Conceived and designed the experiments: AK RMB. Performed the experiments: AK EF IS CVH JVDV. Analyzed the data: AK RMB. Wrote the paper: AK EF RMB.
The authors have declared that no competing interests exist.
The mammalian biological clock, located in the hypothalamic suprachiasmatic nuclei (SCN), imposes its temporal structure on the organism via neural and endocrine outputs. To further investigate SCN control of the autonomic nervous system we focused in the present study on the daily rhythm in plasma glucose concentrations. The hypothalamic paraventricular nucleus (PVN) is an important target area of biological clock output and harbors the pre-autonomic neurons that control peripheral sympathetic and parasympathetic activity. Using local administration of GABA and glutamate receptor (ant)agonists in the PVN at different times of the light/dark-cycle we investigated whether daily changes in the activity of autonomic nervous system contribute to the control of plasma glucose and plasma insulin concentrations. Activation of neuronal activity in the PVN of non-feeding animals, either by administering a glutamatergic agonist or a GABAergic antagonist, induced hyperglycemia. The effect of the GABA-antagonist was time dependent, causing increased plasma glucose concentrations only when administered during the light period. The absence of a hyperglycemic effect of the GABA-antagonist in SCN-ablated animals provided further evidence for a daily change in GABAergic input from the SCN to the PVN. On the other hand, feeding-induced plasma glucose and insulin responses were suppressed by inhibition of PVN neuronal activity only during the dark period. These results indicate that the pre-autonomic neurons in the PVN are controlled by an interplay of inhibitory and excitatory inputs. Liver-dedicated
The role of the CNS in glucoregulation has been recognized since the classical experiments of Bernard in 1849
To investigate possible daily changes in the activation of the pre-autonomic PVN neurons, we first compared the hyperglycemic response evoked by GABAergic and glutamatergic (ant)agonists when administered into the PVN at different moments of the L/D-cycle. Secondly we studied the effect of an SCN-lesion, i.e., removal of the proposed GABAergic projection, on the hyperglycemic effect of BIC applied in the PVN. Thirdly, we inhibited neuronal activity in the PVN during a scheduled meal at different times of the light/dark-cycle in order to investigate whether the parasympathetic input to the pancreas, too, is dependent on (daily changes in) PVN neuronal activity. The results are synthesized into a model explaining how the biological clock is able to somatotopically enforce differentiated autonomic rhythms onto different body compartments.
A total of 280 Wistar rats were used in this study. Histological analysis of probe placement showed that in the majority of the animals, the tip of the microdialysis probes was consistently positioned within 50–100 µm of the borders of the PVN. Typical examples of probe placements can be found in our previous papers
In order to investigate possible daily changes in the activity of GABA- and/or glutamate-containing projections to the PVN we used muscimol (MUS) and NMDA to activate and bicucilline (BIC) and MK801 to antagonize GABAergic and glutamatergic receptors, respectively, and circulating plasma glucose and plasma insulin as the main readout. Mean basal plasma glucose and plasma insulin concentrations at the start of the different experiments, as well as a statistical analysis of their response are indicated in
Only BIC administration showed a strong time-of-day dependency when comparing for all 8 experiments (i.e. BIC, MUS, NMDA and MK801 at either ZT5 or ZT15) the day of drug administration with the control day 1 week later (
Filled symbols indicate the effect of the drug, whereas open symbols show the results of the control experiment in the same animals one week later. Only 3 out of the 8 different treatment protocols showed a significant effect of drug treatment, i.e. NMDA at ZT5 (p = 0.008) and at ZT15 (p = 0.009), and the Bicuculline treatment at ZT5 (p = 0.002).
Plasma insulin concentrations were only significantly affected by the ZT15 NMDA and MUS administration (
Filled symbols indicate the effect of the drug, whereas open symbols show the results of the control experiment in the same animals one week later.
In order to investigate whether the daily change in GABAergic input to the PVN is derived from the biological clock located in the SCN we repeated the PVN administration of BIC in groups of SCN-lesioned and SHAM-lesioned animals. By measuring their diurnal water intake pattern 11 of the 36 animals originally operated upon were identified as having an effective SCN lesion and selected for further experiments. Of the 11 SCN-lesioned and 11 SHAM animals equipped with bilateral PVN probes and a jugular vein catheter, 10 SCN-lesioned animals and 9 SHAM operated animals completed the BIC administration experiment. Eight animals in both groups also completed the control experiment 1 week later. Histological stainings using vasopressin (VP) and vasoactive intestinal polypeptide (VIP) immunocytochemistry were used to check for remaining pieces of SCN tissue at the border of the lesion after finishing the physiological experiments, but in all 10 cases could confirm the completeness of the lesion. Typical examples of such a lesion and the histological stainings can be found in our previous papers
Open symbols show the results of the control experiment in the same animals one week later. Ablation of the SCN caused a profound reduction of the Bicuculline induced hyperglycemia, i.e. there was no significant difference between the Bicuculline treatment and the control day (
Basal plasma glucose and hormone concentrations, i.e. just before opening the door in front of the food hopper and the start of the meal, are indicated in
Open circles show the results of the control experiment in the same animals one week later. Meal feeding caused significant increases of plasma glucose and plasma insulin concentrations on all occasions. Both Muscimol and treatment with the NMDA-antagonist cocktail caused a significant reduction of the glucose and insulin response during the ZT14, but not the ZT8, meal. These changes in glucose and insulin responses were observed despite matching of meal size during drug treatment and control meals. For details of statistical analysis see Tables S6 and S7.
Glutamate and GABA are the most abundant excitatory and inhibitory neurotransmitters in the central nervous system. The present study shows a pronounced time-of-day dependent hyperglycemic effect of the GABA-antagonist bicuculline, but not the glutamate receptor agonist NMDA, when administered in the paraventricular nucleus of the hypothalamus (PVN). Results in SCN-lesioned animals clearly show that this time dependency is derived from the biological clock situated in the hypothalamic suprachiasmatic nuclei (SCN). Combined with our previous liver-denervation experiments
An alternative explanation for the strong time-of-day dependency of the hyperglycemic effect of BIC could be the well-known rhythm in hepatic glycogen stores with its acrophase at light onset
Recently Bando et al
At present it is not clear where the glutamatergic input to the parasympathetic pre-autonomic PVN neurons originates, although the diurnal variation clearly indicates an involvement of the SCN. Strubbe et al
In addition to probe placement, spread of the administered drugs to neighboring areas is always an important concern for the present type of studies. By comparing drug effects of the same drug applied in different hypothalamic nuclei we could calculate in a previous study that when applied by microdialysis the effective spread of BIC was ∼0.5 mm
Next to the autonomic nervous system also circulating epinephrine, glucagon and corticosterone are major players in the regulation of plasma glucose
With regard to the circadian control of the autonomic nervous system the picture that emerges from the present data and our previous experiments is one that shows an important role for a GABAergic-glutamatergic switch, not only with respect to the sympathetic branch but also where it concerns the parasympathetic branch. The different timing of the daily peak in melatonin release (i.e. ZT16–22) and hepatic glucose production (i.e. ZT10–14) indicates that the GABAergic efferents from the SCN differentiate between the pre-autonomic neurons that control the sympathetic input to the pineal and the neurons that control the sympathetic input to the liver. In view of this highly differentiated somatotopic organization it is not surprising that SCN efferents are also able to differentiate between sympathetic and parasympathetic pre-autonomic neurons. Indeed, using a combination of double retrograde viral tracing and selective organ denervation, we found neuro-anatomical evidence for a somatotopic organization in the SCN
In conclusion, the daily rhythm in the activity of the pre-autonomic neurons is predominantly determined by somatotopically organized GABAergic SCN outputs that inhibit selective groups of pre-autonomic neurons at specific times of the L/D-cycle. The abundant presence of GABAergic neurons in the SCN nicely fits with such a prominent role for GABAergic SCN efferents
Male Wistar rats (Harlan, The Netherlands) were housed at a room temperature of 21±1 C with a 12-h light/dark (L/D) cycle (lights on at 07.00 h). For experiments performed during the dark period, animals were housed in a reversed L/D-cycle with lights on at 19.00 h. Animals were allowed to adapt to the new environment for 2 weeks (or 6 weeks in case of housing in a reversed L/D-cycle) before the first experiments. Animals were kept with 4–6 animals per cage until one week before surgery, at which time they were transferred to individual cages (25×25×35 cm). Food and water were available
Experimental animals destined to undergo infusion and blood sampling studies were fitted with bilateral microdialysis probes and an intra-atrial silicone catheter through the right jugular vein when the body weight had reached 300 g, as described previously
A total of 36 rats (180–200 g) were operated upon in order to ensure a sufficient number of effectively lesioned animals. For the SCN lesion procedure animals were mounted with their heads in a David Kopf stereotact (Tujunga, CA) with the toothbar set at +5.0 mm, and sustained a bilateral lesion of their SCN (coordinates 1.4 mm rostral to bregma, 1.1 mm lateral to the midline and 8.3 mm below the brain surface) using bilateral lesion electrodes, 0.2 mm in diameter, that were heated to 85°C for 1 minute (lesion generator, Radionics, Burlington, MA). This temperature/duration combination was found empirically to result in lesion large enough to eliminate the SCN, but small enough to leave surrounding hypothalamic structures, such as the PVN and supraoptic nucleus, intact. Drawback of minimizing the lesion size, however, is the limited yield of completely lesioned animals (∼30%). In order to restrict the number of (false positive) animals to be operated upon an initial screening is made by measuring their diurnal water intake. After a 2-week recovery period the effectiveness of the SCN-lesions was checked by measuring the animals' drinking behavior during a 3-week period
Ringer's perfusion (3 µl/min) was started 2 hours before the start of the 2-h drug infusion period and lasted for 2 more h after the change back to Ringer = s. The 2-h drug infusion periods were initiated at ZT5 or ZT15. Blood samples (0.6 ml) were taken 30 min before (t = −30) and 0, 30, 60, 120 and 180 min after the start of the drug infusion. The following neurotransmitter (ant)agonists were administered to the PVN: muscimol (MUS; a potent GABAA receptor agonist; 100 µM), bicucilline (BIC; a GABAA receptor antagonist; 100 µM), NMDA (a glutamate receptor agonist; 100 µM), and MK801 (a potent, selective and non-competitive NMDA receptor antagonist; 100 µM). In the scheduled feeding experiments (see below) a cocktail of NMDA antagonists was used. This cocktail consisted of MK801 (100 µM), AP5 (500 µM), and DNQX (100 µM). All drug concentrations were chosen on basis of our own previous microdialysis experiments (BIC, MUS, NMDA and MK801
For the scheduled feeding experiment, rats were entrained to a scheduled feeding regimen very much similar to our previously published method
When the experimental protocol was completed animals were anaesthetized with CO2/O2 and decapitated. Brains were subsequently removed, blocked, frozen, sectioned (40 µm) through the hypothalamus, and stained with cresyl violet. In the case of SCN-lesions the sections were processed for vasopressin and VIP immunocytochemistry.
Blood samples were immediately chilled on ice in tubes containing a 10 µl solution of 2.5% EDTA+10% benzamidine hydrochloride (BDH) and centrifuged at 4 C. Plasma was then stored at −80 C until further analysis. Plasma glucose concentrations were determined using a Glucose/GOD-Perid method (Boehringer Mannheim, Mannheim, Germany). Plasma immunoreactive insulin and corticosterone concentrations were measured using radioimmunoassay kits (LINCO Research Inc., Missouri, USA and ICN Biomedicals, Costa Mesa, CA, respectively). All samples were assayed in duplicate. Hepatic glycogen content was measured as described previously
We evaluated the kinetics of the plasma hormone and glucose concentrations as a consequence of the hypothalamic infusions (and the food intake) by using the increments of their plasma concentrations compared with the t = 0 value. The significance of the infusion- (or feeding-)induced variations in plasma glucose and hormone values was assessed using a multivariate analysis of variance (MANOVA) with repeated measures with
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We thank Wilma Verweij for correcting our English and Henk Stoffels for making the figures. The expert assistance of J. Timmer in animal husbandry is gratefully acknowledged.