The purpose of these experiments was to determine the pattern of PER1, PER2 and CLOCK expression within NDNs and ascertain the functional relationship between rhythms of clock gene expression within NDNs and previously determined rhythms of DA turnover within TIDA, THDA and PHDA neurons. Previously, we reported significant circadian rhythms of DA turnover in the OVX rat (58
). We hypothesized that these circadian rhythms of DA release, which dictate the timing of the ovarian steroid induced PRL surge on the afternoon of proestrus, are facilitated by autonomous rhythms of clock gene expression within NDNs. We attempted to validate this hypothesis using antisense knockdown to disrupt the transcriptional feedback loops regulating circadian rhythms of activity in SCN neurons. Given the established relationship between the SCN and DAergic target neurons in the ARN, we hypothesized that disruption of the circadian clock in the SCN would abolish diurnal rhythms of DA release from NDN synaptic terminals.
PER1-, PER2- and CLOCK-IR nuclei are clearly labeled within TH-IR neurons in the DMARN, RARN and PeVN. Generally, circadian clock protein-IR was limited to the nucleus and is readily distinguishable from TH-IR cytoplasmic staining. Only those neurons expressing a clear clock protein-IR nucleus were considered clock protein-IR cells. While only TH-IR and clock protein/TH-double labeled cells were counted in the current experiment, we observed numerous PER1, PER2 and CLOCK-IR nuclei within the analyzed regions that were not TH-IR. Under a standard L:D cycle, PER1-IR within TIDA neurons displayed a diurnal rhythm with a peak late in the subjective day. In addition, PER2 expression displayed a diurnal rhythm of expression with significant peaks at CT6 and CT12. We have previously reported a diurnal rhythm of DA turnover within TIDA neurons with a biphasic pattern, defined by peaks at CT6 and CT12 (57
). Thus, PER2 expression, but not PER1 expression, peaked in parallel with the time of peak DA turnover within these neurons. Like the SCN, TIDA neurons express CLOCK-IR in a constitutive manner under a standard 12:12 L:D cycle. Based on these cumulative data, we can assume that TIDA neurons may act as dampened oscillators, with light-entrained or light-activated rhythms of clock gene expression. Recent findings agree with this conclusion, suggesting that neurons within the ARN are unable to express free-running rhythms of PER expression in isolated cell culture for more than 2-3 cycles (75
Unlike TIDA neurons, THDA neurons failed to exhibit a diurnal rhythm of PER1 expression, but did display a diurnal rhythm of PER2 expression with a significant peak at CT6. Like TIDA neurons, THDA exhibit a diurnal rhythm of DA turnover with a biphasic pattern defined by peaks at CT6 and CT12. Therefore, the rhythm of PER2 expression, but not PER1 expression, within THDA neurons corresponds to the rhythm of DA turnover observed for these neurons under a standard L:D cycle. These data are surprising, given our previous report that THDA neurons exhibit clear diurnal rhythms with significant peaks near CT6 and CT12 (57
). It remains to be seen whether PER1 and PER2 have redundant roles in the regulation of molecular feedback loops of circadian clock genes and/or DA synthetic enzyme expression in NDNs. Additional evidence regarding the molecular interactions of PER proteins in NDNs is required to either confirm or refute this hypothesis. Like TIDA neurons, THDA neurons express CLOCK-IR in a constitutive manner under L:D conditions. In PHDA neurons, both PER1 and PER2 expression displayed diurnal rhythms with significant peaks at CT12 and CT6, respectively. We have shown that DA turnover within the IL peaks between CT10 and CT14, followed by a trough at CT18 (57
). In agreement with data from both TIDA and THDA neurons, PER expression patterns within PHDA neurons correspond to the timing of DA turnover within the IL, both of which occur primarily during the light-phase. Like both TIDA and THDA neurons, PHDA neurons express CLOCK in a constitutive manner. Several experiments have concluded that CLOCK protein is also constitutively expressed within the SCN (7
). Therefore, it would appear that constitutive CLOCK expression is a defining feature of the molecular clock found within both the central circadian oscillator in the SCN and its primary central targets, including the NDNs of the hypothalamus. Thus, our results suggest a potential role for the core molecular feedback loops driving the core circadian clock in the timing of molecular and physiological events in NDNs.
Recently, Kriegsfeld and colleagues (33
) reported a diurnal rhythm of PER1 expression in neuroendocrine cells of the ARN in the female mouse, using the same primary antiserum, with a significantly greater number of PER1::GFP/TH double labeled cells at CT10 than CT22. These data suggest that PER1 expression peaks in the latter portion of the subjective day and reaches a nadir at or near the middle of the subjective night (CT18-22, (33
)). Further, the results of Bae and colleagues revealed that PER1-IR peaked within the SCN at or near CT12 (3
). These data would suggest that the peak in PER1-IR in NDNs parallels the peak of PER1 expression within the SCN. However, experiments indicate that PER1 expression in peripheral tissues peaks approximately 6-12 hours after PER expression within the SCN. Our data show that PER1-IR peaks at CT18, approximately 6-12 hours after the peak of PER1 expression we and others
have observed within the SCN (3
To our best knowledge, we are the first to examine the rhythmic expression of these proteins in the NDNs of steroid-depleted OVX rats. Several experiments suggest that ovarian steroid receptors are expressed in SCN neurons and that steroids exert direct effects on gene expression and physiological activity within the SCN (34
). Therefore, it is difficult to predict what effect removal of endogenous ovarian steroids would have on clock gene expression within the SCN and NDNs. We can assume that light-entrained rhythms of PER expression, which are highly dependent on neural input from the retina, are likely less dependent on the influence of ovarian steroids. Of course, we cannot rule out the dramatic effects of ovarian steroid hormone withdrawal on steroid-sensitive neuronal oscillators within the SCN and/or NDNs. Future experiments using steroid hormone replacement models will address this fundamental issue.
Several hypotheses could be offered to explain the function of the diurnal rhythms of clock protein expression we have observed. We have determined in previous experiments that NDNs express VPAC2 receptors that are affected by the level of circulating ovarian steroid hormones (19
). Further, we have shown that disruption of VIP peptide expression within the SCN affects the activity of NDNs under a standard 12:12 L:D cycle (20
). Numerous studies have shown that VIP peptide displays a diurnal rhythm of expression within the SCN characterized by a significant increase in VIP expression in the late subjective night between CT18 and CT22 (4
). We can conclude from these studies that VIP release from SCN neurons entrains the activity of NDNs in the late subjective night. Moreover, additional evidence suggests VIP induces PER1 and PER2 expression in the SCN during the late subjective night (50
). Therefore, we can assume that VIP, released from SCN afferents within the DMARN, RARN and PeVN, binds to VIP type-2 receptors and differentially activates PER1 and PER2 expression through increased intracellular cAMP and CREB mediated signaling (36
). However, this hypothesis cannot explain the phase relationships between PER1 and PER2 expression in TIDA neurons, or the absence of PER1 expression in THDA neurons. Additional experiments are necessary to verify the functional redundancy of PER1 and PER2 within NDNs. Although we lack the evidence, we cannot rule out a role for arginine vasopressin of SCN origin in our model. Further experiments are necessary to delineate the precise role of both VIP and AVP in the activation and maintenance of clock gene expression within NDNs.
In the present study we have determined the effects of transient per1, per2 and clock
mRNA knockdown on the light-entrained rhythms of PRL secretion, CORT secretion, DA concentration within the anterior lobe of the pituitary and DA turnover within NDN synaptic terminals. We have attempted to ascertain the influence of clock gene-controlled physiological activity and gene expression within the SCN on the rhythm of DA release from NDNs. Using a cocktail of per1, per2
AS-ODN, we have generated a temporary “molecular” lesion of the central circadian oscillator. Based on data from our previous experiments (57
), we hypothesized that NDNs may continue to oscillate with a diurnal rhythm in the absence of photoperiodic cues from the SCN. Given that we have already determined that both PER1 and PER2 display diurnal rhythms of expression within TIDA, THDA and PHDA neurons, we hypothesized that clock gene expression within NDNs may act to facilitate diurnal rhythms of DA release from NDNs in the absence of afferent input from the SCN. A negative effect of clock gene AS-ODN injection in the SCN on diurnal rhythms of DA turnover in NDNs would indicate the capacity of the NDN to behave as a semi-autonomous or damped circadian oscillator.
Injection of AS-ODN into the SCN successfully disrupted the diurnal rhythm of drinking activity in the OVX rat. Interestingly, treatment with AS-ODN appeared to induce a long lasting arrhythmia in drinking behavior for up to 72 hours. Although we observed a long duration effect on drinking behavior, we failed to see a similar decrease in PER1, PER2 and CLOCK expression in SCN tissue extracts beyond 12-18 hours (data not shown). Therefore, we can conclude that our transient molecular knockdown exerted long-lasting effects (up to 72 hours) on downstream targets of the SCN, although the overall level of clock protein expression returned to normal within 24 hours. As we did not measure the rhythm of PER1, PER2 or CLOCK expression in the SCN after AS or RS -ODN injection, we cannot rule out significant long-lasting effects of our ODNs on the phase and/or period of the oscillations.
As in previous experiments, we failed to detect a rhythm of PRL secretion in the OVX rat (12
). Our inability to detect a significant PRL secretory rhythm precludes determination of AS-ODN treatment affects on PRL secretion. Additional experiments, using both steroid-primed and cycling rats, could provide additional insight into the role of clock genes within the SCN in the control of PRL secretion. Unlike PRL, serum CORT exhibited a significant diurnal rhythm in RS-ODN-treated controls that was disrupted by AS-ODN treatment. Data from numerous experiments suggest that AVPergic afferents of SCN origin terminate on CRH neurons within the medial parvicellular paraventricular nucleus (6
). Further evidence suggest that AVP and corticotrophin releasing factor (CRF) mRNA are synthesized in PVN neurons with a distinct circadian rhythms, possibly under the direct control of SCN afferents (74
). Thus, our ability to disrupt the diurnal rhythm of CORT secretion in OVX rats, like the effects we observed on drinking activity, support successful molecular lesion of the SCN with our AS-ODN cocktail.
In agreement with our previous experiments (57
), TIDA and PHDA neurons displayed diurnal rhythms of DA turnover with significant peaks in the early subjective day (CT0 in TIDA and PHDA neurons) and early subjective night (CT15 in TIDA neurons). Treatment with AS-ODN against per1/2 and clock
mRNA significantly adjusted the magnitude of DA turnover, leading to a significant change in the shape of the rhythm. Treatment with AS-ODN eliminated the second peak of DA turnover within TIDA neurons that occurred at CT15 (approximately 2030h) but failed to affect the peak of DA turnover at CT0. AS-ODN treatment advanced the acrophase of DA turnover in the IL from CT6 to CT0. Moreover, AS-ODN treatment increased DA turnover at CT15, such that it no longer represents the absolute nadir of DA turnover, as it does in RS-ODN controls. These data suggest that AS-ODN adjusted the magnitude of DA release but failed to completely eliminate the diurnal rhythm. Further, these results suggest an overall increase in the level of DA release, indicating disruption of a potential DA-release inhibiting factor of SCN or hypothalamic origin. Nonetheless, this effect correlates with our previous experiments, suggesting that treatment with AS-ODN against VIP affected the pattern of immediate early gene expression within NDNs (20
). Several studies suggest that AVP expression within neurons of the SCN shell or dorsomedial SCN displays a free-running endogenous rhythm under the direct control of CLOCK:BMAL1 enhancers, while VIP expression exhibits a light-entrained, but not free-running, circadian rhythm (29
). However, recent studies suggest that VIP mRNA synthesis is under the direct control of the molecular oscillator in the mouse (9
). Although we cannot rule out the potential influence of AVP from the SCN shell, we can assume that, if VIP is the primary neurotransmitter of the SCN-NDN tract, that light-induced or endogenous rhythms of VIP release in the mediobasal hypothalamus are responsible for similar rhythms of DA release from NDNs. Within the AL, we failed to detect a significant rhythm of DA concentration within RS-ODN control animals. However, AS-ODN injection induced a significant diurnal rhythm with peaks between CT6 and CT9. As mentioned above, this response is not expected, given the generally variable, basal level of DA release from all three populations in the OVX rat observed in previous experiments (12
). Variation in the rhythmic release of DA from each individual population, as a result of ovarian steroid hormone withdrawal and the absence of a significant PRL rhythm may result in the distinct, albeit dissociated rhythm we have detected here. Overall, these data suggest that AS-ODN treatment may reveal a significant diurnal rhythm of DA turnover and total DA concentration by removing rhythmic inhibition of DA release from a presently unknown inhibitory pathway.
We can conclude, therefore, from these studies and others that NDNs may function as damped oscillators, expressing clock genes in a L:D cycle under the direct influence of input from the SCN. Additional experiments are needed to gain a more thorough understanding of the role for each clock gene product in NDNs with regards to the synthesis and release of DA. Further, future experiments will attempt to garner a better understanding of the effects of ovarian steroids on the molecular clock in both the SCN and NDNs. Our current report indicated the presence of circadian clock gene products in numerous non-DAergic neurons within the arcuate and periventricular nuclei. Future experiments will provide us with a better understanding of the form and function of these neurons, as well as their role as potential target oscillators in the hypothalamus. For example, we cannot make a definitive claim regarding the function of NDNs as damped oscillators without evidence that they display free-running and cell-autonomous rhythms of gene expression and electrical activity. Our data show that AS-ODN treatment significantly reduced PER1, PER2 and CLOCK expression within NDNs and affected the diurnal rhythms of drinking behavior, CORT secretion and NDN activity in the OVX rat. Therefore, our current data and previous evidence suggest that the diurnal rhythms of DA release in TIDA, THDA and PHDA neurons are semi-dependent on rhythmic output from SCN neurons. Evidence suggests that disruption of clock-controlled genes, such as VIP, either directly via VIP AS-OSN as in previous experiments (20
) or indirectly through disruption of per1, per2
transcription (current report), may modulate, but not abolish, rhythms of DA release. This hypothesis relies on information regarding the relationship between clock gene expression in SCN neurons and VIP mRNA synthesis (20
). Further experiments are needed to strengthen the potential relationship between clock controlled gene expression in NDNs, VIP synthesis and release from SCN afferents and DA release from NDN synaptic terminals.