Hypothalamic Ventromedial Nuclei Amplify Circadian Rhythms: Do They Contain a Food-Entrained Endogenous Oscillator?

Surgery and intracerebral injections

All rats were placed in a stereotaxic apparatus after injections of a rodent anesthetic mixture consisting of ketamine, xylazine, and acepromazine (77, 1.5, and 1.5 mg/ml, respectively; 1 ml/kg, i.p.). The scalp was incised in the midline and retracted, the skull was cleaned, and two small holes were drilled through it. After a 28 gauge needle connected to a microsyringe (Hamilton) was lowered to the appropriate coordinates, sterile saline (0.1 μl) or colchicine (1 μg/0.1 μl) [50:50 colchicine/fluorescein (Molecular Probes, Eugene, OR)/colchicine (Sigma, St. Louis, MO) mixture] in sterile saline was injected into the VMN bilaterally. With the upper incisor bar positioned at −3.3 mm below horizontal zero, injections were made at the following coordinates: 2.6 mm posterior from bregma, 0.7 mm lateral to the midsaggital suture, and 9.2 mm below the surface of the skull (Paxinos and Watson, 1986). All intracerebral injections were made over 1 min followed by a 5 min period before the syringe was removed. The scalp was sewn, and the rats were allowed to recover from anesthesia before returning them to their cages in the animal room. On completion of each experiment, brains were post-fixed in 10% formalin for 24 hr and then stored in a 30% sucrose in 0.1 m PBS solution at 4°C or perfused and fixed with 0.15 m phosphate buffer and ice-cold 4% formalin.

Experiments

Study 1. This study characterized daily rhythms in both core body temperature and locomotor activity after disruption of VMN function with colchicine. After calibration, biotelemetry temperature transmitters (model TM; Mini-Mitter Inc., Sunriver, OR) were inserted intraperitoneally under anesthesia and were monitored for 7 d to ensure a stable baseline of activity. Transmitted temperature and activity signals were picked up every 10 min by an antenna located outside the cage. Subsequently, telemetry data were averaged in 1 hr bins. On day 7, sterile saline or colchicine was injected bilaterally into the VMN (seven rats per group). After surgery, core body temperature and locomotor activity were measured for 15 d. Body weight and food consumption were measured on days 4–7 with a 24 hr fast occurring on day 5. A power outage caused 42 hr of data to be lost.

Study 2a. We examined the circadian responses of behavioral and neuroendocrine systems to VMN disruption with colchicine. Food and water consumption were measured twice daily (at 10 and 14 hr intervals during the light/dark period) for 2 d before and up to 5 d after surgery. Food consumption was calculated by the weight of noningested and spilled food at the end of the measurement period subtracted from the initial weight of the food placed into food bins. On day 5, in both saline- and colchicine-injected animals (16 rats per group), blood samples were taken without anesthesia after lights on in the morning (9:00–10:30 A.M.) or just before lights off in the evening (6:00–7:00 P.M.) from a small incision made in a lateral tail vein (Akana et al., 1992). Blood (∼0.2 ml) was collected in chilled tubes containing 0.3 m disodium EDTA (10 μl/tube). Corticosterone (B) and insulin levels were measured in plasma from blood collected at 0 and 30 min after restraint. The basal blood collection was complete within 60 sec of removing the rat from its cage and placing it in a Plexiglas restraining tube. A second blood sample was obtained after 30 min restraint (Akana et al., 1992). After the restraint period, rats were decapitated, and trunk blood (5 ml) was collected in chilled tubes containing 0.3 m disodium EDTA (100 μl/tube).

Study 2b. To characterize HPA activity after VMN with colchicine in greater detail, another set of rats was injected with saline or colchicine (eight rats per group). Food consumption was measured for 3 d after surgery to be certain of VMN inhibition. Saline- and colchicine-injected rats were equally divided and assigned to two groups. Each group consisted of four saline- and four colchicine-treated animals. At the start of the experiment, a blood sample taken from a tail vein nick was collected from each unanesthetized animal in one of the two groups. Two hours later, blood samples were similarly collected from the second group of rats. Subsequent samples were collected from both groups every 4 hr staggered at 2 hr intervals. This syncopated pattern of blood collection was repeated throughout a 24 hr period. All blood samples during the dark period were collected under dim red light. In the final analysis, values from both groups were pooled to represent corticosterone levels at 4 hr intervals.

Study 3a. The first phase of this experiment assessed c-Fos expression 5 d after saline or colchicine injections into the VMN in rats fed ad libitum. Five days after injections of saline (n = 10) or colchicine (n = 16) into the VMN, blood samples were collected from the tails of unanesthetized rats for measurement of basal levels of B. Half of each group was anesthetized and perfused immediately before the usual time of food presentation during the light cycle; the other half was anesthetized and perfused 3 hr after lights off during the dark cycle on the same day. In samples collected during the dark, rats were anesthetized under dim red light; when asleep, their heads were wrapped loosely with foil before taking them out of the animal room for perfusion that was accomplished in dim light.

Study 3b. The experiment was repeated, but the animals were allowed food for only 2 hr daily, provided 2 hr after lights on (RF). A limited period of food availability during the light period (a time when rats do not normally eat) has been shown to shift rhythms of body temperature, HPA activity, and locomotor activity to peak at the onset of food presentation instead of at the onset of, or during, darkness (Krieger, 1980). Food intake and body weight were measured daily during the protocol. After 16 d of RF, rats were divided into two groups and injected on the 17th day with either saline or colchicine into the VMN as described above. RF was continued, and 5 d after surgery, animals in the light, prefood, and dark phases of the experiment were anesthetized and perfused (described above).

Sections of brain were cut on a freezing microtome at 30 μm, and every section through the VMN was collected and stored in cryoprotectant. Free-floating sections to be reacted with antibodies to Fos protein were incubated with 3% normal goat serum in Tris-PBS (TPBS) and 0.3% Triton X-100 (Sigma) solution for 60 min at room temperature. After this, sections were incubated overnight at room temperature with a rabbit polyclonal antiserum against the N-terminal region of the Fos protein (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA) in TPBS preabsorbed with rat liver powder (Organon Teknika-Cappell, Malvern, PA) to reduce background staining. Sections were subsequently incubated with a biotinylated anti-rabbit IgG in goat (Vector Laboratories, Burlingame, CA) diluted 1:200 in the above TPBS mixture for 1 hr at room temperature, washed, and then incubated with an avidin–biotin–peroxidase complex (Vector) for 1 hr at room temperature. 3,3-Diaminobenzidine tetrahydrochloride (Sigma) with 0.4% NH₄Cl, 20% β-d-glucose, and 1% nickel ammonium sulfate were used to produce a black chromagen.

Hypothalamic and thalamic cell groups were analyzed at the following anterior and posterior levels based on the brain atlas of Paxinos and Watson (1986): SCN (bregma, −1.30 mm), anterior sPVNz (bregma, −1.80 mm), posterior sPVNz (bregma, −2.30 mm), VMN (bregma, −2.56 mm), DMN (bregma, −3.14 mm), parvocellular PVN (parvoPVN) and magnocellular PVN (magnoPVN; bregma, −1.80 mm), mPOA (bregma, −1.30 mm), ARC (bregma, −2.30 mm), and pvTHAL (anterior bregma, −1.40 to −1.80 mm; medial bregma, −2.12 to −2.30 mm; posterior bregma, −2.56 to −2.80 mm) (Bhatnagar and Dallman, 1997). We did not quantify c-Fos counts in any subdivision of these cell groups that has not already been specified. Throughout the analyses, we established particle size limits to distinguish c-Fos immunoreactive cells from staining of non-neuronal particles. Analysis of Fos-like immunoreactivity was measured using NIH Image software (W. Rasband, National Institutes of Health).

Histological analysis

In all animals with injections targeting the VMN, histology was performed to locate the needle tracks and injection site in cresyl violet-stained sections combined with adjacent fluorescent sections. Only rats with bilateral injections into the VMN and visible fluorescence contained within the VMN were included in the analyses and results. Analysis of the fluorescent spot confirmed our previous observation that at 5 d after injection the inactive fluorocolchicine (S. Choi and M. F. Dallman, unpublished observations) and presumably also the colchicine remained localized to the VMN (Choi et al., 1996). These criteria were achieved in 60–70% of the animals overall.

Radioimmunoassays

After centrifugation of the blood at 3000 rpm and 4°C, separated plasma was stored at −20°C and used in radioimmunoassays for B and insulin. Plasma B was measured using an Immuchem double-antibody corticosterone RIA kit for rat (ICN Biochemicals, Costa Mesa, CA). The limit of detection was 1.5 ng of B/ml. Plasma insulin was measured using a rat insulin RIA kit (Linco, St. Charles, MO). The limit of detection was 0.1 ng/ml.

Statistical analysis

Data were analyzed using ANOVA corrected for repeated measures (when required). When main effects were significant, Scheffe analysis was used to test significance of post hoc effects. When only two groups were compared, Student’s unpaired t test was used. Telemetry data were analyzed by fitting a single cosine function to circadian data from each animal. The cosine function used was:

where y is the measured variable (temperature),A is the amplitude, t is the time of day, φ is the phase shift adjustment, and T is the mean temperature.

All statistical analyses were conducted using commercial statistical software packages (StatView, Abacus Concepts, Calabasas, CA; DeltaGraph, Deltapoint Inc.). Statistical significance was established at p ≤ 0.05. In all figures, mean data are accompanied by capped lines indicating SEM.

Ad libitum feeding

The SCN exhibited a pattern in c-Fos immunoreactivity with more protein-expressing cells in the light than dark (Fig.8). By contrast, the number of c-Fos-immunostained cells was significantly higher in the dark than light in the DMN, parvoPVN (Fig. 9,pPVN) and magnoPVN (Fig.10, mPVN) and in all regions of the pvTHAL (Fig. 11). The mPOA also tended to have increased numbers of c-Fos-immunostained cells in the dark (p = 0.057) (Fig. 10). In contrast, Figures 8-10 also show no light/dark differences in c-Fos expression in the sPVNz, the VMN, or the ARC.

Five days after bilateral colchicine injections into the VMN, the light/dark pattern in c-Fos protein expression remained in the SCN but was abolished in the DMN, parvoPVN, magnoPVN, medial preoptic area, and posterior pvTHAL (Figs. 8-11). Furthermore, colchicine injections into the VMN significantly increased c-Fos cell number in the VMN, DMN, and parvoPVN during the light.

Restricted feeding

Restricted feeding in control animals did not affect the phase of patterns in c-Fos-expressing cells in the SCN (Fig. 8), parvoPVN, magnoPVN, mPOA (Figs. 9, 10), or pvTHAL (Fig. 11). However, RF did eliminate the pattern in the DMN (Fig. 9). Interestingly, in RF rats, there was a marked, overall increase in c-Fos-positive cells, and also a pattern emerged in the VMN of control animals with more c-Fos-positive cells in dark than light. This is like all other patterns except those in the SCN. A c-Fos pattern did not exist in the VMN of animals fed ad libitum (Fig. 9). As in animals fedad libitum, the sPVNz and ARC did not exhibit any light/dark differences in c-Fos expression (Figs. 8, 11).

Similar to the results in rats fed ad libitum, colchicine injections into the VMN of RF animals abolished patterns of c-Fos expression in the VMN, parvoPVN, magnoPVN, mPOA, and the entire extent of the pvTHAL (Figs. 9-11). In marked contrast, the sPVNz did express a significant light/dark pattern after colchicine injections in RF animals similar to that in SCN (Fig. 8) (p≤ 0.05).

Missed VMN injections

We have previously shown the lack of effect of injections of colchicine that missed the VMN on food intake and body weight gain (Choi et al., 1996). Furthermore, we have reported that we can clearly distinguish among the effects of bilateral injections of colchicine into the PVN, ARC vs DMN vs VMN (Choi et al., 1997), suggesting strongly that placement and discrete localization is important for the changes that occur after injections of colchicine into the VMN. In Study 3b there were four rats with missed injections in the light group and three rats with missed injections in the dark group that were RF. In the VMN of the missed light and dark groups there were 120 ± 26 and 216 ± 51 c-Fos-stained cells, respectively. The small number of rats does not yield either a significant pattern in VMN (observed in control, RF rats) or a significant difference compared with RF rats with bilateral injections that show equal numbers of c-Fos cells in light and dark (Fig. 9). In these rats, c-Fos in the medial pvTHAL was 285 ± 52 and 368 ± 29 cells in the light and dark, respectively, again suggesting a light/dark pattern in c-Fos cell number, like that in control rats (Fig. 8). The number of cells stained with c-Fos in the anterior sPVNz in these rats was 206 ± 35 in the light and 292 ± 39 in the dark and in the posterior sPVNz was 145 ± 34 in the light and 218 ± 85 in the dark. The lack of significant difference between light and dark counts in the sPVNz demonstrates that colchicine injections that miss the VMN do not cause the gain in neuronal activity pattern that is similar in phase to that of the SCN.