MAP kinases couple hindbrain-derived catecholamine signals to hypothalamic adrenocortical control mechanisms during glycemia-related challenges

Abstract

Physiological responses to hypoglycemia, hyperinsulinemia, and hyperglycemia include a critical adrenocortical component that is initiated by hypothalamic control of the anterior pituitary and adrenal cortex. These adrenocortical responses ensure appropriate long-term glucocorticoid-mediated modifications to metabolism. Despite the importance of these mechanisms to disease processes, how hypothalamic afferent pathways engage the intracellular mechanisms that initiate adrenocortical responses to glycemia-related challenges are unknown. This study explores these mechanisms using network- and cellular-level interventions in in vivo and ex vivo rat preparations. Results show that a hindbrain-originating catecholamine afferent system selectively engages a MAP kinase pathway in rat paraventricular hypothalamic CRH (corticotropin-releasing hormone) neuroendocrine neurons shortly after vascular insulin and 2-deoxyglucose challenges. In turn, this MAP kinase pathway can control both neuroendocrine neuronal firing rate and the state of CREB phosphorylation in a reduced ex vivo paraventricular hypothalamic preparation, making this signaling pathway an ideal candidate for coordinating CRH synthesis and release. These results establish the first clear structural and functional relationships linking neurons in known nutrient-sensing regions with intracellular mechanisms in hypothalamic CRH neuroendocrine neurons that initiate the adrenocortical response to various glycemia-related challenges.

Figure 1.

The relationship between the number of DBH-IR elements and the amount of DBH-IR per element in the PVHmpd of sham (black box), partial (gray box), and complete (white box) lesions is best described by a Boltzmann sigmoidal curve function (solid line). Each PVHmpd (open circles) was designated as having either a sham (intact), partial, or complete lesion of catecholaminergic afferents, as determined by the percentage area occupied by DBH-IR in the PVHmpd at the time of challenge. The area of the PVHmpd occupied by DBH signal in animals injected with the nonspecific mouse IgG-saporin conjugate defined the control range (mean ± 2 SD units) of sham-lesions. For animals injected with the DBH-saporin conjugate, an incomplete lesion was assigned to any PVHmpd where the area occupied by DBH signal was less than 2 SDs below the mean of sham-lesioned animals (topr dashed line) but >20% (bottom dashed line) of the mean of control values. Similarly, complete lesions were assigned when the area of the PVHmpd occupied by DBH signal was ≤20% of the mean of control values (bottom dashed line).

Figure 2.

Primary Crh transcripts show graded responses in the PVH after glycemic challenges. a, Photomicrographs of CRH hnRNA hybridization signals show that levels increase in the PVHmpd after intravenous injections of insulin (Ins) or 2-DG but not saline (Sal). bi–biii, No AVP hnRNA hybridization was seen in the PVHmpd after any treatment). biv, In situ hybridization signal for AVP hnRNA in an adrenalectomized (Adx) rat (for comparison). Note the presence of robust signal in both PVH compartments of the adrenalectomized rat, whereas those in bi–biii lack detectable AVP hnRNA signal in the PVHmpd. c, CRH hnRNA responses were significantly greater for 2-DG than for insulin. ***p < 0.0001. Numbers below each bar are the samples per group.

Figure 3.

Phospho-(p)ERK1/2 show graded responses in the PVHmpd after glycemic challenges. a, bi–biii, pERK1/2-IR increases in the PVHmpd after intravenous injections of insulin (Ina) or 2-DG, but not saline (Sal). pERK1/2-IR responses were significantly greater for 2-DG than for insulin. The white square (biii) is the region depicted at higher magnification in biv, showing pERK1/2-IR in the somata and dendrites of PVHmpd neurons. **p < 0.001, ***p < 0.0001. Numbers below each bar are the samples per group. Scale bar: bi–biii, 100 μm (bi-iii); biv, 20 μm.

Figure 4.

Phospho-(p)ERK1/2 levels in the PVHmpd are tightly correlated to ACTH release and Crh transcription following glycemic challenges. a, b, Incremental increases (Δ0–30 min) in plasma ACTH concentrations are significantly correlated with the number of pERK1/2-IR elements (a) and the amount of pERK1/2-IR per element (b) in the PVHmpd after intravenous injections of either saline (open circles), insulin, or 2-deoxy-d-glucose (solid circles). c, CRH hnRNA and pERK1/2-IR were strongly correlated in control (open circles) and stimulated rats (insulin, 2-deoxy-d-glucose, or anesthesia followed by hypertonic saline injections; solid circles). d, The relationship between the number of pERK1/2-IR elements and the amount of pERK1/2-IR per element. The dashed line is the regression for the control samples; the solid line is the regression for those stimulated rats. a, b, Only intact animals or those with bilaterally symmetric incomplete or complete lesions were included in the analyses. c, d, Each data point represents a single PVHmpd. Gray boxes show control ranges (mean ± 2 SDs) for each variable in saline-injected rats. See text for levels of statistical significance.

Figure 5.

Increased Crh transcription following glycemic challenges is dependent on intact catecholaminergic inputs to the neuroendocrine PVH. a, Insulin or 2-DG significantly increased mean (+SEM) CRH hnRNA levels compared with saline (Sal) in the PVHmpd of sham (MSAP)-lesioned rats (black bars). Catecholaminergic denervation of the PVH completely abolished the Crh transcriptional response to insulin (white bars) and significantly attenuated the response after 2-DG, whereas partial lesions (gray bars) had no effect on Crh transcriptional responses. **p < 0.001, ***p < 0.0001. ns, Not significant. Numbers below the bars are the samples per group. b, Representative photomicrographs of the CRH hnRNA in situ hybridization signals in animals with sham or complete lesions following insulin or 2-DG injections.

Figure 6.

Increased ERK1/2 phosphorylation following glycemic challenges is dependent on intact catecholaminergic inputs to the neuroendocrine PVH. a–c, Insulin (a, c) or 2-DG (b, c) significantly increased mean (+SEM) pERK1/2-IR compared with saline (Sal) in the PVHmpd of sham (MSAP)-lesioned rats. Catecholaminergic denervation of the PVH (as assessed by DBH immunocytochemistry) completely abolished pERK1/2 responses to both challenges (a, c). Partial lesions abolished the pERK1/2 response to insulin (a, c) but not 2-DG (b, c). An asymmetric lesion in the PVH of an insulin-injected animal (a) produced a correspondingly asymmetric pERK1/2 response. **p < 0.001, ***p < 0.0001. ns, Not significant. Numbers below the bars (c) are the number of samples per group. Scale bars, 100 μm.

Figure 7.

Mechanisms that phosphorylate ERK1/2 in the neuroendocrine PVH remain fully functional after catecholaminergic denervation. a, A multimodal stimulus (A+HS) increases pERK1/2 in the PVHmpd in intact rats (solid bars) and rats with complete catecholaminergic denervation of the PVH (open bar). b, DBH-IR and pERK1/2-IR from representative animals. Robust pERK1/2 is seen in both sham- and complete lesioned rats, including the PVHpm. Scale bar, 100 μm. c, d, Complete CA denervation significantly attenuated but did not abolish Crh transcriptional responses to A+HS. **p < 0.001, ***p < 0.0001; ns, not significant. Numbers below each bar show the number of samples per group.

Figure 8.

Phospho-(p)CREB and phospho-(p)ERK1/2 colocalize in the PVHmpd after stressful challenges. a–c, Intravenous insulin, 2-DG, or a mixed modality stressor (A+HS) all significantly increased pCREB- (a) and pERK1/2-IR (b) in the PVHmpd compared with saline-injected controls (c). pCREB immunoreactivity (green) was found exclusively in nuclei after injections of insulin, 2-DG, or A+HS, with extensive colocalization with cytoplasmic pERK1/2-IR (red). White squares in a and b show the regions that are depicted at higher magnification in c. Scale bar: a, b, 100 μm; c, 20 μm.

Figure 9.

a, Intravenous insulin (Ins), 2-DG, or a mixed modality stressor (A+HS) significantly increased pCREB in sham-lesioned rats (solid bars) compared with saline-injected rats, but responses to 2-DG and A+HS were significantly greater than after insulin. Complete catecholaminergic denervation of the PVH (open bars) abolished pCREB responses to insulin and 2-DG, and significantly attenuated responses to A+HS. *p < 0.01, **p < 0.001, ***p < 0.0001. ns, Not significant. Numbers below each bar show the samples per group. b, c, The number of pCREB-containing neurons in the PVHmpd was significantly correlated with the number of pERK1/2 immunoreactive elements (b) and with Crh transcription (c) in control (open circles) and stimulated (solid circles) rats. See text for levels of significance.

Figure 10.

Inhibiting MEK activity abolishes norepinephrine (NE)-evoked increases in PVH neuroendocrine neuronal firing rates in ex vivo hypothalamic slices. a, Representative spike trains from electrophysiologically identified parvicellular neuroendocrine neurons in the PVHmp in hypothalamic slices after control or application of 100 μm norepinephrine. b, Norepinephrin increased in the firing rate of PVHmp neurons, which persisted through the washout period. c, Preapplication of U0126 (a MEK inhibitor), but not U0124 (the inactive analog of U0126) significantly inhibited the ability of 100 μm norepinephrine to increase spike frequency. Numbers below each bar shows the number of samples per group.

Figure 11.

Inhibiting MEK activity abolishes ERK1/2 and CREB phosphorylation in ex vivo hypothalamic slices in response to norepinephrine treatment. a, Norepinephrine (NE; 100 μm) increased both pERK1/2 (top) and pCREB (middle) in the PVHmpd compared with control slices (aCSF). This effect was blocked by U0126 (third column), but not U0124 (fourth column). The proximity of the PVH to the third ventricle meant that it was not always possible to obtain flat-field illumination across every field, particularly in aCSF-treated slices. To confirm that each slice contained the PVH, the third row shows the vasopressin gene product, copeptin, in the same field as pERK1/2 and pCREB. Scale bar, 40 μm. b, An orthogonal view of a two-channel-merged confocal image showing colocalization of pCREB and pERK1/2 in PVHmpd neurons. Scale bar, 10 μm. c, U0126 but not U0124 significantly reduced the number of neurons double-labeled with pCREB and pERK1/2 following norepinephrine treatment. Numbers below each bar shows the samples per group.