unit with summary histogram (c) illustrating light-evoked neuronal activity. d–f, In a YFP-microinjected animal, firing rates unchanged between the unilluminated (d) and 473 nm illuminated (e) recording periods, which are summarized in a histogram (f). Blue bars indicate 5 ms laser pulse.

Figure 6.

Phenotypic characterization of avBST neurons and terminal fields in PVH. a, b, Dark-field photomicrograph showing in situ hybridization of GAD67 (a) and VGLUT (b) mRNA in avBST and its vicinity. c–f, Confocal laser-scanning microscopic image displaying the distribution of YFP-fluorescent terminals (green) that originated from avBST (c) and corresponding images in the same z-section showing GAD-65 (d, red) and CRF (e, cyan) immunoreactivity (f, composite). Scale bar, 400 μm (a, b); 20 μm (c–f).

Figure 7.

Selective consequences of avBST-to-PVH pathway photoinhibition on HPA output. a, b, Sagittal schematic diagram depicting AAV-Arch microinjection into avBST (a) and bilateral fiber optic probe implantation dorsal to avBST terminal fields in PVH (right, coronal view; b). c, Dark-field photomicrograph depicting fiber-optic termination (green outline) above YFP-fluorescent avBST terminals in the medial parvicellular (mp) and posterior magnocellular (pm) PVH. d, e, Photomicrographs showing immunoperoxidase labeling of Fos in PVH from a YFP rat subjected to TS and the marked increase in immunoreactivity after avBST terminal photoinhibition. Scale bar, 200 μm (c–e). f, Quantification of Fos-immunoreactive nuclei reveals a significant induction as a result of stress exposure (*p < 0.05) and further enhancement after photoinhibition of avBST neuron axons (†p < 0.05). n = 2 YFP + control, n = 5 YFP + stress, n = 5 Arch + stress. g, ACTH levels in YFP and Arch groups before and after 10 min TS coincident and followed by 561 nm illumination of avBST terminals in PVH, with significant elevations observed 30, 60, and 90 min after stress onset (*p < 0.05). h, CORT levels from the same groups in e showing significant elevations at 30 and 60 min in the Arch group versus YFP controls (*p < 0.05). i, Immobility during TS was not significantly different between groups (p = 0.2). n = 9 YFP, n = 7 Arch (f–i).

Figure 8.

Selective behavioral consequences after avBST-to-vlPAG pathway photoinhibition. a, Schematic diagram depicting AAV-Arch microinjection into avBST and fiber-optic probe placement above its terminal fields in the ventrolateral periaqueductal gray (vlPAG). b, Dark-field photomicrograph showing fiber-optic probe (green outline) placement above a YFP-fluorescent terminal plexus originating from avBST. ca, Cerebral aqueduct. c–e, Digital reconstructions of YFP-fluorescent axonal and terminal fields in vlPAG (green, c) and colocalization with GAD-65 immunofluorescent terminals (red, d; overlap in e). Scale bar, 200 μm (b); 40 μm (c–e). f–h, Bilateral photoinhibition of avBST neuron axons in vlPAG during TS (f) led to a marked increase in immobility behavior (*p < 0.05), whereas radioimmunoassay of ACTH (g) and CORT levels (h) in YFP and Arch animals failed to reveal any significant differences in HPA activation. n = 5 YFP, n = 6 Arch (f–h).

Figure 9.

Consequences of avBST-to-vlPAG photoinhibition generalize to FS behavior, but not locomotor activity. a, Behaviorally naive animals bearing AAV-YFP or AAV-Arch microinjections in avBST and fiber-optic placements above vlPAG were subjected to the FS test and 561 nm illumination of avBST terminals in vlPAG, revealing a significant increase in immobility in Arch animals relative to YFP rats (*p < 0.05). b, c, Assessment of locomotor activity for 10 min concurrent with 561 nm illumination revealed no differences between groups in either mean velocity (b) or total distance traveled (c) (p = 0.88 and 0.97, respectively). n = 6 per group (a–c).

Figure 10.

Summary diagram highlighting the role of avBST in the coordination of behavioral and neuroendocrine responses to stress. The data support the pathways highlighted in red, with avBST providing inhibitory control over (1) HPA effector neurons in PVH and (2) passive coping circuits centered within the vlPAG.