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Try out PMC Labs and tell us what you think. Learn More. Oscillations of gene expression and physiological activity in suprachiasmatic nucleus SCN neurons result from auto-regulatory feedback loops of circadian clock gene transcription factors.
Dopamine DA of hypothalamic origin exerts tonic inhibitory control over prolactin PRL secretion for review see DA is released directly into hypothalamo-hypophyseal portal blood from three populations of neuroendocrine dopaminergic neurons NDNs, 5 These subpopulations include the tuberoinfundibular dopaminergic TIDA and tuberohypophyseal neuroendocrine dopaminergic THDA,A12 neurons with cell bodies in the arcuate nucleus and periventricular hypophyseal dopaminergic PHDA,A14 neurons with cell bodies in the periventricular region 5 TIDA axons terminate on fenestrated capillary beds within the external zone of the median eminence that drain into long portal vessels LPVtransporting DA to the anterior lobe of the pituitary gland 8 In mammals, circadian rhythms are coordinated by the master biological clock located in the suprachiasmatic nucleus SCN of the anterior hypothalamus.
The SCN contains several thousand individual autonomous oscillators shown to express circadian rhythms of gene expression and physiological activity.
Journal of biological chemistry
Several circadian clock gene transcription factors have been cloned in mammals, including the period gene family, clock, and bmal1 15 Recent data suggest that neural targets of the SCN, the primary circadian oscillator, express the putative clock genes with a circadian rhythm 1222375 Therefore, in the absence of SCN input, these tissues cannot sustain a circadian rhythm of clock gene expression for more than days, or alternatively, display desynchronized rhythms of gene expression masked by SCN-mediated coordination.
However, a notable exception to this pattern is the olfactory bulb, which appears to maintain free-running oscillations of PER1 expression in SCN lesioned rats A corresponding rhythm of clock gene expression within NDNs would suggest that NDNs may act as semi-autonomous or damped circadian oscillators. Using Western blot and immunocytochemistry, we have attempted The gift of Fair Grove mm for mw verify the expression of several of the clock gene products within tissues of the hypothalamo-pituitary-gonadal axis and to further characterize the rhythmic expression of these gene products within each of the three populations of hypothalamic DA neurons in the OVX rat.
Given the potential relationship between diurnal rhythms of clock gene expression in SCN neurons and rhythms of DA turnover in NDNs, it is reasonable to assume that a molecular lesion of the SCN may lead to transient disruption of clock-controlled gene expression in SCN neurons. We believe that our will add to our general understanding of the functional role for clock genes in target oscillators within the brain that may or may not depend on the SCN for their synchronization.
Standard rat chow and water were available ad libitum. The room was illuminated with four 40 W fluorescent bulbs, producing a minimum illumination of lux at cage level. Animals were anesthetized with halothane and OVX bilaterally. All animals were placed under a standard L:D cycle lights on hh for days for habituation to the home cage.
Feeding and drinking patterns are established methods for determining circadian time CTa subjective measure of time based on the activity of the animal, independent of the L:D cycle 1445 Drinking was measured over the hour day with an automated device Dilog Instruments, Tallahassee, FL. CT12 was used as a reference time for tissue collection under a standard L:D cycle.
The use of the CT scale allowed us to make direct comparisons between data collected The gift of Fair Grove mm for mw the current experiments and analyses of DA turnover performed under constant conditions in published reports 57 Double plotted actograms of drinking activity hour moving average of drinking activity around a central peak of activity were produced with Circadia software ver. Animals were deeply anesthetized with Halothane and transcardially perfused through the descending aorta with 60 mls of pre-wash 0. Louis, MO in 0. Sections were collected in 12 well plates containing cryoprotectant solution 71 and stored at C.
Each series of sections was rinsed three times for 15 min in 0.
Louis, MO. Polyclonal primary antibodies against mouse PER1 rabbit host, polyclonal used at , kind gift of Dr. All primary and secondary antibodies were diluted in PBX. Sections were washed three times for 10 min with PBX between each step. Sections were then rinsed with PBX three times for 15 min, mounted and coverslips were applied with diluted aqua-polymount Polysciences, Warrington, PA. After several hours the edges of the coverslips were sealed with nail polish.
Controls included sections wherein primary antibody was excluded or PER1, PER2 and CLOCK primary antibodies were pre-absorbed with a substantial amount fold higher concentration of the peptide fragments they were The gift of Fair Grove mm for mw against data not shown. Although clock proteins translocate back to the nucleus, like most proteins they are formed in the cytoplasm and require phosphorylation and dimerization to reach the nucleus Clock gene expression within the nucleus of neuroendocrine dopaminergic neurons.
An absence of granular staining within the nucleolus is a clear indicator of nuclear staining. Bilateral 33 gauge solid steel mandrils were placed inside the guide tubes and dust caps were used to secure the apparatus. Animals were allowed to recover on a heated pad and returned to their home-cage for days. After thorough recovery, animals were transferred, during the light portion of the L:D cycle between h and h, to the lickometer device and allowed to habituate for a minimum of 24h. Animals were placed in the Lickometer device in a L:D cycle with lights on from hh for 5 days.
On the fifth day under a L:D cycle animals were injected intra-SCN with clock gene antisense cocktail. Briefly, animals were anesthetized with Halothane and 33 gauge bilateral mandrils were removed.
Bilateral internal cannulae 33 gauge, Sequences for mper1 Genebank accession : NM and mper2 Genebank accession : NM AS-ODN were slightly modified from those used by Akiyama and colleagues 270 in order to slightly increase the GC content of the oligonucleotide. Per1, per2 and clock mRNA levels recovered to control values by 12h post-injection and remained at normal levels at 36 and 48h after infusion of AS-ODN data not shown. Therefore, in order to verify the acute effects of clock gene mRNA knockdown all experimental animals were injected 6h before sacrifice.
Individual 2 mm thick sections containing the SCN or the medial basal hypothalamus including the ARN and median eminence were placed on a diethylpyrocarbonate DEPC treated sterile glass slide. The aqueous phase was removed from each tube and pooled according to sample. An equal volume of isopropanol was added to each tube and all tubes were placed in a C freezer. After 6 hours at C, precipitated RNA was pelleted by centrifugation at 13, rpm for 20 minutes at 4C.
Samples were stored at C until use. Gels were stained by immersion in ethidium bromide solution 0. Proteins were extracted from neuroendocrine tissues according to the method of Lee and colleagues The brain and pituitary gland were rapidly removed and the brain was The gift of Fair Grove mm for mw in a chilled brain-sectioning matrix ASI Instruments Inc. Two mm thick frontal sections of the brain including the SCN and the ARN were made with a sterile razor blade and placed on a chilled glass slide.
Using a scalpel, a 3mm x 3mm cube including the SCN and a 3mm x 4mm angled dissection of the ARN were removed, as well as the cerebellum and a majority of the piriform cortex from both hemispheres. Following dissection, tissues were rapidly frozen in liquid nitrogen and stored at C until protein extraction. After centrifugation at 12, x g for 15 min, supernatant was transferred to a fresh tube and re-centrifuged.
Supernatant was removed and extracted protein concentration was determined by the micro-modified bichonchoninic acid protein detection system Pierce, Rockford IL. As clock proteins vary ificantly in MW i.
Samples were briefly centrifuged at 12,g and cooled to room temperature. Electroblotting was carried out with standard buffers. Blots were rinsed three times for 10 min with Tris-buffered saline with 0. Choogon Lee, data not shown.
The brain and pituitary gland were quickly removed and placed on a chilled glass slide. The median eminence, neural, intermediate and anterior lobes of the pituitary gland were carefully dissected, placed in homogenization buffer 0.
On the day of analysis for catecholamines, tissue samples were thawed and processed for HPLC-EC analysis as ly described The concentrations of DA and DOPAC, a primary metabolite of DA, were measured in tissue extracts from the pituitary gland and mediobasal hypothalamus as ly described The amount of catecholamine in each sample was estimated by direct comparison to the area under each peak for known amounts of catecholamine. Parlow as ly described For antisense knockdown experiments, four adult female OVX Sprague-Dawley were housed individually in cages attached to the automated drinking device under L:D conditions for 5 days.
Moreover, as samples were obtained from each animal following decapitation or perfusion with fixative at individual time points over a 24 h period, it is difficult to extrapolate accurate phase and period measures. While it is preferable when performing circadian studies to collect serial samples of individual animals, analyses of recovered tissue preclude such an approach in our experiments.
To facilitate direct comparisons between current and experiments, all data points regardless of treatment condition were aligned by circadian time. Circadian clock protein immunoreactivity within NDNs is characterized by dense, granular nuclear staining and an absence of staining within the nucleolus see Fig. However, without functional evidence, we cannot assume that the circadian clock genes drive circadian rhythms in NDNs in the absence of als from the SCN.
To verify a functional circadian clock and to determine a reference point for AS-ODN injection and tissue collection, animals were placed in our lickometer device following stereotaxic surgery. The beginning of the hour activity period, identified as CT12, was determined on the two days prior to tissue collection for each animal and averaged to predict the onset of activity on the following day.
We predicted CT12 under entrained conditions with an assumed error of minutes, given a variance in activity onset among our rats generally minutes from cycle to cyclewhich we consider acceptable with a sampling frequency of hours.
Animals were sacrificed at approximately ZT8 under hypercapnic conditions. Individual 2 mm thick tissue punches containing the suprachiasmatic nucleus SCN or the medial basal hypothalamus including the arcuate nucleus ARN and median eminence and the entire anterior lobe of the pituitary were dissected and used for total RNA extraction.
Gene specific primers for per1, per2, clock and bmal were used to amplify cDNA. PCR products were separated with agarose gel electrophoresis and stained with ethidium bromide.