Noradrenergic innervation of the dorsal medial prefrontal cortex modulates hypothalamo-pituitary-adrenal responses to acute emotional stress

Abstract

The medial prefrontal cortex (mPFC) has been proposed to play a role in the inhibition of hypothalamo-pituitary-adrenal (HPA) responses to emotional stress via influences on neuroendocrine effector mechanisms housed in the paraventricular hypothalamic nucleus (PVH). Previous work also suggests an involvement of the locus ceruleus (LC) in behavioral and neuroendocrine responses to a variety of acute stressors. The LC issues a widespread set of noradrenergic projections, and its innervation of the prefrontal cortex plays an important role in the modulation of working memory and attention. Because these operations are likely to be critical for stimulus selection, evaluation, and comparison with past experience in mounting adaptive responses to emotional stress, it follows that the LC-to-mPFC pathway might also be involved in regulating HPA activity under such conditions. Therefore, in the present study, we assessed the effects of selectively ablating noradrenergic inputs into the mPFC, using the axonally transported catecholamine immunotoxin, saporin-conjugated antiserum to dopamine-beta-hydroxylase, on acute restraint stress-induced activation of HPA output. Immunotoxin injections in the dorsal mPFC (centered in the prelimbic cortex) attenuated increments in restraint-induced Fos and corticotropin-releasing factor mRNA expression in the neurosecretory region of PVH, as well as HPA secretory responses. Stress-induced Fos expression in dorsal mPFC was enhanced after noradrenergic deafferentation and was negatively correlated with stress-induced PVH activation, independent of lesion status. These findings identify the LC as an upstream component of a circuitry providing for dorsal mPFC modulation of emotional stress-induced HPA activation.

Figure 1.

Noradrenergic denervation after anti-DBH-saporin injections in mPFCd. Dark-field photomicrographs showing DBH immunostaining as a function of lesion status. Top row, Immunotoxin or sham injections were centered in the prelimbic area of the mPFC. Anti-DBH-saporin injections virtually eliminate all noradrenergic fibers and terminals throughout the mPFCd, whereas control injections of the untargeted toxin (IgG-saporin) leave these inputs intact. Middle, bottom rows, A partial depletion of noradrenergic fibers and terminals in adjacent cortical structures was observed after anti-DBH-saporin injections into the prelimbic area, consistent with reports of collateralization of prelimbic-projecting LC neurons to adjacent cortical fields. AId, Agranular insular cortex, dorsal subdivision. Scale bar, 250 μm (applies to all).

Figure 2.

Neurochemical specificity of immunotoxin-mediated denervation in mPFCd. The specificity of noradrenergic deafferentation of the mPFCd was assessed by evaluating the extent to which immunotoxin injection affected dopaminergic fibers and terminals. Confocal images of dual immunofluorescence preparations show TH (green, left column) and DBH (red, middle column) fiber and terminal labeling in the prelimbic area. Because TH is required for the synthesis of DA and NE, the immunolabeling profile for this enzyme represents both DA and NE fibers and terminals, whereas immunolabeling for DBH is specific to NE. In sham-lesioned animals (top row), the overlay of TH- and DBH-stained fibers (right, yellow) represents the subset of inputs that are dopaminergic, whereas the DA fibers display only TH labeling (green). After anti-DBH-saporin injections into the prelimbic area, the density of fibers and varicosities labeled for TH only is comparable with controls, whereas there is a near elimination of DBH staining in the mPFCd (middle), reflected in the absence of elements that would normally be double labeled for both enzymes (right). Scale bar, 250 μm (applies to all).

Figure 3.

Effects of noradrenergic denervation of mPFCd on restraint-induced activational responses in PVH. Top, Bright-field photomicrographs showing comparable immunoperoxidase staining for DBH in the PVH receiving control (sham) or immunotoxin (lesion) injection in mPFCd. Middle, Bright-field photomicrographs showing stress-induced Fos immunoreactivity in the PVH. Relative to sham-lesioned controls, rats receiving anti-DBH-saporin injections centered in the prelimbic area show an attenuation of restraint-induced Fos expression in the hypophysiotropic zone of the PVH (dashed outline). Bottom, Mean + SEM number of Fos-immunoreactive neurons in the PVH of treatment groups. *p < 0.05, differs significantly from unstressed controls; ^†p < 0.01, differs significantly from sham-lesioned stressed animals. n = 6 per group. Scale bar, 250 μm (applies to all).

Figure 4.

Effects of noradrenergic denervation of mPFCd on restraint-induced upregulation of CRF mRNA in PVH. Top, Dark-field photomicrographs showing CRF mRNA expression in the PVH as a function of treatment status. Relative to sham-lesioned unstressed animals, acute restraint exposure results in increased relative levels of CRF mRNA in the hypophysiotropic zone of the PVH. No such enhancement was seen in animals that received anti-DBH-saporin injections into mPFCd 14 d before stress. Bottom, Relative levels (mean + SEM) of CRF mRNA expression in the PVH in treatment groups. Stress exposure increases relative levels of CRF mRNA, whereas this effect is not observed after immunotoxin-mediated denervation of mPFCd. *p < 0.05, differs significantly from unstressed controls; ^†p < 0.01, differs significantly from sham-lesioned stressed animals; ns, nonsignificant. n = 4 per group. Scale bar, 200 μm (applies to all).

Figure 5.

Noradrenergic denervation of mPFCd attenuates pituitary-adrenal secretory responses to acute restraint. Mean ± SEM plasma ACTH (top) and corticosterone (bottom) levels in sham and immunotoxin-injected animals before (0 min) and at varying intervals after acute restraint exposure. Stress exposure significantly increased plasma levels of both hormones in both sham- and immunotoxin-lesioned animals. Although mean values did not differ reliably at any poststress time point (p values >0.05), the overall integrated response of each hormone of the lesion group (areas under the curve) was significantly lower than those of sham controls (see Results). *p < 0.05, differs significantly from basal (0 min) values from within each group. n = 5 per group.

Figure 6.

Acute stress-induced activational responses of mPFCd and PVH are inversely correlated. Top, Mean + SEM number of Fos-immunoreactive neurons in dorsal (PL; left) and ventral (IL; right) parts of the mPFC as a function of treatment status. Exposure to a single 30 min restraint session results in an increased number of Fos-immunoreactive neurons in PL, which was reliably enhanced in animals receiving immunotoxin lesions of mPFCd. No such effect of immunotoxin lesions was seen in IL. *p < 0.05, differs significantly from unstressed controls; ^†p < 0.05, differs significantly from sham-lesioned stressed animals. n = 6 per group. Bottom, Relationship between Fos activational responses in mPFC subdivisions, PL and IL, as a function of Fos activation in the PVH after acute restraint stress. Plots of individual values from sham- and immunotoxin-lesioned animals subjected to a single 30 min restraint session indicated that the degree of Fos activation in PL is inversely correlated with that in the PVH (r = −0.71; p < 0.05). In contrast, there is no reliable correlation between Fos activation in IL and PVH (r = −0.05; p = 0.9).

Figure 7.

Effects of immunotoxin on stress-induced activational responses in the LC. Top, middle, Lower- and higher-magnification bright-field photomicrographs showing Fos-immunoreactive (black nuclei) and DBH-immunoreactive (brown cytoplasm) labeling in LC as a function of treatment. Relative to sham-lesioned, unstressed controls, restraint stress (30 min) results in a marked increase in Fos expression in DBH-labeled noradrenergic neurons in LC. In contrast, animals with immunotoxin lesions of mPFCd show an attenuation of stress-induced Fos immunoreactivity in LC noradrenergic neurons. Arrows mark examples of double-labeled neurons. Bottom, Mean ± SEM number of double-labeled neurons in treatment groups. Although there is almost a complete absence of Fos activation in sham-lesioned, unstressed animals, stress results in a significant activation, and anti-DBH-saporin lesions attenuate the responsiveness of DBH-stained LC neurons. The degree of reduction (37%) is similar to the extent of immunotoxin-induced cell loss (23%). *p < 0.01, differs significantly from sham-lesioned unstressed animals; ^†p < 0.05, differs significantly from sham-lesioned stressed animals. Average of n = 4 per group. Scale bar: top row, 125 μm; bottom row, 50 μm.

Figure 8.

Circuitry providing for LC modulation of mPFCd influences on HPA output. Previous studies support the idea that, during acute emotional stress, the mPFCd exerts inhibitory effects on PVH neurons governing ACTH secretion by way of GABAergic neurons in the bed nucleus of the stria terminalis (BST) and/or hypothalamus (HYP). The present findings suggest that stress-induced activation of LC projections facilitates PVH/HPA output via a net inhibitory influence on mPFC. cc, Corpus callosum; Glu, glutamatergic projection; Pit., pituitary gland; ot, optic tract.