Abstract
The stress response is crucial for allowing adaptation to an ever-changing external environment. The neuroendocrine stress response is controlled by corticotropin-releasing hormone (CRH) neurons located within the paraventricular nucleus (PVN) of the hypothalamus. During stress, noradrenaline (NA) is released within the PVN to activate CRH neurons and drive stress hormone secretion. This study aimed to investigate the mechanism through which NA regulates activity within the CRH neuronal network activity. In vitro GCaMP6f calcium imaging and on-cell electrophysiology was performed on brain slices containing the PVN from CRH-GCaMP6f mice.
Here, we show that NA (10 M) induces a robust increase in CRH neuron excitability, with a large proportion of CRH neurons switching into a bursting pattern of activity. NA-induced excitation was significantly reduced when the adrenergic 1 receptor was blocked with prazosin (10 M) (n = 14 slices). In contrast, NA-induced excitation was significantly increased when the adrenergic 2 receptor was blocked with yohimbine (10 µM) (n = 11 slices) (One-way ANOVA with Tukey’s post-hoc analysis, p < 0.0001).
We also demonstrated that the excitatory effects of NA on CRH neurons were unchanged if either ionotropic glutamate receptors were blocked with CNQX (10 µM) and DL-AP5 (40 µM) (n = 13 slices), ionotropic GABA receptors were blocked with picrotoxin (50 µM) (n = 13 slices) or action potential firing was blocked with TTX (1 µM) (n = 8 slices) (One-way ANOVA with Tukey’s post-hoc analysis, p > 0.05). Interestingly, when all synaptic inputs and action potential firing were blocked together (n = 14 slices), the excitatory effects of NA on CRH neurons significantly increased (One-way ANOVA with Tukey’s post-hoc analysis, p < 0.001).
Furthermore, we showed using simultaneous calcium imaging and on-cell electrophysiology that when action potential firing is blocked in CRH neurons, a subset of neurons fire small amplitude, long duration calcium spikes (n = 4 cells). Additionally, we demonstrated that opening of voltage-gated calcium channels (VGCCs) plays an important role in NA-induced excitation of CRH neurons, as blocking all VGCCs with CdCl2 (100 µM) (n = 10 slices) or just L-type VGCCs with nifedipine (100 µM) (n = 11 slices) led to a significant reduction in excitation of CRH neurons by NA (One-way ANOVA with Tukey’s post-hoc analysis, p < 0.0001).
Lastly, we showed that release of intracellular calcium does not appear to play an important role in NA-induced calcium elevations in CRH neurons, and blockade of intracellular calcium re-uptake with CPA (30 µM) (n = 9 slices) appears to enhance CRH neuron excitation.
These results provide a pathway by which NA acts to excite CRH neurons, as well as demonstrating novel mechanisms by which CRH neuron excitation and activity patterns are controlled. Specifically, this is the first time a bursting pattern of activity has been identified in mammalian CRH neurons. We have also shown new evidence that calcium spikes and opening of VGCCs may play an imperative role in CRH neuron excitation. Overall, these results lead us one step closer to understanding central control of the stress response.