The secretion of glucocorticoid hormones is a highly regulated process. One of the important regulators of the HPA axis is circadian information originating in the SCN. This temporal information is critical for coordination of whole body physiology, preparing the body for daily changes between activity and rest. The SCN can be divided into two anatomically distinct portions based on the expression patterns of two neuropeptides: VIP and vasopressin (AVP). VIP-ir fibers make up the “core”, which receives input from the retinohypothalamic tract (RHT) and in all likelihood signals to the “shell”, made up of AVP-ir neurons. It is postulated that the AVP-neurons, situated next to the third ventricle, play a neurosecretory role. Both VIP-ir and AVP-ir fibres have been found to innervate various hypothalamic nuclei [20
]. In particular, VIP-ir fibres in the PVN show a similar distribution to transcripts of the VPAC2
]. This anatomical distribution of VIP and VPAC2
R strongly suggests the importance of VIP in SCN to PVN communication, and may be critical for transmission of circadian signals to the HPA axis.
The necessity of VIP and its receptor in the SCN, VPAC2
R, for rhythms in electrical activity in SCN neurons and behavioural rhythms has been previously demonstrated [12
]. We now demonstrate that diurnal and circadian rhythms of serum concentration of ACTH and corticosterone are lost in VIP-deficient mice. As such, we propose that VIP, and likely its receptor VPAC2
R, are critical for the daily rhythms of glucocorticoids by their action to maintain rhythmicity and synchrony within the SCN.
R knockout mice show a lack of rhythms in expression levels of clock genes [14
] and glucocorticoid rhythmicity [26
]. We would predict a similar loss in clock gene rhythmicity in VIP-deficient mice. Mutants in clock genes, e.g. the Period
genes, show a variety of circadian defects. In particular, the Per2
double knockout model shows a lack of glucocorticoid rhythms [27
]. In this instance, the authors determined that a functional oscillator in the SCN is necessary for the rhythms in ACTH and that a circadian rhythm in corticosterone is dependent on the oscillator in adrenals. They suggest the adrenals can modulate production of corticosterone in a manner dependent on time of day. Their data and ours highlight the importance of a functional/rhythmic SCN in determining rhythmicity in the periphery. It remains to be seen if the VIP-deficient mice have functional peripheral oscillators and if such oscillators require a daily rhythm in glucocorticoids for synchrony with the SCN.
Other dimensions of VIP action in the HPA axis could be within the adrenals, where VIP has been shown to be synthesised in adrenal glands [28
]. VIP-ir fibres innervate both the cortex and medulla of adrenal glands and originate from the splanchnic nerve or from ganglion cells in the medulla [30
]. Both receptors for VIP, VPAC1
R and VPAC2
R, are also found in the adrenal medulla and capsule [32
]. The role of VIP in the adrenal glands is not entirely clear; functional studies have demonstrated a response by VIP to ACTH administration and splanchnic nerve stimulation as well as effects of VIP on glucocorticoid secretion by the adrenal glands [28
]. Splanchnic denervation in rats has been shown to influence the diurnal rhythm of corticosterone production, either by sympathetic signals to directly induce corticosterone production or in adapting the adrenal responsiveness to ACTH [36
]. This suggests some form of interaction between the HPA axis and the autonomic nervous system controls corticosterone rhythmicity.
In our study, VIP-deficient mice maintain their capacity to produce corticosterone; basal concentrations, while not rhythmic, are similar to non-peak concentrations in WT mice, suggesting that VIP signalling is not critical for normal adrenal production of corticosterone. Alternatively, if VIP does play a role within the adrenals in interpretation of the ACTH or splanchnic nerve signal, other neuropeptides, like PACAP or AVP, may play a compensatory role in the mutant mice. While PACAP has been implicated in a number of stress responses (e.g. 37, 38), recent data makes it less likely that an up-regulation PACAP may compensate for the loss of VIP (39). The basal concentration of ACTH is significantly higher in the VIP-deficient mice at all time points tested. This raises the question of whether the loss of the ACTH rhythm is due to a breakdown in its circadian control or if VIP is necessary for the repression of ACTH from the pituitary. Adrenalectomy normally results in the loss of the major source of circulating corticosterone and a corresponding increase in ACTH. Prior to adrenalectomy, ACTH concentration in VIP mutants is already significantly higher than in WT mice, and remains high following adrenalectomy. The high basal concentration of ACTH may indicate defects in the ability of corticosterone to feedback on CRF-releasing neurons in the PVN.
One of the key components driven by the HPA axis is the rapid release of ACTH and adrenal corticosterone into the circulation in response to a variety of stressors (like noise, restraint, footshock, immune challenge, hypoglycaemia, haemorrhage, novel environment, predator exposure). This responsiveness of the HPA axis has a diurnal rhythm, and the magnitude of the corticosteroid response to stress is stressor specific. Stress by ether and novel environment has been reported to produce a greater corticosterone response at the start of the active phase [40
], with restraint stress inducing a larger response in the early day [6
]. Lesioning the SCN causes a loss in the diurnal difference in response, but not the ability to respond [6
]. Furthermore, application of anti-sense VIP transcripts by intracranial infusion into the hypothalamic region transiently blunts the corticosterone rhythm without affecting corticosterone production in response to acute stress [42
]. In support of this, our study shows that mice lacking VIP not only in the hypothalamus but also in the adrenals are capable of mounting a normal corticosterone response to acute stress. Taken together with the ability of VIP-deficient mice to maintain normal basal concentration of corticosterone, we can conclude that VIP is not essential for non-circadian functioning of the HPA axis.
Light acts to entrain the circadian clock and synchronises it to the environment. The term circadian is literally translated to “about a day”, and the free-running period of nocturnal animals tends to be just under 24 hours. As such, under conditions of LD of 12:12 or normal changing LD patterns in the wild, light synchronises the circadian clock daily by shifting the phase of the oscillator [43
]. The ability of light to shift the endogenous clock is well documented, and even brief pulses of light (from seconds to minutes) can effect a change in the rhythm of locomotor behaviour and shift peripheral molecular oscillators [e.g. 44
]. The phase shifting effect of light has been shown to pass through the RHT to VIP neurons in the SCN [45
], modifying the concentration of one of the clock genes, Per1
], and changing the phase of the molecular oscillator. The SCN is necessary for light-induced changes in the adrenals: in SCN-lesioned rodents, there was no corticosterone induction nor Per1
up regulation [19
]. This adrenal response is also dependent on the integrity of the splanchnic nerve [19
]. In response to exposure to light in the early night, we observed an increase in corticosterone concentration in WT mice, in agreement with evidence from Ishida and colleagues [19
]. In contrast, VIP mutant mice are deficient in this light-induced response, suggesting that VIP is critical for interpretation of light input.
We also showed that we can measure a significant up regulation of Per1 expression following light treatment in WT mice. This response is lost in VIP KO mice, providing further evidence of a defect in response to light. The Per1 response to a different acute stressor, footshock, remains present, if blunted, in VIP KO mice, again confirming that some aspects of the HPA axis are unaffected by the loss of VIP. The selective deficits in behavioural, hormonal and gene expression response to light continue to be the most intriguing aspects of VIP loss. It remains to be determined if this deficiency is due to loss of VIP in the SCN or a breakdown of signalling in the rest of the HPA axis that may be dependent on VIP.
There are many peripheral organs in which rhythms in gene expression have been demonstrated, including the liver and adrenal glands [49
]. For the SCN to synchronise the molecular oscillators in the periphery, there must be an entraining or resetting signal originating from the SCN. This could take a number of forms, including direct secretion from secretory AVP-ir neurons next to the third ventricle, neurotransmitters through the autonomic nervous system, or activation of the HPA axis [52
]. There is a growing body of evidence for the HPA axis as an output of the SCN. A glucocorticoid receptor agonist, dexamethasone, can induce Per1
expression in the liver with subsequent phase shifting of the liver oscillator [3
]. Further evidence comes from food-restriction studies, where loss of endogenous corticosterone by adrenalectomy has been found to speed up synchronisation of the liver oscillator to food-restriction regimes [53
]. Indications of the possible roles corticosterone could play as an output signal were teased out in a study where the rhythmicity of genes involved in glycogenesis, gluconeogenesis and cholesterol synthesis, amongst other pathways, in the liver were affected by adrenalectomy [54
]. Another striking effect of adrenalectomy on circadian rhythms in non-SCN tissues is the loss of PER2 rhythms in the oval nucleus of stria terminalis and the central nucleus of the amygdala in rats, which can be rescued by rhythmic application of corticosterone [55
The VIP knockout mouse provides a unique model in which the circadian information from the SCN is lost or not transmitted. So far, we have determined that rhythms in glucocorticoids are lost even though the ability of the HPA axis to respond to acute stress is intact. It is reasonable to assume that other endocrine rhythms are lost as well. This unique dissociation of circadian and responsive aspects of the HPA axis thus makes VIP-deficient mice a good model for dissecting out downstream physiological processes that require signals from the SCN. Conversely, it may also prove a useful model in which to study the HPA axis without the confounding effect of circadian regulation. Furthermore, the deficient corticosterone response to light provides an interesting avenue to explore the mechanisms by which light signals from the environment change physiological state.