A cholecystokinin-mediated pathway to the paraventricular thalamus is recruited in chronically stressed rats and regulates hypothalamic-pituitary-adrenal function

Abstract

Chronic stress alters hypothalamic-pituitary-adrenal (HPA) responses to acute, novel stress. After acute restraint, the posterior division of the paraventricular thalamic nucleus (pPVTh) exhibits increased numbers of Fos-expressing neurons in chronically cold-stressed rats compared with stress-naive controls. Furthermore, lesions of the PVTh augment HPA activity in response to novel restraint only in previously stressed rats, suggesting that the PVTh is inhibitory to HPA activity but that inhibition occurs only in chronically stressed rats. In this study, we further examined pPVTh functions in chronically stressed rats. We identified afferent projections to the pPVTh using injection of the retrograde tracer fluorogold. Of the sites containing fluorogold-labeled cells, neurons in the lateral parabrachial, periaqueductal gray, and dorsal raphe containing fluorogold also expressed cholecystokinin (CCK) mRNA. We then examined whether these CCKergic inputs to the pPVTh were involved in HPA responses to acute, novel restraint after chronic stress. We injected the CCK-B receptor antagonist PD 135,158 into the PVTh before restraint in control and chronically cold-stressed rats. ACTH responses to restraint stress were augmented by PD 135,158 only in chronically stressed rats but not in controls. In addition, CCK-B receptor mRNA expression in the pPVTh was not altered by chronic cold stress. We conclude that previous chronic stress specifically facilitates the release of CCK into the pPVTh in response to acute, novel stress. The CCK is probably secreted from neurons in the lateral parabrachial, the periaqueductal gray, and/or the dorsal raphe nuclei. Acting via CCK-B receptors in pPVTh, CCK then constrains facilitated ACTH responses to novel stress in chronically stressed but not naive rats. These results demonstrate clearly that chronic stress recruits a new set of pathways that modulate HPA responsiveness to a novel stress.

Fig. 1.

A representative injection of fluorogold in the posterior PVTh [−3.30 mm from bregma based on Paxinos and Watson (1986) in a] and visualized immunocytochemically inb (see Materials and Methods). In the experiments presented here, fluorogold injections were limited to the middle/posterior divisions of the PVTh.

Fig. 2.

Fluorogold-labeled cells in the dorsal (a, b; approximately −9.3 mm from bregma) and central (c, d; approximately −8.72 mm from bregma) divisions of the lateral parabrachial and in the periaqueductal gray (e, f; approximately −7.64 mm from bregma) after fluorogold injection into the pPVTh. Pictures on theright (b, d, f) are higher magnifications of the boxedareas shown on the left (a, c, e). Coordinates are based on the atlas of Paxinos and Watson (1986). scp, Superior cerebellar peduncle; 4v, fourth ventricle.

Fig. 3.

Fluorogold-labeled cells in the caudal (approximately −9.16 mm from bregma) levels of the dorsal raphe after fluorogold injection into the pPVTh. b is a higher magnification of a. Coordinates are based on the atlas of Paxinos and Watson (1986). mlf, Medial longitudinal fasciculus.

Fig. 4.

Characterization of CCK-expressing afferents to the pPVTh using fluorogold injections into the pPVTh and in situ hybridization for CCK mRNA. Left, Dark-field photomicrographs illustrating that CCK mRNA is expressed with varying degrees of prominence in the central and dorsal lateral parabrachial nuclei (a, c, respectively), the ventrolateral periaqueductal gray (PAG; e), and the caudal dorsal raphe (g). Right, Higher magnification bright-field views of the regions on theleft (see arrows) showing the distribution of the CCK transcript relative to that of retrogradely labeled fluorogold afferents. Many fluorogold-labeled neurons in the parabrachial (b, d) and caudal dorsal raphe nuclei (h) are CCK positive (seearrowheads). Only occasional instances of fluorogold–CCK colocalization were encountered in the ventrolateral PAG (f). See Results for more details.4v, Fourth ventricle.

Fig. 5.

Dark-field (a) and bright-field (b) images of the CCK receptor mRNA distribution in the posterior division of the PVTh (approximately −3.30 mm from bregma). Neurons in b are lightly stained with cresyl violet. Chronic stress did not alter CCK-B receptor mRNA in any division of the PVTh. Coordinates are based on the atlas of Paxinos and Watson (1986).

Fig. 6.

Representative photomicrographs of the track of an injector cannula placed in the pPVTh (a) and in the ventricle (b) and a missed placement lateral to the PVTh (c).

Fig. 7.

ACTH responses during and after a 30 min period of restraint in control animals and in animals exposed to chronic, intermittent cold stress. Control and chronically stressed animals had PVTh injections of either vehicle (VEH) or the CCK-B receptor antagonist PD 135,158 (CCK-B ANT) or injections of the antagonist into areas other than the PVTh (CCK-B ANT MISSED). For control groups,VEH, n = 6–7; CCK-B ANT, n = 10; CCK-B ANT MISSED, n = 4–5. For chronic stress groups, VEH, n = 6–7;CCK-B ANT, n = 7; CCK-B ANT MISSED, n = 4–7. *p ≤ 0.05.

Fig. 8.

ACTH responses during and after a 30 min period of restraint in control animals and in animals exposed to chronic, intermittent cold stress. Control and chronically stressed animals had PVTh injections of either vehicle (VEH) or the CCK-B receptor agonist unsulfated CCK-8 (CCK-8) or injections of the agonist into areas other than the PVTh (CCK-8 MISSED). For control groups, VEH,n = 6; CCK-8, n= 6; CCK-8 MISSED, n = 3. For chronic stress groups, VEH,n = 6–7; CCK-8, n = 5; CCK-8 MISSED, n = 6.

Fig. 9.

ACTH responses during and after a 30 min period of restraint in control animals and in animals exposed to chronic, intermittent cold stress. Control and chronically stressed animals had PVTh injections of either vehicle (VEH) or the CCK-A receptor agonist sulfated CCK-8 (CCK-8S) or injections of the agonist into areas other than the PVTh (CCK-8S MISSED). For control groups, VEH,n = 7; CCK-8S, n= 6; CCK-8S MISSED, n = 4. For chronic stress groups, VEH,n = 5–6; CCK-8S, n = 7; CCK-8S MISSED, n = 3.