The functional significance of biochemical alterations in streptozotocin-induced diabetes

Physiology & Behavior, Vol. 50, pp. 973-981. ©Pergamon Press plc, 1991. Printed in the U.S.A.

0031-9384/91 $3.00 + .00

The Functional Significance of Biochemical Alterations in Streptozotocin-Induced Diabetes L. L. BELLUSH,1 S. G. R E I D A N D D. N O R T H Department of Psychology, Ohio University R e c e i v e d 4 M a r c h 1991 BELLUSH, L. L., S. G. REID AND D. NORTH. The functional significance of biochemical alterations in streptozotocin-induced diabetes. PHYSIOL BEHAV 50(5) 973-981, 1991.--These experiments examined the effects of restraint stress on dopamine (DA) and 5-hydroxytryptamine (5-HT) and their principal metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA), respectively, in 4 brain regions, as well as on plasma corticosterone concentration (CORT) and behavior in streptozotocin-induced diabetic rats and nondiabetic controls. Diabetic rats had widespread reductions in DA and 5-HT turnover (DOPAC/DA and 5-HIAA/5-HT ratios). Restraint led to equivalent increases in DA turnover in diabetics and nondiabetics, but attenuated increases in 5-HT turnover in diabetic rats. CORT concentration of diabetics and nondiabetics measured in complete quiet did not differ. Relative to these measures, only diabetics had elevated CORT when either restrained or kept in the same room with restrained rats with food and water removed. Open-field exploration was suppressed by restraint in diabetics only. All diabetic rats showed decreased locomotion in a novel environment which was normalized during a second exposure to the apparatus. Together, these results suggest that diabetes-induced disruptions in open-field activity are related to anxiety rather than to motor or energy deficits, and may be related to impaired 5-HT and CORT systems. Dopamine turnover Plasma corticosterone

5-Hydroxytryptamine turnover Restraint stress

Open-field activity

A number of hormonal and neurochemical alterations have been characterized in various rodent models of insulin-dependent diabetes mellitus which may have behavioral relevance (21,30). The hormonal alterations which have been consistently reported include elevations in plasma corticosterone concentration (CORT) during the inactive circadian phase (27,33), elevations in plasma epinephrine (EPI), norepinephrine (NE) and dopamine-beta-hydroxylase (7,20), and elevated urinary NE excretion (4,6). Diabetes-induced changes in neurochemistry include reduced turnover of NE (35), dopamine [DA; (34)] and serotonin [5-HT; (37)] in many brain regions. These alterations have been equated with a state of chronic stress (11,41). Elevations in basal CORT concentration are consistent with the effects of chronic stressors such as restraint (29) and cold (40). With regard to the neurochemical changes, reduced activity of monoamines is comparable to the depletions that have been noted to occur following severe stress (3). Chronic stress has been implicated in the development of physiological disorders such as ulcers (28) and cardiovascular disease (26), as well as major depression (19). Furthermore, individuals with diabetes mellitus have enhanced responsiveness to stress and are at greater risk for developing depression (22). Therefore, it is important to determine what biochemical factors contribute to these stress-related problems. The biochemical responses of diabetic rodents to several stressors have been examined. Exaggerated rises in plasma CORT in diabetic mice (21) and in NE and EPI in diabetic rats (20) were noted in response to acute stressor presentation. The en-

Radial ann maze

hanced responsiveness of CORT to acute stressors is consistent with enhanced CORT responses to footshock following chronic cold stress (40). Less work has been published comparing neurochemical responses to stress in diabetics versus nondiabetics (30). Moreover, only a few studies have addressed the behavioral significance of any of the biochemical alterations in diabetics. STZ-diabetic rats (6) and mice (21) both showed enhanced retention for passive avoidance training. Genetically diabetic BB-Wistar rats groomed more than nondiabetics following placement in a novel environment (1). These findings indicate a behavioral sensitization to stressors in diabetic rodents. While Leedom et al. (21) related enhanced passive avoidance in diabetic mice to greater increments in CORT following footshock, there was no difference in CORT of diabetic and nondiabetic rats 10 min after footshock (6). Relating reduced monoamine activity in diabetic rodents to behavioral alterations has been more problematic. No clear neurocbemical basis for enhanced passive avoidance was established (6). However, enhanced grooming in diabetic BB-Wistar rats was attributed to the upregulation of D~ receptors in the brain (1). This upregulation (23) is thought to result from reduced DA turnover, which has in turn been linked to reduced amphetamineinduced stereotypy in diabetic rats (31). In sum, altered passive avoidance and stress-induced grooming remain the only clear behavioral changes demonstrated in the diabetic rodent, and the evidence for neurochemical correlates of the altered behaviors remains inconclusive.

tRequests for reprints should be addressed to Dr. Linda L. Bellush, Dept. of Psychology, Ohio University, Athens, OH 45701.

973

974

BELIA'SH ET Ai

Bellush and Henley (4) recently examined neurochemical correlates of cold stress and hypoxia and found that, although diabetic rodents had reduced turnover of DA and 5-HT relative to nondiabetics in several brain regions, they showed similar changes in turnover of DA and 5-HT in response to both stressors. The only exception was in the bralnstem, where hypoxia caused a reduction in 5-HT turnover in nondiabetics but no change in diabetics. Subsequently, immobilization was shown to produce increased 5-hydroxyindoleacetic acid (5-HIAA) in the hypothalamus in nondiabetic but not in long-term (5 weeks duration) female streptozotocin (STZ)-diabetic rats (10). Thus, as with hypoxia, 5-HT activity in diabetics was unresponsive to immobilization stress. Immobilization also produced smaller CORT increments in diabetic rats (10). These authors suggested that the attenuated 5-HT and CORT responses were related to each other, and that these alterations may underlie the enhanced vulnerability of diabetics to depression. However, no behavioral measures were taken to assess the impact of the diabetes-related biochemical alterations on subsequent behavior. This would seem to be a critical step in establishing a functional role of 5-HT and CORT in this regard. The present experiments were conducted to extend the investigation of the effects of restraint stress in diabetic rats. Neurochemical assessment was broadened to include several brain regions, rather than just the hypothalamus. In addition, because of the possibility that DA could be involved in diabetes-related behavioral disruption (1,25), both DA and 5-HT were assessed. CORT responses to restraint were also measured. Finally, the effects of restraint on food intake and open-field activity were assessed, since both of these behaviors have also been reported to be reduced by restraint (18). We used a milder form of restraint than previously employed (10,18), based on the finding that diabetic rats show enhanced responsivity to milder stress but not to more severe stress (1). EXPERIMENT 1. NEUROCHEMICAL RESPONSES TO RESTRAINT STRESS METHOD Animals Adult male Sprague-Dawley rats weighing about 300 g were given intraperitoneal injections of either STZ (Sigma Chemical Co., St. Louis, MO), 55 mg/kg diluted in citrate buffer (0.1 M, pH=4.5) or buffer alone. Diabetes was verified 48 h later by measuring blood glucose concentration (Johnson and Johnson One Touch glucometer). Any animal that did not become diabetic was reinjected, and all diabetics included in the experiment had blood glucose concentrations ->300 mg/dl. Restraint was carded out 3 weeks after induction of diabetes. Throughout the experiment, rats were singly housed in hanging stainless steel cages with food and water freely available, an artificial 12-h light/12-h dark cycle with lights on at 7:00 a.m., and ambient temperature maintained at 23-+ 2°C. Restraint Equal numbers of diabetic and nondiabetic rats (n = 8) were assigned to restraint (RES) or control (CTRL) conditions. Restraint consisted of carrying the rat to a room near the vivarium and confining it in a Plexiglas cylinder (17 cm diameter) for 30 min. Control rats were carded to this room also, but remained undisturbed in their cages. Neurochemical Determinations At the end of 30 min, the RES and CTRL rats were taken in turn to a third room and killed by decapitation. Brains were rap-

idly removed and placed on an ice-chilled plate. The frontal cortex, hypothalamus, amygdala and brainstem were quickly dissected as previously described (4), wrapped in foil, and frozen, first on dry ice and then at -80°C, until assayed for monoamine and metabolite concentrations using high-performance liquid chromatography (HPLC). The HPLC system consisted of a Bioanalytical Systems (BAS) isocratic solvent delivery system with glassy carbon electrode and a C~8 stationary phase, applied potential= +700 mV. The mobile phase consisted of monochloracetic acid (0.1 M, pH = 3.1), containing sodium octyl sulfate (120 mg/l), Na2 EDTA (500 mg/l) and 3% acetonitrile. Tissues were homogenized in 0.1 M perchloric acid containing isoproterenol as internal standard. They were then centrifuged at 10,000 rpm at 4°C for 10 min, and the supernatant was filtered and injected onto the HPLC column. DA, dihydroxyphenylacetic acid (DOPAC), 5-HT and 5-HIAA were determined on the basis of comparison with internal and external standards, normalizing content to tissue wet weight. As an additional estimate of monoamine activity, ratios of DOPAC/DA and 5-HIAA/ 5-HT were also calculated ("turnover"). Data Analysis Monoamine and metabolite concentrations were calculated using a commercial spreadsheet (SuperCalc4) and statistics package (CSS), and separate two-way analyses of variance (ANOVA's) were used to compare each monoamine and metabolite as well as turnover in each of the 4 brain regions. When a significant effect was obtained, individual pairwise comparisons were made using LSD tests. RESULTS Indices of regional DA and 5-HT activity are shown in Tables 1 and 2, respectively. Restraint led to increased DA concentrafion in the brainstem, F(1,24) = 19.54, p = 0.0004. Restrained rats had significantly higher DOPAC concentration in the brainstem, F(1,24) = 24.25, p =0.0002, frontal cortex, F(1,26) = 7.80, p=0.009, and hypothalamus, F(1,26)=6.84, p=0.01; restraint was associated with significantly higher DOPAC/DA ratios in the amygdala, F(1,26) = 5.89, p =0.02, and brainstem, F(1,24) = 7.76, p=0.01. There were also significant effects of diabetes on DOPAC concentration in the brainstem, F(1,24) = 12.07, p = 0.002, frontal cortex, F(1,26)=14.93, p = 0 . 0 0 1 , and hypothatamus, F(1,26)= 19.48, p=0.0003, and on DOPAC/DA ratios in the amygdala, F(1,26) = 13.83, p =0.001, brainstem, F(1,24) = 10.27, p=0.004, and frontal cortex, F(1,26)= 14.26, p=0.001. In all cases, diabetics had significantly lower values than nondiabetics. There were restraint-induced increases in 5-HIAA concentration in the frontal cortex, F(1,26) = 4.89, p = 0.03, and in 5-HIAA/ 5-HT ratios in the amygdala, F(1,26)=7.60, p=0.001, and frontal cortex, F(1,26) = 8.20, p = 0.008. Diabetics had lower 5-HT concentrations in the brainstem, F(1,24) = 4.40, p = 0.04, frontal cortex, F(1,26) = 6.82, p = 0.01, and hypothalamus, F(1,26)=5.17, p=0.03. They had lower 5-HIAA concentrations in the arnygdala, F(1,26)=31.02, p=0.00005, brainstem, F(1,24)= 18.0, p=0.0005, frontal cortex, F(1,26)= 19.26, p = -0.0003, and hypothalamus, F(1,26) = 14.58, p=0.001. They had lower 5-HIAA/5-HT ~ o s in the amygdala, F(1,26)=56.4, p<0.000001, brainstem, F(1,24)= 23.37, p=0.0002, frontal cortex, F(1,260)=11.1, p=0.003, and hypothalamus, F(1,26)= 15.19, p=0.00009. In none 0fthe statistical analyses were there any significant interactions.

BEHAVIORAL DISRUPTIONS IN DIABETIC RATS

975

TABLE 1 EFFECTS OF RESTRAINT ON REGIONAL DOPAMINE lvlETABOLISM

DOPAC Amygdala N-CTRL N-RES D-CTRL D-RES

0.027 0.031 0.022 0.027

± ± ± ±

0.003 0.003 0.004 0.003

Dopamine (DA)

DOPAC/DA

0.129 0.121 0.132 0.138

0.22 0.26 0.16 0.20

ns

-+ -+ -+ ±

0.014 0.010 0.022 0.006

ns

--- 0.02 ± 0.02 ± 0.02 ± 0.02 *t

Brainstem N-CTRL N-RES D-CTRL D-RES

0.025 0.036 0.019 0.028

± ± ± ± *t

0.001 0.003 0.002 0.002

0.050 0.062 0.046 0.051

-+ 0.002 ___ 0.003 --- 0.003 +-- 0.003 *

0.50 0.58 0.42 0.49

--- 0.03 --- 0.03 --- 0.02 --- 0.03 *t

Frontal Cortex N-CTRL N-RES D-CTRL D-RES

0.037 0.046 0.027 0.034

± 0.003 ± 0.003 --- 0.002 ± 0.003 *t

0.096 0.105 0.087 0.100

+_. 0.008 ± 0.010 ± 0.003 _+ 0.008 ns

0.39 0.45 0.31 0.34

± 0.04 --- 0.02 ± 0.03 ± 0.02 *t

Hypothalamus N-CTRL N-RES D-CTRL D-RES

0.082 0.089 0.060 0.075

-+ 0.004 --- 0.005 ___ 0.000 ± 0.006

0.503 0.449 0.378 0.438

_+ 0.038 _+ 0.034 -+ 0.034 ± 0.034

0.17 0.20 0.16 0.17

_ 0.02 ± 0.02 --- 0.01 ± 0.01

*t

ns

ns

Shown are mean _+ SE (p,g/g). N=nondiabetic; D=diabetic; CTRL=unstressed controls; RES =restrained rats. *Significant effect of restraint, tSignificant effect of diabetes.

DISCUSSION The present results confirm previous findings of increased 5-HT activity associated with restraint stress (10,18). Dopamine turnover was also increased in some brain regions by restraint. Interestingly, we did not find an effect of restraint stress on 5-HT turnover in hypothalamus. In the amygdala and frontal cortex, there were significant main effects of restraint indicated by the ANOVA. Although no statistically significant interactions were demonstrated, responses of 5-HT systems to stress in diabetics were somewhat attenuated in comparison with those of nondiabetics (amygdala--5% vs. 16%; b r a i n s t e m - - 2 % vs. 11%; frontal c o r t e x - - 1 2 % vs. 23%; and hypothalarnus--7% decrease vs. 4% increase). Stress-induced changes in DA systems were equivalent in diabetics and nondiabetics. Increased 5-HT activity in the hypothalamus after restraint was noted in at least 2 other reports besides that of Chaouloff et ai. (10, 13, 32). However, in one case (32), severe restraint stress similar to that of Chaouloff's (10) procedure was used. The other study involved mice (13). Thus differences in stressor intensity as well as in species may account for the discrepancy. Indeed, it has been noted that 5-HT responses to stress are more variable than are those of the other monoamines and that severity of stress is one source of variability (3). The effects of restraint on DA turnover are consistent with previous findings of activation by various stressors, including immobilization (9,15). However, in the present study, diabetics and nondiabetics had equivalent increases in DA turnover following stress, although in absolute terms, diabetics had consistently lower turnover rates than nondiabetics. Although the stress-induced changes in both DA and 5-HT

turnover were parallel in diabetics and nondiabetics, it should be kept in mind that both stressed and nonstressed diabetics had significantly lower turnover than their nondiabetic counterparts. The diabetic may therefore always have a relative deficiency in 5-HT and DA turnover which may, in turn, be expected to impact on adaptive behavior. In the following experiments, the behavioral consequences of restraint were investigated. We hypothesized that the restraint procedure employed would be mild enough that nondiabetic rats would not express long-lasting effects as had been noted after severe immobilization (18), whereas stress-induced effects would be measurable in diabetics. EXPERIMENT 2. H O R M O N A L A N D BEHAVIORAL CORRELATES OF RESTRAINT STRESS IN DIABETIC AND NONDIABETIC RATS METHOD

Experiment 2a Animals. Adult male Sprague-Dawley rats weighing about 300 g initially were made diabetic or given buffer, as described in Experiment 1. They were maintained throughout the experimental period in an identical fashion as well. Equal numbers of diabetic and nondiabetic rats were assigned to one of two conditions: Restraint (RES) or Control (CTRL) 3 weeks after induction of diabetes. Restraint. For this experiment, the restraint period was extended to 2 hours for compatibility with the immobilization procedure of Chaouloff et al. (9). In addition, rather than move

976

BELLUSH E'F AI,

TABLE 2 EFFECTS OF RESTRAINT ON REGIONAL 5-HT METABOLISM

5-HIAA

5-HT

5-HIAA/5-HT

Amygdala N-CTRL N-RES D-CTRL D-RES

0.301 0.323 0.210 0.224

± ± -+ ± +

0.015 0.027 0.015 0.011

0.622 0.569 0.569 0.574

--_ 0.032 -_+ 0.042 -+ 0.037 ± 0.036 ns

0.49 0.57 0.37 0.39

--- 0.02 ± 0.03 --- 0.02 ± 0.01 *t

Brainstem N-CTRL N-RES D-CTRL D-RES

0.512 0.544 0.394 0.426

± 0.017 __. 0.044 ± 0.024 ± 0.025 t

0.710 0.669 0.611 0.652

-- 0.040 ± 0.030 ± 0.022 _+ 0.026 t

0.73 0.81 0.64 0.65

_ 0.03 - 0.03 ± 0.02 +-- 0.02 t

Frontal Cortex N-CTRL N-RES D-CTRL D-RES

0.335 0.401 0.250 0.285

± 0.021 ± 0.039 ± 0.013 --- 0.011 *+

0.712 0.696 0.611 0.623

___ 0.041 ± 0.047 --_ 0.024 ± 0.024 *

0.47 0.58 0.41 0.46

___ 0.02 ± 0.05 +_ 0.01 ± 0.02 *t

Hypothalamus N-CTRL N-RES D-CTRL D-RES

0.364 0.405 0.284 0.279

_ ± ± ±

0.674 0.725 0.595 0.612

± 0.050 - 0.046 --+ 0.045 ± 0.040

0.54 0.56 0.48 0.45

- 0.02 --- 0.03 ± 0.03 --- 0.01

0.035 0.032 0.024 0.022

Shown are mean _ SE (p,g/g). N=nondiabetic; D=diabetic; CTRL=unstressed controls; RES =restrained rats. *Significant effect of restraint, tSignificant effect of diabetes.

animals from the colony as in Experiment 1, the restraint procedure was carded out in the colony. Nonrestrained control rats remained in their home cages during the restraint period in this experiment. However, food and water were removed for the 2 hours of restraint, since restrained rats would not have access to either during confinement. Restraint was initiated between 1:30 and 2:00 p.m. Food intake measurement. Pelleted chow was replaced with powdered chow contained in glass jars and affixed to the cages with springs 5 days prior to restraint procedures. After a 4-day acclimation period, baseline food intake was measured during the 24 h preceding restraint by weighing jars at the beginning and end of the measurement interval. A second measurement was taken, beginning at the conclusion of the restraint period and ending when open-field activity was measured. Intake was normalized to body weight and expressed as g/kg. Analysis of variance with 2 between factors (diabetic/nondiabetic and restraint/control) and one within factor (baseline/poststress) was used to assess food intake. Open-field activity. Twenty-four hours after restraint, rats were taken individually to a nearby room and observed for 5 min in an open field, which consisted of a rectangular plywood box ( 6 6 . 3 × 5 5 . 6 x 4 5 . 3 cm) with a removable Plexiglas floor made opaque with shelf covering. The floor was divided into 20 sections (13.5 x 12.9 cm), and it rested on 4 glass jars positioned beneath holes (3.8 cm diameter) located in each comer 2.5 cm from the sidewalls. Each jar contained a unique o b j e c t - - a brass weight, a sliver of soap, dried flowers and galvanized joints-intended to elicit exploratory behavior [nosepoking; based on the

procedure of File (14)]. The only light in this room was that of a 60-W red light suspended 72 cm above the open field. Two observers scored the behaviors. Locomotion--number of squares entered with all 4 p a w s - - w a s assessed by one observer, while exploration--number of holepokes-- and number of rears were counted by a second observer who was blind to the experimental treatment conditions of individual animals. Hand-held tally counters were used to count frequency of behaviors. Each of the behavioral categories was analyzed statistically using Kruskal-Wallis analyses of variance by ranks, and when significance occurred, Mann-Whitney U-tests were used to identify differences between individual pairs. CORT assessment. At the end of the restraint period, 200 p,l of blood was quickly collected from a tail nick into ice-chilled heparinized tubes. Blood was taken from home cage control rats within 2 rain of removing them from the cage, and from restrained rats while they were in the cylinders, just before returning them to the home cage. A second sample was taken from each rat at the end of the open-field test 24 h later, Blood was centrifuged for 10 min at 5,000 rpm in a refrigerated centrifuge (Jouan, Inc.) and the plasma removed and frozen at - 2 0 ° C until assayed for plasma CORT concentration. Radioimmunoassays were carded out to assess CORT concentration in 10-tzl plasma samples using a commercially available kit (rodent corticosterone kit; ICN Bioehemicals, Inc., Costa Mesa, CA). Briefly, the p r o c ~ u r e involved a double antibody technique, whereby anti-CORT antiserum was incubated with 125I-CORT and unlabeled CORT standards or plasma sam, pies. A mixture containing goat anti-rabbit gamma globulins was

BEHAVIORAL DISRUPTIONS IN DIABETIC RATS

used to precipitate the antibody-bound CORT. Following centrifugation and decanting of the supernatant, the residual radioactivity in the precipitate was counted in a gamma counter. Sensitivity of the assays was 0.8 l~g/dl; cross-reactivity with other steroids was -<1%; interassay and inwaassay coefficients of variability were 0.11 and 0.12, respectively. Postrestraint CORT was determined in only 6 rats from each group due to a laboratory error in which the remaining samples were lost. However, post-open-field samples from all rats were analyzed. Due to the discrepancy in group composition, separate 2-way ANOVAs were therefore used to analyze the results.

Experiment 2b This experiment was conducted to determine if different durations of confinement in small cylinders would comprise different severities of stress. Our contention was that our procedure of confinement in cylinders was less severe than that of taping the limbs of the rats to a countertop (9,16). However, in Experiment 1, we measured neurochemical responses after 30 min of restraint, whereas in Experiment 2a, a 2-h restraint period was used 24 h prior to behavioral assessment. In order to determine if the duration of restraint by our technique might have an impact on subsequent behavior, Experiment 2b was conducted, in which groups of rats were restrained for various intervals and assessed 24 h later in the open field. Animals. Adult male Sprague-Dawley rats were given either streptozotocin or buffer as previously described. They were housed and maintained exactly as in Experiment 2a, and diabetes was allowed to develop for 3 weeks. Procedures. Diabetics and nondiabetics were separately divided into 3 groups (n = 7) which were restrained for either 30, 60 or 120 min in hardware cloth cylinders. At the end of the restraint periods, blood samples were taken for measurement of plasma corticosterone. Twenty-four hours later, each animal was placed in the open field described in Experiment 2a for a 5-min period. In this experiment, the open field was brightly lit with 2 75-W light bulbs placed 90 cm above the field. Behavior was videotaped and later scored by two individuals as indicated in Experiment 2a. Separate Kruskal-Wallis analyses of variance were used to compare each of the 3 restraint-interval groups (diabetics and nondiabetics). Separate one-way ANOVAs were used to assess differences in CORT concentrations as a function of restraint duration. Diabetics and nondiabetics were not compared directly, since the manipulations were conducted separately. RESULTS

Experiment 2a Food intake. Table 3 shows food intakes. As expected, diabetic rats consumed considerably more food than nondiabetic rats. Comparing the two measurement periods, all rats reduced food intake during the 24 h following restraint, and the difference was significant, F(1,36) = 14.76, p = 0.0008. Open-field activity. Figure la shows the number of holepokes (means ---SE are shown). There were significant differences among the groups, H(3, N = 4 3 ) = 16.6, p=0.0008. Pairwise comparisons indicated the restrained diabetic group (D-RES) differed significantly from all other groups. The two nondiabetic groups showed no difference in the number of holepokes. Also, exploration in unrestrained diabetics (D-CTRL) did not differ from that of the nondiabetic groups. Locomotor activity (number of squares entered) is shown in Fig. lb. There were significant differences among groups in this behavior too, H(3, N = 4 3 ) = 10.2, p=0.02. In this case, D-RES

977

~ s

Group N-CTRL N-RES D-CTRL D-RES

TABLE 3 OF 2-H RESTRAINTON FOODINTAKE 24-h Food Intake (g/kg body wt.) Baseline Poststress 64.5 61.0 149.0 149.8

_+ 1.9 -+ 2.0 -+ 7.0 -+ 5.8 t

58.6 ___3.5 56.6 _+ 1.8 142.9 -*- 6.7 136.6 • 6.6 *t

Shown are mean _+ SE. N=nondiabetic; D--diabetic; CTRL=unstressed controls; RES =restrained rats. *Significanteffect of restraint. tSignificant effect of diabetes.

were significantly different from the two nondiabetic groups, but did not differ significantly from the D-CTRL group. However, the D-CTRL group did not differ from the nondiabetic groups, either. CORT assessment. Mean plasma CORT concentrations taken at the end of restraint and at the end of the 5-min open-field test are shown in Fig. 2. The ANOVAs indicated main effects of diabetes on both postrestraint CORT, F(1,20) = 13.26, p =0.002, and post-open-field CORT, F(1,39) = 4.95, p =0.03.

Experiment 2b Exploratory behavior and locomotor activity are shown in Fig. 3. There were no differences among either the 3 diabetic or the 3 nondiabetic groups in either exploratory behavior or locomotor activity. CORT concentration (shown in Fig. 4) did not vary significantly as a function of restraint duration in either the diabetics or the nondiabetics. DISCUSSION Food intake dropped in all rats during the 24 h following the restraint procedure. This could reflect a response to the general activity in the colony, including removal of food and water bottles from unrestrained controls. Alternatively, it has been suggested that intraspecific communication of distress can occur during restraint which may disturb control rats if they are housed in the same room (29). In any case, mild restraint per se did not lead to anorexia, as was the case with the more severe procedure (18). In the open-field test (Experiment 2a), only restrained diabetics displayed significantly suppressed behavior. Thus diabetics had an enhanced responsiveness to the mild restraint. This finding is consistent with previous studies, in which diabetic rats (6) and mice (21) both showed enhanced retention for passive avoidance training, which was attributed to greater responsivity to footshock. As we predicted, open-field behavior in nondiabetic rats was not affected by the milder restraint stress employed. In addition, Experiment 2b demonstrated that manipulation of the duration of the milder restraint we employed had no differential effect on subsequent open-field behavior, since activity measures were virtually identical in groups restrained 30, 60 and 120 minutes. Of potential importance in the present study was the intermediate locomotor activity of unrestrained diabetics (Experiment 2a). It is possible that the general activity during restraint procedures in the colony were sufficiently arousing to affect the activity level of even the unrestrained diabetic rats (29). Alternatively, as noted by Ahmad and Merali (1), the novel environment itself

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FIG. 1. Number of holepokes (a) and number of squares entered (b) in a novel open field during a 5-min measurement taken 24 h after a 2-h restraint session (mean +- SE). CTRL = unstressed controls; RES = restrained groups. *Significantly different from both nondiabetic groups. **Significantly different from all other groups. may have been more stress-inducing in diabetics. Indeed, the open field may actually be aversive rather than simply novel, given that it consists o f a large open area (and hence the thigmotaxis noted frequently in rodents in this apparatus) (39). A totally different explanation is also plausible, viz., there may be either an energy deficit in diabetic rats, due to impaired caloric utilization, or a motor deficit (23,31) making them less active behaviorally.

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60 120 DIABETIC

FIG. 3. Number of holepokes (a) and number of squares entered (b) during a 5-min measurement taken 24 h after a 30- 60- or 120-min restraint session (mean_+ SE). There were no significant differences as a function of restraint duration in either diabetic or nondiabetic groups.

The C O R T data bear on the stressfulness o f the restraint procedure. First, the only significant differences in C O R T concentration were between diabetics and nondiabetics in Experiment 2a. It was quite surprising that the restraint procedure per se appeared to induce no increment in C O R T relative to the unstressed controls. It is possible that the " b a s e l i n e " CORT measures reflected some arousal in all rats, and that we do not in fact have a true afternoon " b a s e l i n e " C O R T measure here. This

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FIG. 2. Plasma corticosterone concentration measured at the end of a 2-h restraint period (clear bars) in rats confined in Plex_iglas cylinders and unstressed controls which remained in the homecage without access to food or water, and 24 h later (hatched bars) at the end of a 5-min open-field measurement. Shown are means ± SE. N = nondiabetics; D = diabetics;' CTRL = unstressed controls; RES = restrained groups. *Significantly different from nondiabeties.

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30 60 120 NONDIABETIC

30 60 120 DIABETIC

FIG. 4. Plasma corticosterone concentration (CORT) measured at the end of 30- 60- or 120-rain restraint sessions in hardware cloth cylinders (mean __ SE). There were no significant differences as a function of restraint duration in either diabetic or nondiabctic groups.

BEHAVIORAL DISRUPTIONS IN DIABETIC RATS

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1 2 - [ ] TRIAL 1

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DIABETIC

FIG. 5. Number of arms entered during 2 5-min trials in an 8-arm radial maze separated by 24 h (means---SE). *Significantly different from all other groups.

possibility hampers the establishment of a relationship between biochemical responses and behavioral adaptations to stress. Also problematic is the locomotor activity in unrestrained diabetic rats, which was intermediate, not differing statistically from either nondiabetics or restrained diabetics. To clarify both of these issues, two additional experiments were conducted. EXPERIMENT 3. ACTIVITY LEVELS UPON REPEATED EXPOSURE TO A BEHAVIORAL ARENA IN DIABETIC AND NONDIABETIC RATS This experiment was designed to determine if the intermediate locomotion in unstressed diabetics represented a diabetesspecific reduction in activity, and if so, if the reduction was due to stress invoked by the open field, or alternatively to a motor or energy deficit. It was hypothesized that, if diabetic rats were more reactive to the open-field environment, their performance would be less affected in a different apparatus, the radial arm maze, which lacks the large open area. In addition, if behavioral suppression was a response to a novel environment, then activity should increase to normal levels during a second exposure to the apparatus. A motor or energy deficit due to the diabetic state would be expected to cause consistently reduced activity in the diabetics, irrespective of apparatus or number of exposures. METHOD Animals Twenty Sprague-Dawley rats were used, 10 of which received STZ, the other 10 citrate vehicle, 3 weeks prior to behavioral testing exactly as in Experiments 1 and 2. They were housed singly in hanging stainless steel cages, with food and water available ad lib and a 12:12 light:dark cycle maintained throughout the experiment. Procedure Exploration was assessed on 2 consecutive days in a radial 8-arm maze. The arms measured 8 0 x 1 7 . 5 x 1 8 cm and consisted of plywood floors and sidewalls painted gray with removable clear Plexiglas covers. The arms were positioned on the floor of a testing room with arms abutting such that they formed a center arena (45 cm diameter). The arms were numbered 1 through 8. The room was kept darkened except for a single red light positioned 145 cm above the maze during all testing. For consistency with the open-field experiments, rats were handled

DIABETIC

FIG. 6. Plasma corticosterone concentration (CORT) in diabetic and nondiabetic rats from blood samples collected under quiet conditions in the colony (clear bars) or at the end of 2 h without food or water available with restraint procedures occurring in the colony (hatched bars). Shown are means__ SE. *Significantly different from nondiabetic counterpart. + Significantly different from "quiet" counterpart.

only to measure body weight weekly prior to the behavioral assessments. Testing consisted of two 5-min sessions spaced 24 h apart and conducted between 2:30 and 5:00 p.m. Each rat was carded in its home cage to the testing room and placed in the center arena of the radial ann maze. An observer recorded the number of each ann entered by the rat. The total number of arms entered was counted for each rat. Performance of the two groups was assessed first using a Kruskal-Wallis nonparametric analysis of variance, followed by Mann-Whitney U-tests to make pairwise comparisons. RESULTSAND DISCUSSION Figure 5 shows the number of arms entered by diabetic and nondiabetic rats during each trial (shown are the means_+ SE). There was a significant difference among the groups, H(3, N = 4 0 ) = 9 . 6 9 , p = 0 . 0 2 . Mann-Whitney U-tests indicated that the performance of diabetics in Trial 1 differed from all other groups. The performance of nondiabetics in the two trials did not differ significantly, nor did the Trial 2 performance of diabetics differ from the performance of nondiabetics. In the radial ann maze, an apparatus which may be less aversive for rats than is the open field, diabetics showed reduced activity relative to nondiabetics during the initial exposure, but not on a subsequent return to the maze. The test session duration in the radial arm maze was identical to that in the open field, so that any effects of fatigue or habituation would be identical. Since session 2 activity levels of diabetics and nondiabetics were indistinguishable in the radial arm maze, it is not likely that diabetics have an energy deficit of relevance to our behavioral testing. However, since behavior of diabetics was suppressed in Trial 1 in the radial ann maze, it would appear that a novel environment has a disruptive effect on diabetics not seen in nondiabetics, even in the absence of prior stress experience. EXPERIMENT 4. CORT CONCENTRATIONS IN UNDISTURBED DIABETIC AND NONDIABETIC RATS METHOD Animals Eighteen rats were used in this experiment. Half the rats had been made diabetic 3 weeks earlier, and all housing and mainte-

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BELLUSH ET AL.

nance parameters were identical to those in Experiments l and 2. Procedure

Blood sampling was carried out between 2:30 and 3:00 p.m. when no other activity was occurring in the colony. It consisted of removing each rat from the home cage and collecting blood from a tail nick within 2 min. Diabetic and nondiabetic rats were sampled alternately. Plasma was prepared as described above, and radioimmunoassays were conducted using all methodological details described in Experiment 2. A two-way ANOVA was used to compare the CORT concentrations in this manipulation with those measured in the nondiabetic and diabetic baseline groups in Experiment 2a. RESULTSAND DISCUSSION Mean CORT concentrations (--. SE) of diabetic and nondiabetic rats are shown in Fig. 6. ANOVA indicated main effects of both diabetes, F(1,26)= 13.0, p=0.002, and measurement period, F(1,26)= 55.1, p=0.000001, as well as a significant interaction, F(1,26)=22.6, p=0.0002. Pairwise comparisons indicated no difference between diabetics and nondiabetics measured under "quiet" conditions, and no difference between "quiet" and baseline measures in nondiabetics. However, the difference between diabetics and nondiabetics measured during activity in the lab (baseline) and between "quiet" and baseline measures in diabetics were highly significant. These findings conf'Lrm that the ongoing activity in the colony during the restraint manipulation was sufficient to produce increments in baseline CORT. However, it is also apparent that the effects were much greater in diabetics than in nondiabetics. Indeed, when care was taken to create minimal disturbance during blood sampling, afternoon CORT did not differ in diabetics and nondiabetics (27,33). GENERAL DISCUSSION The present studies confirmed previous observations of widespread reduction of DA and 5-HT turnover in chronic diabetes (3 weeks duration). Moreover, although there were no statistically significant interactions between diabetes and restraint, there was a tendency for diabetics to show a smaller increment in 5-HT turnover following restraint. No such attenuation in DA activation by restraint occurred. Exploration (nosepoking) was suppressed by restraint stress in diabetics but not in nondiabetics, while behavioral suppression in a novel environment was seen in all diabetic rats. In addition, plasma CORT concentration was increased to a greater extent in diabetics than nondiabetics by ongoing activity in the colony during restraint procedures. Our neurochemical findings are in partial agreement with those of Chaouloff et al. (10), who noted attenuated increases in 5-HIAA following 2 h of immobilization in diabetic rats. In addition, we previously found a significant attenuation in brainstem 5-HT responses to hypoxia in diabetic rats (4). All of these findings suggest that diabetics have a reduced capacity for adaptation of central 5-HT systems to stressors. A relationship between central 5-HT and nosepoking has been proposed (14), strengthening our hypothesis that attenuated 5-HT activation in diabetics following restraint was associated with reduced exploration. Further support for the relationship of 5-HT to altered activity comes from a previous report of reduced sensitivity of diabetics to 5-HT agonist-induced behaviors (36). The present f'mdings are inconsistent with previous demonstrations that restraint-induced increases in 5-HT activity per se

cause behavioral suppression (13,17). If such a relationship existed, then diabetics should have shown less suppression than nondiabetics, since 5-HT responses were somewhat attenuated. In fact, the restrained nondiabetics showed no subsequent behavioral suppression. These inconsistencies may reflect differences in restraint procedures (17), in species or timing of behavioral testing, relative to stress (13). The finding of attenuated responses to 5-HT agonists in diabetics (36) is consistent with the general decreases in 5-HT turnover found in diabetic rats, irrespective of stress. Although both DA and 5-HT turnover were compromised in diabetics, we hypothesize that 5-HT is more likely involved in the behavioral suppression observed in the radial ann maze in all diabetics. Thus locomotion in an open field was reduced following lesions of ascending 5-HT fibers (16). In addition, DA has previously been associated with general motor activation (31), rather than with affective responses in rats. Because reduced activity in diabetics subsided upon reexposure to the radial arm maze, this reduction is best explained by heightened reactivity to novelty, rather than reduced motor activation. One important factor not addressed in the present studies was whether streptozotocin itself caused any of the alterations in the diabetics. There is ample documentation that streptozotocin-induced neurochemical alterations are reversible with insulin replacement (5, 23, 35), as is attenuation of apomorphine- and amphetamine-induced stereotypy (5,31). However, reversal of locomotor changes in diabetics remains to be demonstrated. The CORT finding was surprising. First, restraint in the present study led to no increment in CORT measures relative to unrestrained controls housed in the room with the restrained rats. Pitman et al. (29) suggested that intraspecific communication by stressed animals may affect control animals housed in the same environment. While this could have occurred in the present experiment, diabetics still evidenced significantly higher CORT than nondiabetics. We hypothesized that this represented the greater sensitivity of adrenocortical responses in diabetics (11,40). In addition, there have been reports of higher baseline CORT in diabetic rats beginning in the late afternoon (27,33). However, when we took additional CORT samples, being careful to cause minimal disturbance in the colony, absolutely no afternoon difference between diabetics and nondiabetics could be detected. The elevations in diabetics, both in Experiment 2 and in the previous studies, could thus be the result of greater responsivity of diabetics to general ongoing activity, which is hard to avoid during execution of measurement procedures. Whether the enhanced CORT sensitivity of diabetics was associated in any way with altered 5-HT neurotransmission cannot be determined from the present data, but such a relationship between 5-HT, at least in the hypothalamus, and CORT responses has been proposed (2,12). The present results do not support a role of CORT in exploration, since CORT was not differentially affected by restraint stress, while exploration was (8). However, a role of CORT in locomotion is not ruled out. The finding that even exposure to novel environments is anxiety-inducing in diabetics is important with regard to a general enhancement in diabetic humans of the perceived stressfulness of life events (22). This enhanced perception of stress has, in turn, been linked with problems in metabolic regulation, suggesting a biochemical basis for the stress-related problems. In sum, chronic hyperglycemia leads to sensitization of the CORT response to environmental perturbation, reduced turnover of DA and 5-HT, and loss of plasticity in 5-HT responses to stress. The outcome of these biochemical alterations is a greater behavioral reactivity to these environmental perturbations. Although no direct correlations can be drawn between this scenario

BEHAVIORAL DISRUPTIONS IN DIABETIC RATS

981

and the increased vulnerability of human diabetics to depression (24), there is certainly evidence of a connection between distur-

bances of brain 5-HT metabolism and adrenocortical activity on the one hand and vulnerability to depression on the other (38).

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