Altered hormone levels and circadian rhythm of activity in the WKY rat, a putative animal model of depression

Departments of ¹ Psychiatry and Behavioral Sciences and ² Neurobiology and Physiology, Northwestern University, Chicago, Illinois 60611

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Wistar Kyoto (WKY) rat is hyperreactive to stress and exhibits depressive-like behavior in several standard behavioral tests. Because patients with depressive disorders often exhibit disruptions in the circadian rhythm of activity, as well as altered secretory patterns of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid hormones, we tested the hypothesis that these phenomena occur in the WKY rat. Plasma ACTH and corticosterone levels remained significantly higher after the diurnal peak for several hours in WKY rats relative to Wistar rats. Also, plasma levels of thyroid-stimulating hormone were significantly higher in WKY relative to Wistar rats across the 24-h period, despite normal or slightly higher levels of 3,5,3'-triiodothyronine. In addition, under constant darkness conditions, WKY rats exhibited a shorter free running period and a decreased response to a phase-delaying light pulse compared with Wistar rats. In several ways these results are similar to those seen in other animal models of depression as well as in depressed humans, suggesting that the WKY rat could be used to investigate the genetic basis for these abnormalities.

hypothalamic-pituitary-adrenal axis; hypothalamic-pituitary-thyroid axis; forced swim test

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

SEVERAL DIFFERENT LABORATORIES have shown that the Wistar Kyoto (WKY) rat, a proposed animal model of depression, exhibits a hyperresponsiveness to stress and depressive-like behavior in multiple behavioral tests, including the forced swim test (FST; e.g., Refs. 3, 8, 13, 14, 19, 21, 39, 40, 44, 47, 51-54, 56, 62, 70, 77). These depressive-like behaviors are reversed after treatment with antidepressant drugs (39, 40, 56). Recent studies also indicate that WKY rats exhibit a 50% increase in paradoxical sleep during the normal sleep phase, as well as increased sleep fragmentation, relative to Wistar rats (18), paralleling some sleep abnormalities in depressed humans (11). As these depressive-like behaviors and sleep abnormalities are inherent to the animal, this strain may be useful as a genetic animal model of depression.

Although only single time point determinations have been made, WKY rats also appear to exhibit hormonal abnormalities of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid (HPA and HPT, respectively) axes, two axes that are often disrupted in depressed patients. For example, WKY rats show increased plasma ACTH after both chronic and acute stress relative to several other rat strains, including the Wistar rat (24, 56, 62), and have higher basal thyroid-stimulating hormone (TSH) and 3,5,3'-triiodothyronine (T₃) levels relative to Wistar rats (63).

In human depression, the daily sleep/wake cycle and several hormonal rhythms, especially those of the HPA and HPT axes, are often disrupted (for reviews, see Refs. 20, 50, 69, 78). Due to these circadian rhythm alterations in depressed humans, we tested the hypothesis that the WKY rat would exhibit similar hormonal and activity rhythm alterations.

To test this hypothesis we examined the diurnal secretory patterns of ACTH, corticosterone, and TSH in freely moving WKY rats kept on a long-day (LD) cycle, relative to their outbred progenitor strain, the Wistar rat (45). We also measured plasma levels of T₃ at a single time point in these two strains. In addition, we monitored activity rhythms in these strains under both LD and constant dark (DD) conditions. To investigate the intrinsic properties of the master circadian pacemaker, we measured the free running rhythm and the response of the activity rhythm after a light pulse while rats were under DD conditions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Experimental Protocol

Eleven male Wistar and eleven male WKY rats were obtained from Harlan Sprague Dawley at 10-11 wk of age. Immediately on arrival, rats were placed into individual cages in a light-tight room on a 14:10-h light-dark cycle (lights on at 0700, off at 2100 Central Standard Time). Animals were kept under constant ambient temperature (21 ± 1°C) with food and water available ad libitum. Rats were handled approximately four times a week to accustom them to the investigator in an effort to decrease stress during the 24-h period of blood sampling. Two weeks after arrival, animals were weighed and implanted with a jugular cannula to allow for serial blood sampling over a 24-h period. The 24-h bleed took place 2 days after the surgery.

At 6 mo of age, eight of the previously cannulated Wistar and nine WKY rats were placed into light-tight boxes to monitor daily activity rhythms. The light intensity in these boxes was ~300 lx (white light). Animals were maintained under a 14:10-h light-dark cycle for a 3-wk period and were then transferred to DD for the remainder of the experiment. After 2 wk in DD, animals were exposed to a 15-min light pulse (white light) 1 h after activity onset. Exposure to light at this time is known to cause phase delays in the activity rhythm of rats (57). Activity rhythms were monitored in DD for a 2-wk period after the light pulse.

Two months after the 24-h bleed and before monitoring activity rhythms, an FST was administered to verify a depressive-like state in the WKY rat. Previous studies have shown that WKY rats exhibit high levels of immobility in the FST relative to several other rat strains, which has been interpreted as a high level of despair in this strain (e.g., Refs. 44, 56).

To obtain sufficient plasma volume for measuring T₃ levels, eight male Wistar and eight male WKY rats were killed by decapitation at 1700 in a separate study. Trunk blood was collected on ice in 15-ml conical tubes with EDTA (15.6 mg EDTA/tube). Plasma was collected and stored at 80°C. Levels of T₃ were determined by radioimmunoassay.

Jugular Cannulation and 24-h Blood Sampling

Before jugular cannulation, animals were anesthetized with an injection of ketamine (80 mg/kg ip) and xylazine (12 mg/kg ip). A small incision was then made in the neck to expose the jugular vein, and the short end of the cannula was inserted into the vein and securely tied off. The long end of the cannula was drawn out through a small incision in the back of the animal to enable serial sampling with minimal disturbance to the animal. After surgery, 0.5 ml of gentamicin antibiotic was administered to each animal to prevent infection. To prevent clogging, cannulas were flushed the following day with heparinized saline (5 U/ml).

On the following day (2 days postsurgery), 1 h before the first blood sample on the morning of the serial bleed, all cannulas were flushed with heparinized saline and small blood samples (0.1 ml) were taken to obtain hematocrit readings. These initial readings were compared with hematocrit readings taken at the end of the 24-h bleed. Animals had a starting hematocrit of 45.1% (±0.77) and an ending hematocrit of 42.0% (±0.33).

During the 24-h bleed, blood samples were collected at the following time points: 0800, 1100, 1400, 1700, 2000, 2030, 2100, 2130, 2200, 2430, 0300, 0600, and 0800. The more frequent blood samples between 2000 and 2200 were taken to ensure the detection of the diurnal peaks for ACTH and corticosterone, which normally occur in this time frame. When samples were taken every 30 min, 0.3 ml of blood was taken, whereas at all other time points, 0.5 ml of blood was taken. Blood samples were collected on ice into EDTA coated tubes (2.5 mg EDTA per tube). After each sample, the same amount of donor blood (0.3 or 0.5 ml) was given to each animal. Donor blood was prepared as previously described (76), with donor blood cells reconstituted in sterile saline to reach a hematocrit of 45-47%. Plasma was collected and stored at 80°C for subsequent determination of ACTH, corticosterone, and TSH hormone levels by RIA.

RIAs

All assays were done in duplicate.

Corticosterone. The RIA was carried out as described previously (62). Briefly, 1-2 µl of plasma was incubated overnight with the primary corticosterone antibody raised against corticosterone 3-carboxymethyloxime-bovine serum albumin, with ¹²⁵I-labeled corticosterone conjugate as the tracer (ICN Pharmaceuticals, Costa Mesa, CA). The assay sensitivity was 16.7 pg/tube. The intra- and interassay coefficients of variation were 11.6 and 7.5%, respectively.

ACTH. As described previously (62), 25 µl of unextracted plasma was incubated for 2 days with a primary ACTH antibody that recognizes ACTH-(1-24) and -(1-39) on an equimolar basis (1:8,000 dilution, INCSTAR, Stillwater, MN) and incubated for 1 day with ¹²⁵I-labeled ACTH as the tracer (Amersham, Piscataway, NJ). The assay sensitivity was 1.5 pg/tube. The intra- and interassay coefficients of variation were 9.6 and 10%, respectively.

TSH. As described previously (64), 25 µl of unextracted plasma was incubated for 2 days with the primary TSH antibody (1:500,000 dilution; National Institute of Diabetes and Digestive and Kidney Diseases, Baltimore, MD), and incubated for 1 day with ¹²⁵I-labeled TSH tracer (rat TSH RP-2, iodinated by the Chloramine-T method). The assay sensitivity was 23.3 pg/tube (0.9 ng/ml). The intra- and interassay coefficients of variation were 8.1 and 6.7%, respectively.

T₃. T₃ RIAs were performed using ImmunoChem-coated tubes purchased from ICN Pharmaceuticals (Costa Mesa, CA), using 100 µl of unextracted serum and incubating with ¹²⁵I-labeled T₃ as tracer (provided) at 37°C for 1 h. The assay sensitivity was 24.7 ng/dl. The intra-assay coefficient of variation was 3.6%.

Activity Rhythm Data Collection and Analysis

An infrared sensor (Petite 101, Newark, NJ) was placed over each cage to monitor daily activity rhythms. The number of infrared beam crossings was continuously recorded and sent to an online data-acquisition system (Chronobiology Kit, Stanford Software Systems). Clocklab (Actimetrics, Evanston, IL), a rhythm analysis program, was used to analyze the activity rhythm data. While rats were kept on an LD cycle, the phase angle of entrainment was measured. In DD, the free-running period of the activity rhythm of the rats was measured, using the ² periodogram, 10 days before and 14 days after administration of the light pulse. The onset of activity was defined by the Clocklab program as follows: Clocklab searches for a best fit between the activity record and an activity pattern consisting of 6 h of inactivity followed by 6 h of activity. To find the most likely activity onset time, the program replaces each point in the activity record with "1" if it is in the top 80% of non-zero counts, or "1" otherwise. The results are then convolved with a square wave that consists of 1 for the first 6 h and 1 for the next 6 h. The local maximum of this convolution within each 24-h period is taken as the activity onset time. To calculate phase shifts in the activity rhythm after the light pulse, Clocklab fit a line to the onsets of locomotor activity for 10 days before the light pulse and for 14 days after the light pulse. Then, a second line was retro-projected to the day of the light pulse, and the magnitude of the phase shift was calculated as the difference between the two lines.

FST

The FST procedure used was similar to the one described by Porsolt et al. (60). Briefly, animals were placed into a large cylinder (30 cm × 45 cm) of 25°C water for a 15-min period. Twenty-four hours later, the rats were again placed into the cylinder of water for a 5-min period. All testing took place between 1100 and 1430. Activity during the posttest was video recorded for subsequent scoring. We used a time sampling technique for scoring, as previously described by Detke et al. (16), where time immobile, swimming, and climbing, measured every 5 s, were labeled as bins.

Statistical Analyses

A repeated measures one-way ANOVA was used to determine the statistical significance in hormone levels across the 24-h period between WKY and Wistar rats. Differences between the two strains at specific time points were also determined using Bonferonni's post hoc test. A two-way ANOVA was used to measure statistical differences in free running period between Wistar and WKY rats both before and after the light pulse. A Newman-Keuls post hoc test was used to make pairwise comparisons where significant differences were seen. A one-way ANOVA was used to measure statistical differences in the magnitude of the phase shift between the two strains. A one-way ANOVA was also used to determine statistical significance in body weight (at the time of jugular cannulation) as well as immobility, climbing, and swimming behaviors in the FST between WKY and Wistar rats. Differences in plasma levels of T₃, taken from trunk blood, were also determined by a one-way ANOVA. Means are reported with standard error of measurement.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Confirming data from several other laboratories, including our own (i.e., Refs. 3, 39, 51), WKY rats exhibited increased immobility (WKY = 9.7 ± 1.3 bins vs. Wistar = 3.7 ± 0.87 bins, F_1,19 = 14.33, P < 0.01) and decreased climbing (WKY = 5.8 ± 1.2 bins vs. Wistar = 11 ± 2.2 bins, F_1,19 = 5.86, P < 0.05) in the FST, a behavioral test for despair. No differences were seen in time spent swimming between the two strains. WKY rats also weighed significantly less than Wistar rats (WKY = 272.1 ± 3.0 g vs. Wistar = 397.5 ± 10.7 g, F_1,22 = 126.2, P < 0.0001), similar to weights shown previously in these strains (55).

Diurnal Profile of Plasma ACTH and Corticosterone

Plasma levels of ACTH were similar between the two strains during the daylight hours. However, plasma levels of ACTH remained significantly higher for several hours after the peak in WKY rats relative to Wistar rats (see Fig. 1A). With the use of a one-way repeated measures ANOVA on the 24-h hormonal profile between the two strains, there were main effects of time (F_12,211 = 11.38, P < 0.001) and strain (F_12,211 = 13.07, P < 0.01), as well as a strain × time interaction (F_12,211 = 3.06, P < 0.001). WKY rats exhibited significantly higher levels of ACTH relative to Wistar rats at 2430 (P < 0.01, Newman Keuls) and 0300 (P < 0.05, Newman Keuls), demonstrating a sustained peak of ACTH in this strain.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Twenty-four-hour profiles of plasma ACTH (A) and corticosterone (Cort; B) levels in Wistar Kyoto (WKY) and Wistar rats. Values are means ± SE of 8-11 animals of each strain at each time point. For plasma ACTH, only 5 or 6 animals are represented at 2030 and 2130. The dark bar along the x-axis represents time of lights out (2100) (*P < 0.05, **P < 0.01, WKY vs. Wistar).

Plasma levels of corticosterone were also similar between the two strains during the daylight hours. Similar to plasma ACTH, plasma levels of corticosterone remained significantly higher for several hours after the peak in WKY rats relative to Wistar rats (see Fig. 1B). Using a repeated-measures one-way ANOVA on the 24-h hormonal profile between the two strains, there were main effects of time (F_12,243 = 38.72, P < 0.001) and strain (F_12,243 = 6.98, P < 0.01) and a strain × time interaction (F_12,243 = 3.75, P < 0.001). A sustained peak of corticosterone in WKY rats is indicated by significantly higher levels of corticosterone in this strain relative to Wistar rats at 2200 and 0300 (P < 0.01, Newman Keuls).

Diurnal Profile of TSH and Single Time Point Levels of T₃

Levels of TSH were significantly higher in WKY rats relative to Wistar rats at several time points sampled during the 24-h period (see Fig. 2). With the use of a repeated measures ANOVA, there were main effects of time (F_12,211 = 8.45, P < 0.001) and strain (F_12,211 = 46.17, P < 0.001) and a strain × time interaction (F_12,211 = 3.69, P < 0.001). WKY rats exhibited a significant peak of plasma TSH at 1100. There was a highly significant increase in levels of plasma TSH at 0800, 1100, 1400, 1700, and 0600 in WKY rats relative to Wistar rats (P < 0.01, Newman Keuls).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Twenty-four-hour profile of plasma thyroid-stimulating hormone (TSH) levels in WKY and Wistar rats. TSH values are means ± SE of 8-11 animals of each strain at each time point. At 2100 and 2130 only 6 animals are represented. The dark bar along the x-axis represents time of lights out (2100) (**P < 0.01, WKY vs. Wistar).

Plasma levels of T₃ in WKY rats, measured from trunk blood taken at 1700, were significantly higher relative to Wistar rats (WKY = 168.8 ± 6.5 vs. Wistar = 120.2 ± 9.8 ng/ml, F_1,14 = 17.16, P < 0.001).

Activity Rhythms

While on an LD cycle, no differences were seen in the phase angle of entrainment between WKY and Wistar rats. However, while under DD conditions, WKY rats had a significantly shorter free running period relative to Wistar rats, and this difference was enhanced after the phase-delaying light pulse (see Figs. 3 and 4). With the use of a two-way ANOVA on free running period both before and after administration of the light pulse, there were significant main effects of strain (F_1,13 = 35.63, P < 0.001), light pulse (F_1,13 = 63.95, P < 0.001), and a strain × light pulse interaction (F_1,13 = 9.15, P < 0.01). The initial free running period for the WKY rat was 24.0 h (±0.02), significantly shorter than that of the Wistar rat, 24.1 h (±0.03) (P < 0.05, Newman Keuls). After the phase-shifting light pulse, this difference in free running period increased as the free running period of WKY rats lengthened by only 9.6 min to 24.16 h (±0.03), whereas the period of Wistar rats lengthened by 20.4 min to 24.44 h (±0.04) (P < 0.01, Newman Keuls). WKY rats also exhibited a smaller phase delay in the activity rhythm after the light pulse: 40.2 (±7.8) min phase delay in WKY rats as opposed to 83.4 (±10.8) min delay in Wistar rats (F_1,16 = 10.83, P < 0.01) (see Figs. 3 and 5).

View larger version (78K): [in this window] [in a new window]   Fig. 3.   Representative activity records for Wistar (A-C) and WKY (D-F) rats while in constant darkness (DD). The 15-min light pulse, marked by an arrow, was administered at circadian time (CT) 13 on day 11.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Mean free running period (±SE) of Wistar and WKY rats while in DD, both before and after the light pulse was administered. Data were analyzed over a 10-day period before administration of the light pulse (prelight pulse) and over a 14-day period after the light pulse (postlight pulse). *P < 0.05, **P < 0.01.

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Mean phase-shift, in hours (±SE), in response to a 15-min light pulse presented at CT 13 in WKY and Wistar rats free-running in DD. Phase shifts were calculated as described in MATERIALS AND METHODS. Negative values represent phase delays. **P < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have demonstrated that WKY rats show prolonged plasma ACTH and corticosterone diurnal peaks, as well as hypersecretion of plasma TSH throughout the 24-h period and of plasma T₃ sampled at a single afternoon time point relative to Wistar rats. In addition, WKY rats exhibit a shorter free running period, as well as smaller phase shifts and an attenuated lengthening of the free running period after a light pulse relative to Wistar rats while in DD.

Hypersecretions of ACTH and Corticosterone in WKY Rats

Both WKY and Wistar rats exhibited diurnal rhythms of plasma ACTH and corticosterone secretion similar to those described previously for the rat (e.g., Refs. 2, 4). In WKY rats, the peaks of both ACTH and corticosterone, which occur after lights off, were prolonged, as plasma levels remained high for at least 5 h in WKY rats while the diurnal peaks of ACTH and corticosterone began to decline immediately after lights off in Wistar rats.

The differences in plasma levels of ACTH and corticosterone between WKY and Wistar rats occurred during the active dark period following the diurnal peak. Depressed patients, particularly the melancholic subtypes, exhibit increased levels of plasma ACTH and cortisol across the 24-h period (17, 43). A closer examination of the plasma cortisol profile in these studies reveals that the greatest difference is seen after the diurnal peak, because the peak is prolonged in depressed patients relative to normal controls, similar to what we see in the WKY rat. A phase advance of the diurnal cortisol peak has also been seen in depressed patients (43), which was not mirrored in the WKY rat.

The increased and prolonged peaks of ACTH and corticosterone in the WKY rat are more likely due to defective glucocorticoid negative feedback (79) than to increased corticotropin-releasing hormone (CRH) at this time (12, 49), as CRH mRNA binding and peptide content measured at single time points are not different in WKY rats relative to several other rat strains (24, 41, 62). However, although WKY rats seem to respond normally to dexamethasone treatment (23) and have hippocampal glucocorticoid or mineralocorticoid receptor binding and mRNA levels (24) similar to several other rat strains, adrenalectomy and corticosterone replacement have little or no effect on several glucocorticoid-dependent measures (62), including the dramatically elevated pituitary pro-opiomelanocortin mRNA in WKY relative to Wistar rats (61, 62). These data suggest that WKY rats may have a decreased sensitivity to glucocorticoids, which may be responsible, in part, for the prolonged diurnal peak of ACTH.

Hypersecretion of TSH and T₃ in WKY Rats

WKY rats, whose plasma TSH levels were much higher throughout the 24-h period relative to Wistar rats, exhibited a clear diurnal peak of plasma TSH, similar to that reported in other rat strains (e.g., Refs. 34, 65, 84). The finding that Wistar rats showed no discernible diurnal rhythm could be due to their low-to-normal TSH levels, which were close to the assay sensitivity. Interestingly, the elevated plasma TSH levels in WKY rats were not caused by a relative hypothyroidism, as T₃ levels were also slightly elevated in this strain.

Depressed humans exhibit alterations of the HPT axis, including both subclinical hyper- and hypothyroidism (7, 32). Subclinical hypothyroidism is manifested in slightly elevated plasma TSH levels and normal plasma levels of the thyroid hormones (25, 33) comparable to the elevated TSH and somewhat elevated T₃ found in WKY rats. In addition, depressed patients with subclinical hypothyroidism also tend to be resistant to treatment with antidepressants (27, 42, 58, 80). Similarly, WKY rats have been suggested as a model of treatment-resistant depression due to the unresponsiveness of this strain to acute treatment of antidepressants (39, 40). A lack of the nocturnal plasma TSH peak has been noted in some depressed patients (6, 22), which was not mirrored in the WKY rat.

The hypersecretion of TSH, together with slightly elevated T₃, is not likely to be due to increased stimulation of the thyrotroph, as a recent study found no differences in prepro-thyrotropin releasing hormone mRNA levels in several brain regions between WKY and Wistar rats (75). However, WKY rats may harbor a resistance to thyroid hormones, because similar thyroid function profiles have been seen in humans (for a review, see Ref. 36) and in animals with thyroid hormone resistance (1, 9, 82). Furthermore, decreased body weight has also been found in mutant thyroid hormone ₁-receptor transgenic mice, an animal model for thyroid hormone resistance (85), similar to the decreased body weight of WKY rats relative to Wistar rats. The assumption of thyroid hormone resistance in WKY rats is further supported by the finding that relative to Wistar rats, WKY rats require much higher doses of T₃ before exhibiting a behavioral response (63).

Altered Activity Rhythms Under DD Conditions in WKY Rats

No differences were noted in the activity profile of WKY relative to Wistar rats while under LD conditions. However, WKY rats did exhibit activity rhythm differences while in DD relative to Wistar rats. WKY rats exhibited a free running period of ~24 h, similar to what has been found previously in this strain (57, 66). The Wistar rat, on the other hand, had a free running period of 24.1 h, similar to previous findings in both Wistar and Sprague-Dawley rats (e.g., Refs. 10, 28). This difference suggests that the master pacemaker in WKY rats is running more quickly than that of the Wistar. In addition, the Flinders Sensitive Line, another genetic rat model of depression, has shown a similar difference in free running period relative to its control, the Flinders Resistant Line (71), suggesting a short free running period may be a common phenotype in animal models of depression. The difference in free running period between the two strains was enhanced after administration of a light pulse, inasmuch as the free running period in WKY rats increased by only 10 min, whereas the free running period of Wistar rats increased by >20 min. In addition, the phase delay of the activity rhythm in response to a light pulse was significantly smaller in WKY rats relative to Wistar rats. These differences suggest that the circadian pacemaker of WKY rats may be less responsive to light, thereby causing possible alterations in daily rhythm patterns of this strain.

It is difficult to measure activity rhythms in depressed patients, as these studies require temporal isolation to exclude environmental influences on the properties of the circadian clock. However, although controversial, there are some data demonstrating that patients with depression may have an altered sensitivity to light (see Refs. 20, 37).

Previous studies have found that spontaneously hypertensive rats (SHR) have a similar or shorter free running period than the normotensive WKY rats (57, 66, 68). Interestingly, when the hypertensive and hyperactive traits are separated by the selective breeding of progeny from a WKY and SHR cross (26), the hypertensive substrain exhibits a shorter free running period than the hyperactive substrain (67). These data provide an indirect link between free running period and hypertension and may offer a possible explanation for the short free running period in SHR. In addition, given that SHR and WKY rats are genetically related (45), it is not surprising that the free running period of both WKY rats and SHR is shorter than other rat strains.

The physiological basis for the smaller phase delays of the activity rhythm, as well as a decreased lengthening of the free running period after a light pulse in WKY rats relative to Wistar rats, is unknown. Rosenwasser and Plante (68) found that with increased light intensity, the free running period of WKY rats increased significantly more than that of the SHR, and thus have suggested that WKY rats have an increased sensitivity to light compared with SHR. In addition, an increase in light intensity results in a decreased lengthening of the free running period in SHR relative to WKY rats (66). However, in the present study, the WKY rat exhibited a decreased responsiveness to the phase-shifting light pulse compared with Wistar rats, suggesting that both SHR and WKY rats may have a decreased sensitivity to light. Although not confirmed, the decreased response of the WKY rat to a phase-shifting light pulse may be due to a difference in the shape of the phase response curve, because the delay/advance region of the phase response curve is related to the length of the free running period (15). The attenuated lengthening of the free running period in WKY relative to Wistar rats may be due to the observed smaller phase delays of the activity rhythm of this strain. In support of this hypothesis, light pulses resulting in large phase delays of the activity rhythm consistently lead to a lengthening of the free running period in several species, including the rat, whereas small phase delays do not consistently result in period lengthening (59, 74).

Activity Rhythm and Hormonal Alterations in the WKY Rat are Similar to Other Animal Models of Depression

HPA dysregulation has been demonstrated in several animal models of depression. For example, rats exposed to chronic mild stress exhibit several depressive-like behaviors (83) and have increased levels of basal corticosterone 24 h after termination of the stress procedure relative to levels before chronic mild stress exposure (5). In addition, the olfactory bulbectomy model of depression exhibits increased plasma levels of corticosterone in the early morning hours, right after lights turn on (46). Prenatal stress, another animal model of depression, results in a phase advance of the corticosterone rhythm (35). To our knowledge, thyroid function has not been assessed in other animal models of depression.

The depressive-like behavior in the FST has been shown to be dependent on both glucocorticoids and thyroid hormones (30, 31). Removal of glucocorticoids by adrenalectomy in the rat decreases immobility in the FST, which is reversed by treatment with glucocorticoids (29). In contrast, thyroidectomy and a state of mild hypothyroidism increase immobility in the FST, which is reversed by L-thyroxine treatment (38).

A shorter free running period can also be observed in other animal models of depression. For example, the Flinders Sensitive Line, a genetic line bred for anticholinesterase sensitivity, has a free running period of ~24 h, similar to our results in the WKY rat, significantly shorter than the control Flinders Resistant Line (71). In addition, rats exposed to prenatal stress exhibit depressive-like behavior in adulthood and also have a shorter free running period relative to control rats not exposed to prenatal stress (81). On the other hand, chronic or severe stress-induced depression models, in which only short-term behavioral changes are seen (i.e, chronic mild stress, defeat stress, learned helplessness), result in no change or in a lengthening of the free running period (i.e., Refs. 48, 72, 73), suggesting that a shortened free running period may be associated with long-term endogenous depressive-like behavior in rodents.

In conclusion, the present study is the first to demonstrate prolonged ACTH and corticosterone peaks, as well as both hypersecretion of TSH and T₃, in WKY relative to Wistar rats. In addition, WKY rats exhibited a shorter free running activity rhythm, an attenuated lengthening of the free running rhythm, and smaller phase shifts of the activity rhythm after a light pulse relative to Wistar rats. The sustained peak of ACTH in the presence of high levels of glucocorticoids and the increased levels of TSH in the presence of elevated levels of T₃ suggest that the glucocorticoid and thyroid negative feedback systems are not functioning properly in WKY rats. Smaller phase shifts of the activity rhythm and an attenuated lengthening of the free running period after a light pulse in WKY rats suggest that these animals may be less sensitive to the effects of light. Future work in this animal model will determine whether the hormonal or activity rhythm abnormalities are causally related to the well-established depressive-like behavior of the WKY rat. In addition, this strain can be used to elucidate the connection between HPA and HPT dysregulation. Inasmuch as many of the hormonal and activity rhythm characteristics found in the WKY rat are homologous to those found in other animal models of depression, as well as in depressed humans, our data provide further support for the WKY rat as a valid animal model of depression.

Perspectives

Although no animal model can represent an exact replica of human affective disorders, the biological alterations we describe here, together with the well-established behavioral deficits found in the WKY rat, provide strong evidence that this strain is a valid animal model of depression. Furthermore, because the WKY is an inbred strain, it may be particularly useful for determining if shared genetic elements underlie the behavioral, hormonal, and rhythm phenotypes seen in the WKY rat. Although premature, the discovery of the genetic bases for these abnormalities in the WKY rat may provide preliminary information regarding possible genetic abnormalities in human depression.

	ACKNOWLEDGEMENTS

We thank B. Olson and E. Naylor for technical assistance.

	FOOTNOTES

This work was supported by the National Alliance for the Research on Schizophrenia and Depression; the Berman Foundation; and National Institutes of Health Grants HL-59598, P01-AG-11412, and R01-AG-18200.

Address for reprint requests and other correspondence: L. Solberg, Northwestern Univ., 303 E Chicago Ave., Ward 9-246, Chicago, IL 60611 (E-mail: leahs{at}northwestern.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 November 2000; accepted in final form 10 May 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1.   Abel, ED, Kaulbach HC, Campos-Barros A, Ahima RS, Boers ME, Hashimoto K, Forrest D, and Wondisford FE. Novel insight from transgenic mice into thyroid hormone resistance and the regulation of thyrotropin. J Clin Invest 103: 271-279, 1999[ISI][Medline].

2.   Allen-Rowlands, CF, Allen JP, Greer MA, and Wilson M. Circadian rhythmicity of ACTH and corticosterone in the rat. J Endocrinol Invest 3: 371-377, 1980[ISI][Medline].

3.   Armario, A, Gavalda A, and Marti J. Comparison of the behavioural and endocrine response to forced swimming stress in five inbred strains of rats. Psychoneuroendocrinology 20: 879-890, 1995[ISI][Medline].

4.   Atkinson, HC, and Waddell BJ. Circadian variation in basal plasma corticosterone and adrenocorticotropin in the rat: sexual dimorphism and changes across the estrous cycle. Endocrinology 138: 3842-3848, 1997[Abstract/Free Full Text].

5.   Ayensu, WK, Pucilowski O, Mason GA, Overstreet DH, Rezvani AH, and Janowsky DS. Effects of chronic mild stress on serum complement activity, saccharin preference and corticosterone levels in Flinders lines of rats. Physiol Behav 57: 165-169, 1995[Medline].

6.   Bartalena, L, Placidi GF, Martino E, Falcone M, Pellegrini L, Dell'Osso L, Pacchiarotti A, and Pinchera A. Nocturnal serum thyrotropin (TSH) surge and the TSH response to TSH-releasing hormone: dissociated behavior in untreated depressives. J Clin Endocrinol Metab 71: 650-655, 1990[Abstract].

7.   Bauer, MS, and Whybrow PC. Thyroid hormones and the central nervous system in affective illness: interactions that may have clinical significance. Integrative Psych 6: 75-100, 1988.

8.   Berton, O, Ramos A, Chaouloff F, and Mormede P. Behavioral reactivity to social and nonsocial stimulations: a multivariate analysis of six inbred rat strains. Behav Genet 27: 155-166, 1997[ISI][Medline].

9.   Brown, NS, Smart A, Sharma V, Brinkmeier ML, Greenlee L, Camper SA, Jensen DR, Eckel RH, Krezel W, Chambon P, and Haugen BR. Thyroid hormone resistance and increased metabolic rate in the RXR-alpha-deficient mouse. J Clin Invest 106: 73-79, 2000[ISI][Medline].

10.   Buttner, D, and Wollnik F. Strain-differentiated circadian and ultradian rhythms in locomotor activity of the laboratory rat. Behav Genet 14: 137-152, 1984[ISI][Medline].

11.   Buysse, DJ, Frank E, Lowe KK, Cherry CR, and Kupfer DJ. Electroencephalographic sleep correlates of episode and vulnerability to recurrence in depression. Biol Psychiatry 41: 406-418, 1997[ISI][Medline].

12.   Carnes, M, Lent SJ, Erisman S, Barksdale C, and Feyzi J. Immunoneutralization of corticotropin-releasing hormone prevents the diurnal surge of ACTH. Life Sci 45: 1049-1056, 1989[ISI][Medline].

13.   Castanon, N, Hendley ED, Fan XM, and Mormede P. Psychoneuroendocrine profile associated with hypertension or hyperactivity in spontaneously hypertensive rats. Am J Physiol Regulatory Integrative Comp Physiol 265: R1304-R1310, 1993[Abstract/Free Full Text].

14.   Courvoisier, H, Moisan MP, Sarrieau A, Hendley ED, and Mormede P. Behavioral and neuroendocrine reactivity to stress in the WKHA/WKY inbred rat strains: a multifactorial and genetic analysis. Brain Res 743: 77-85, 1996[ISI][Medline].

15.   Daan, S, and Pittendrigh CS. A functional analysis of circadian pacemakers in nocturnal rodents. II. The variability of phase response curves. J Comp Physiol 106: 253-266, 1976.

16.   Detke, MJ, Rickels M, and Lucki I. Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants. Psychopharmacology (Berl) 121: 66-72, 1995[Medline].

17.   Deuschle, M, Schweiger U, Weber B, Gotthardt U, Korner A, Schmider J, Standhardt H, Lammers CH, and Heuser I. Diurnal activity and pulsatility of the hypothalamus-pituitary-adrenal system in male depressed patients and healthy controls. J Clin Endocrinol Metab 82: 234-238, 1997[Abstract/Free Full Text].

18.   Dugovic, C, Solberg LC, Redei E, Van Reeth O, and Turek FW. Sleep in the Wistar Kyoto rat, a genetic animal model for depression. Neuroreport 11: 1-5, 2000[ISI][Medline].

19.   Dumont, EC, Deschepper CF, and Drolet G. HPA responsivity to psychogenic stressor in rat strains that display susceptibility to stress (Wistar Kyoto and WKHA rats). Soc Neurosci Abstr 26: 423, 2000.

20.   Duncan, WC, Jr. Circadian rhythms and the pharmacology of affective illness. Pharmacol Ther 71: 253-312, 1996[ISI][Medline].

21.   Gilad, GM, and Shiller I. Differences in open-field behavior and in learning tasks between two rat strains differing in their reactivity to stressors. Behav Brain Res 32: 89-93, 1989[ISI][Medline].

22.   Golstein, J, Van Cauter E, Linkowski P, Vanhaelst L, and Mendlewicz J. Thyrotropin nyctohemeral pattern in primary depression: differences between unipolar and bipolar women. Life Sci 27: 1695-1703, 1980[ISI][Medline].

23.   Gomez, F, De Kloet ER, and Armario A. Glucocorticoid negative feedback on the HPA axis in five inbred rat strains. Am J Physiol Regulatory Integrative Comp Physiol 274: R420-R427, 1998[Abstract/Free Full Text].

24.   Gomez, F, Lahmame A, de Kloet ER, and Armario A. Hypothalamic-pituitary-adrenal response to chronic stress in five inbred rat strains: differential responses are mainly located at the adrenocortical level. Neuroendocrinology 63: 327-337, 1996[ISI][Medline].

25.   Haggerty, JJ, Jr, Stern RA, Mason GA, Beckwith J, Morey CE, and Prange AJ, Jr. Subclinical hypothyroidism: a modifiable risk factor for depression? Am J Psychol 150: 508-510, 1993.

26.   Hendley, ED, and Ohlsson WG. Two new inbred rat strains derived from SHR: WKHA, hyperactive, and WKHT, hypertensive, rats. Am J Physiol Heart Circ Physiol 261: H583-H589, 1991[Abstract/Free Full Text].

27.   Hickie, I, Bennett B, Mitchell P, Wilhelm K, and Orlay W. Clinical and subclinical hypothyroidism in patients with chronic and treatment-resistant depression. Aust NZ J Psychiatry 30: 246-252, 1996[ISI][Medline].

28.   Honma, K, Honma S, and Hiroshige T. Response curve, free-running period, and activity time in circadian locomotor rhythm of rats. Jpn J Physiol 35: 643-658, 1985[ISI][Medline].

29.   Jefferys, D, Copolov D, Irby D, and Funder J. Behavioural effect of adrenalectomy: reversal by glucocorticoids or [D-Ala2,Met5]enkephalinamide. Eur J Pharmacol 92: 99-103, 1983[ISI][Medline].

30.   Jefferys, D, and Funder JW. Thyroid hormones and the acquisition and retention of behavioural responses. Endocrinology 125: 1103-1105, 1989[Abstract].

31.   Jefferys, DE, and Funder JW. The thyroadrenal axis, food deprivation and retention of a behavioural response. Eur J Pharmacol 239: 189-193, 1993[ISI][Medline].

32.   Joffe, RT, Roy-Byrne PP, Udhe TW, and Post RM. Thyroid function and affective illness: a reappraisal. Biol Psychiatry 19: 1685-1691, 1984[ISI][Medline].

33.   Joffe, RT, and Sokolov ST. Thyroid hormones, the brain, and affective disorders. Crit Rev Neurobiol 8: 45-63, 1994[ISI][Medline].

34.   Jordan, D, Rousset B, Perrin F, Fournier M, and Orgiazzi J. Evidence for circadian variations in serum thyrotropin, 3,5,3'-triiodothyronine, and thyroxine in the rat. Endocrinology 107: 1245-1248, 1980[Abstract].

35.   Koehl, M, Barbazanges A, Le Moal M, and Maccari S. Prenatal stress induces a phase advance of circadian corticosterone rhythm in adult rats which is prevented by postnatal stress. Brain Res 759: 317-320, 1997[ISI][Medline].

36.   Kopp, P, Kitajima K, and Jameson JL. Syndrome of resistance to thyroid hormone: insights into thyroid hormone action. Proc Soc Exp Biol Med 211: 49-61, 1996[Abstract].

37.   Kripke, DF. Light treatment for nonseasonal depression: speed, efficacy, and combined treatment. J Affect Disord 49: 109-117, 1998[ISI][Medline].

38.   Kulikov, A, Torresani J, and Jeanningros R. Experimental hypothyroidism increases immobility in rats in the forced swim paradigm. Neurosci Lett 234: 111-114, 1997[ISI][Medline].

39.   Lahmame, A, and Armario A. Differential responsiveness of inbred strains of rats to antidepressants in the forced swimming test: are Wistar Kyoto rats an animal model of subsensitivity to antidepressants? Psychopharmacology (Berl) 123: 191-198, 1996[Medline].

40.   Lahmame, A, del Arco C, Pazos A, Yritia M, and Armario A. Are Wistar-Kyoto rats a genetic animal model of depression resistant to antidepressants? Eur J Pharmacol 337: 115-123, 1997[ISI][Medline].

41.   Lahmame, A, Grigoriadis DE, De Souza EB, and Armario A. Brain corticotropin-releasing factor immunoreactivity and receptors in five inbred rat strains: relationship to forced swimming behaviour. Brain Res 750: 285-292, 1997[ISI][Medline].

42.   Lasser, RA, and Baldessarini RJ. Thyroid hormones in depressive disorders: a reappraisal of clinical utility. Harvard Rev Psych 4: 291-305, 1997[ISI][Medline].

43.   Linkowski, P, Mendlewicz J, Kerkhofs M, Leclercq R, Golstein J, Brasseur M, Copinschi G, and Van Cauter E. 24-Hour profiles of adrenocorticotropin, cortisol, and growth hormone in major depressive illness: effect of antidepressant treatment. J Clin Endocrinol Metab 65: 141-152, 1987[Abstract].

44.   Lopez-Rubalcava, C, and Lucki I. Strain differences in the behavioral effects of antidepressant drugs in the rat forced swimming test. Neuropsychopharmacology 22: 191-199, 2000[ISI][Medline].

45.   Louis, WJ, and Howes LG. Genealogy of the spontaneously hypertensive rat and Wistar-Kyoto rat strains: implications for studies of inherited hypertension. J Cardiovasc Pharmacol 16: S1-S5, 1990.

46.   Marcilhac, A, Maurel D, Anglade G, Ixart G, Mekaouche M, Hery F, and Siaud P. Effects of bilateral olfactory bulbectomy on circadian rhythms of ACTH, corticosterone, motor activity and body temperature in male rats. Arch Physiol Biochem 105: 552-559, 1997[ISI][Medline].

47.   Marti, J, and Armario A. Forced swimming behaviour is not related to the corticosterone levels achieved in the test: a study with four inbred rats strains. Physiol Behav 59: 369-373, 1996[Medline].

48.   Meerlo, P, van den Hoofdakker RH, Koolhaas JM, and Daan S. Stress-induced changes in circadian rhythms of body temperature and activity in rats are not caused by pacemaker changes. J Biol Rhythms 12: 80-92, 1997[Abstract/Free Full Text].

49.   Muglia, L, Jacobson L, Weninger S, Luedke C, Bae D, Jeong K, and Majzoub J. Impaired diurnal adrenal rhythmicity restored by constant infusion of corticotropin-releasing hormone in corticotropin-releasing hormone-deficient mice. J Clin Invest 99: 2923-2929, 1997[ISI][Medline].

50.   Musselman, DL, and Nemeroff CB. Depression and endocrine disorders: focus on the thyroid and adrenal system. Br J Psychiatry Suppl 30: 123-128, 1996.

51.   Pare, WP. Open field, learned helplessness, conditioned defensive burying, and forced-swim tests in WKY rats. Physiol Behav 55: 433-439, 1994[Medline].

52.   Pare, WP. Passive-avoidance behavior in Wistar-Kyoto (WKY), Wistar, and Fischer-344 rats. Physiol Behav 54: 845-852, 1993[Medline].

53.   Pare, WP. The performance of WKY rats on three tests of emotional behavior. Physiol Behav 51: 1051-1056, 1992[Medline].

54.   Pare, WP. Stress ulcer and open-field behavior of spontaneously hypertensive, normotensive, and Wistar rats. Pavlovian J Biol Sci 24: 54-57, 1989[ISI][Medline].

55.   Pare, WP, and Kluczynski J. Differences in the stress response of Wistar-Kyoto (WKY) rats from different vendors. Physiol Behav 62: 643-648, 1997[Medline].

56.   Pare, WP, and Redei E. Depressive behavior and stress ulcer in Wistar Kyoto rats. J Physiol (Paris) 87: 229-238, 1993[ISI][Medline].

57.   Peters, RV, Zoeller RT, Hennessey AC, Stopa EG, Anderson G, and Albers HE. The control of circadian rhythms and the levels of vasoactive intestinal peptide mRNA in the suprachiasmatic nucleus are altered in spontaneously hypertensive rats. Brain Res 639: 217-227, 1994[ISI][Medline].

58.   Pies, RW. The diagnosis and treatment of subclinical hypothyroid states in depressed patients. Gen Hosp Psychiatry 19: 344-354, 1997[ISI][Medline].

59.   Pittendrigh, CS, and Daan S. A functional analysis of circadian pacemakers in nocturnal rodents. I. The lability and lability of spontaneous frequency. J Comp Physiol 106: 223-252, 1976.

60.   Porsolt, RD, Le Pichon M, and Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature 266: 730-732, 1977[Medline].

61.   Redei, E, Halasz I, and Aird F. Discordant in vitro regulation of ACTH secretion in anterior pituitary corticotrophs of Wistar Kyoto (WKY) rats. Soc Neurosci Abstr 19: 761, 1993.

62.   Redei, E, Pare WP, Aird F, and Kluczynski J. Strain differences in hypothalamic-pituitary-adrenal activity and stress ulcer. Am J Physiol Regulatory Integrative Comp Physiol 266: R353-R360, 1994[Abstract/Free Full Text].

63.   Redei, EE, Solberg LC, Kluczynski JM, and Pare WP. Paradoxical hormonal and behavioral responses to hypothyroid and hyperthyroid states in the Wistar Kyoto rat. Neuropsychopharmacology 24: 632-639, 2000.

64.   Rittenhouse, PA, and Redei E. Thyroxine administration prevents streptococcal cell wall-induced inflammatory responses. Endocrinology 138: 1434-1439, 1997[Abstract/Free Full Text].

65.   Rookh, HV, Azukizawa M, DiStefano JJI, Ogihara T, and Hershman JM. Pituitary-thyroid hormone periodicities in serially sampled plasma of unanesthetized rats. Endocrinology 104: 851-856, 1979[ISI][Medline].

66.   Rosenwasser, AM. Circadian drinking rhythms in SHR and WKY rats: effects of increasing light intensity. Physiol Behav 53: 1035-1041, 1993[Medline].

67.   Rosenwasser, AM, Pellowski MW, and Hendley ED. Circadian timekeeping in hyperactive and hypertensive inbred rat strains. Am J Physiol Regulatory Integrative Comp Physiol 271: R787-R796, 1996[Abstract/Free Full Text].

68.   Rosenwasser, AM, and Plante L. Circadian activity rhythms in SHR and WKY rats: strain differences and effects of clonidine. Physiol Behav 53: 23-29, 1993[Medline].

69.   Rosenwasser, AM, and Wirz-Justice A. Circadian rhythms and depression: clinical and experimental models. In: Handbook of Experimental Pharmacology: Physiology and Pharmacology of Biological Rhythms, edited by Redfern PH, and Lemmer B.. Heidleberg: Springer-Verlag, 1997, vol. 125, p. 457-486.

70.   Sands, SA, Gould G, Frazer A, and Morilak DA. Behavioral inhibition in Wistar-Kyoto rats associated with a lack of brain noradrenergic reactivity to stress and reduced benzodiazepine binding in limbic forebrain. Soc Neurosci Abstr 26: 1433, 2000.

71.   Shiromani, PJ, and Overstreet D. Free-running period of circadian rhythms is shorter in rats with a genetically upregulated central cholinergic system. Biol Psychiatry 36: 622-626, 1994[ISI][Medline].

72.   Solberg, LC, Horton TH, and Turek FW. Circadian rhythms and depression: effects of exercise in an animal model. Am J Physiol Regulatory Integrative Comp Physiol 276: R152-R161, 1999[Abstract/Free Full Text].

73.   Stewart, KT, Rosenwasser AM, Hauser H, Volpicelli JR, and Adler NT. Circadian rhythmicity and behavioral depression: I. Effects of stress. Physiol Behav 48: 149-155, 1990[Medline].

74.   Summer, TL, Ferraro JS, and McCormack CE. Phase-response and Aschoff illuminance curves for locomotor activity rhythm of the rat. Am J Physiol Regulatory Integrative Comp Physiol 246: R299-R304, 1984.

75.   Suzuki, S, Solberg L, Redei E, and Handa RJ. Differential expression of preprothyrotropin-releasing hormone (prepro-TRH) 178-199 in Wistar Kyoto (WKY) rat strains: implication for underlying mechanism for vulnerability to depression. Soc Neurosci Abstr 24: 1926, 1998.

76.   Swann, JM, and Turek FW. Transfer from long to short days reduces the frequency of pulsatile luteinizing hormone release in intact but not in castrated male golden hamsters. Neuroendocrinology 47: 343-349, 1988[ISI][Medline].

77.   Tizabi, Y, Aguilera G, and Gilad GM. Age-related reduction in pituitary corticotropin-releasing hormone receptors in two rat strains. Neurobiol Aging 13: 227-230, 1992[ISI][Medline].

78.   Van Cauter, E, and Turek FW. Depression: a disorder of timekeeping? Perspect Biol Med 29: 510-519, 1986[ISI][Medline].

79.   Van Haarst, AD, Oitzl MS, Workel JO, and de Kloet ER. Chronic brain glucocorticoid receptor blockade enhances the rise in circadian and stress-induced pituitary-adrenal activity. Endocrinology 137: 4935-4943, 1996[Abstract].

80.   Van Praag, H. Treatment of refractory primary depression. Ned Tijdschr Geneeskd 141: 1375-1379, 1997[Medline].

81.   Van Reeth, O, Koehl M, Weibel L, Lemoal M, and Maccari S. Effects of prenatal stress on circadian synchronization in adult rats (Abstract). J Sleep Res 7: 287, 1998.

82.   Weiss, RE, Forrest D, Pohlenz J, Cua K, Curran T, and Refetoff S. Thyrotropin regulation by thyroid hormone in thyroid hormone receptor beta-deficient mice. Endocrinology 138: 3624-3629, 1997[Abstract/Free Full Text].

83.   Willner, P. Validity, reliability and utility of the chronic mild stress model of depression: a 10-year review and evaluation. Psychopharmacology (Berl) 134: 319-329, 1997[Medline].

84.   Wong, CC, Dohler KD, Geerlings H, and von zur Muhlen A. Influence of age, strain and season on circadian periodicity of pituitary, gonadal and adrenal hormones in the serum of male laboratory rats. Horm Res 17: 202-215, 1983[ISI][Medline].

85.   Wong, R, Vasilyev VV, Ting YT, Kutler DI, Willingham MC, Weintraub BD, and Cheng S. Transgenic mice bearing a human mutant thyroid hormone beta 1 receptor manifest thyroid function anomalies, weight reduction, and hyperactivity. Mol Med 3: 303-314, 1997[ISI][Medline].