Cortical involvement of GHRH-GHRHR in sleep regulation

Growth hormone releasing hormone (GHRH) is involved in the regulation of non-rapid eye movement sleep (NREMS). The hypothalamus is a well-characterized sleep-promoting site of action for GHRH. However, GHRH may also act in the cortex to influence sleep; application of GHRH to the surface of the cortex changes electroencephalogram (EEG) delta power, a measure of the NREMS intensity. GHRH and the GHRH receptor (GHRHR) and their mRNAs are detectable in the rat cortex and the expression of cortical GHRHR is activity-dependent. Here we microinject a GHRH antagonist or GHRHR siRNA (siGHRHR) onto the surface of somatosensory cortex of the rat brain. The unilateral application of the GHRH antagonist ipsilaterally decreased EEG delta wave power during NREMS, but not wakefulness, during the initial 40 min after injection. Insufficient REMS epochs occurred during this 40 min period to allow power analyses during REMS. Similarly, injection of siGHRHR reduced the expression of GHRHR in the cortex and suppressed EEG delta wave power of NREMS during 20-24 hr after injection. Using the Fura-2 calcium imaging technique, we detected cultured cortical cells that respond to GHRH with an increase of intracellular calcium. This increase was due to the influx of extracellular calcium. Approximately 18% of these GHRH responsive cells are GABAergic as illustrated by GAD67 immunostaining. Double labeling for GAD67 and GHRHR in vitro and in vivo indicated that 32% and 20% of GHRHR containing cells are GAD-immunoreactive, respectively. Therefore, in the cortex, the number of GHRH responsive cells that are GABAergic are fewer than in the hypothalamus, where most of these cells are GABAergic. Our data suggest that endogenous cortical GHRH activates local cortical cells to affect EEG delta wave power state-specifically. Results are also consistent with the hypothesis that GHRH contributes to local network state regulation