Physiology& Behavior. Vol. 47. pp. 705-708. ©PergamonPress plc, 1990. Printedin the U.S.A.
0031-9384/90$3.00 + .00
Behavioral and Neurochemical Differences in an Inbred Strain of Rats N. P R A D H A N , S. A R U N A S M I T H A A N D H. B. U D A Y A
Department o f Psychopharmacology, National Institute o f Mental Health and Neurosciences Bangalore 560029, India Received 4 M a y 1989
PRADHAN, N., S. ARUNASMITHA AND H. B. UDAYA. Behavioral and neurochemical differences in an inbred strain of rats. PHYSIOL BEHAV 47(4) 705-708, 1990.--The spontaneous motility of 75 Sprague-Dawley rats obtained from an inbred strain was found to vary widely. To test the hypothesis that heterogeneity in motility is accompanied by heterogeneity in neurochemical and vegetative function variables, the median motility score was used to divide the animals into high and low motility groups. Subgroups of high and low motile animals were compared on fecal pellet scores and on dopamine, dihydroxyphenyl acetic acid and homovanillic acid content of the corpus striatum. Significant differences were obtained between the two groups, indicating that even in an inbred strain considerable heterogeneity (possibly representing further subtypes) may exist. The theoretical and experimental implications of these findings are discussed. Sprague-Dawley rats Fecal pellet scores
Motility
Dopamine
Dihydroxyphenyl acetic acid
THE spontaneous motor behavior shown by rats in a novel environment is dependent on (amongst other factors) the integrity of the forebraln dopaminergic system (9,21). Depletion of dopamine reduces the spontaneous motor behavior, while enhancement of dopaminergic transmission has the opposite effect (5, 6, 12, 17). Thus, spontaneous motor behavior (baseline motility) can be, and has been, used to study the dopaminergic system in the brains of intact animals (3, 5, 13). The nigrostriatal dopaminergic system is of special relevance in this regard (7,20). A large negative genetic correlation between open field activity and open field defecation has been reported in mice (10,11). In an inbred strain of mice, it has been reported that animals showing lesser activity have higher defecation scores, and animals with greater activity have lower defecation scores (2). Previous researchers have found that 20% of the additive genetic covariance of open field activity and fecal pellet scores is attributable to the involvement of a particular genetic locus. In our laboratory, motility measurements using automated devices showed consistent heterogeneity in male Sprague-Dawley rats obtained from an inbred strain. It was hypothesised that this heterogeneity in motility is accompanied by heterogeneity in other behavioral parameters, such as fecal pellet score, and by heterogeneity in nigrostriatal dopamine content as well. The present investigation was designed to test this hypothesis.
Homovanillic acid
Institute of Mental Health and Neurosciences, Bangalore, India) were used in the experiment. The rats were fed with standard Hindustan Lever chow pellets and water ad lib. They were housed in pairs in Plexiglas cages and reared in a 12/12 hour light-dark cycle, temperature controlled (23 -+ 1°C), soundproof animal house, one week prior to commencing the experiments. Motility monitoring was conducted by use of computerised methods. Taking the median value of the distance travelled in cm, the rats were segregated into two groups. From each group, rats were taken randomly for fecal pellet scoring and neurochemical experiments.
Behavioral Experiments At the start of the behavioral experiments, the animals were acclimatized to the monitoring cage for 30 minutes every day, for three consecutive days. Thereafter, the spontaneous motility of the animals was measured using the Optovarimax Apple II system (Columbus Instruments, International Corp., OH). The measurement consisted of five trials per session for a duration of 21.7 minutes. The monitoring began 5 min after the animal was placed in the Optovarimax monitoring cage. All monitoring was done between 9 a.m. and 1 p.m. to reduce the confounding influence of diurnal variation in motility. Seven parameters of motility were obtained by this procedure. These were: a) total distance travelled, in cm, b) total time spent in resting, in sec, c) time spent in stereotypic movement, in sec, d) time spent in ambulatory behavior, in sec, e) number of bursts of stereotypic movements, f) number of clockwise rotations, and g) number of counter clockwise rotations. The rats were segregated into two groups on the basis of the first parameter, i.e., total distance travelled, in cm. The rats above the median value were
METHOD
Subjects Male Sprague-Dawley rats weighing 180-200 grams from an inbred strain (Central Animal Research Laboratory of the National
705
706
PRADHAN, ARUNASMITHA AND UDAYA
categorised into a high motile group, and those below, as low motile.
TABLE 1 MEAN± SEM MOTILITY SCORES IN LOW AND HIGH MOTILE RATS
Biochemical Analysis The animals were randomly chosen from the high motile group and the low motile group for neurochemical estimations. The rats were sacrificed between 10 a.m. and 11 a.m. every time, to reduce the influence of diurnal variation. The brain was dissected, the corpus striatum separated on ice and homogenized in ice cold 0.1 M PCA with an internal standard (Isoproterenol) at a concentration of 1 ~,g/ml. The homogenates were centrifuged in cold, the supernatant separated and ultrafiltered through an Amicon (MPS) micorpartition system. The clear ultrafiltrate was used for the quantification of dopamine (DA) and its metabolites by HPLC with electrochemical detection (8). An isocratic HPLC system comprised of a HPLC pump (Model 302 Gilson, France) with the pulse dampener, Rheodyne injector with a 100 Ixl loop (Rheodyne Corp.) and an Amperometric detector (LC 4B Bioanalytic System), online solvent filter, prefilters and C-18 guard columns (Supelco) were used. The glassy carbon electrode potentials were set to 0.65 V for DA and DOPAC and 0.9 V for HVA. The compounds were eluted with TEA phosphate buffer system (pH 2.80) containing 0.1 mM EDTA and 50 mM/litre of heptane sulphonic acid as an ion-pairing agent on a 5 cm C-18, 3-~m packing (Supelco) column with a flow rate of 0.8 ml/min, at ambient temperature.
Variable
Low Motile Rats (n = 37)
High Motile Rats (n = 38)
Distance travelled (cm)
1250.24 -4-84.01
3051.34 --+152.32
t
Time resting (sec)
1037.35 -+42.30
819.52 _+38.11
p
0031-9384/90$3.00 + .00
Behavioral and Neurochemical Differences in an Inbred Strain of Rats N. P R A D H A N , S. A R U N A S M I T H A A N D H. B. U D A Y A
Department o f Psychopharmacology, National Institute o f Mental Health and Neurosciences Bangalore 560029, India Received 4 M a y 1989
PRADHAN, N., S. ARUNASMITHA AND H. B. UDAYA. Behavioral and neurochemical differences in an inbred strain of rats. PHYSIOL BEHAV 47(4) 705-708, 1990.--The spontaneous motility of 75 Sprague-Dawley rats obtained from an inbred strain was found to vary widely. To test the hypothesis that heterogeneity in motility is accompanied by heterogeneity in neurochemical and vegetative function variables, the median motility score was used to divide the animals into high and low motility groups. Subgroups of high and low motile animals were compared on fecal pellet scores and on dopamine, dihydroxyphenyl acetic acid and homovanillic acid content of the corpus striatum. Significant differences were obtained between the two groups, indicating that even in an inbred strain considerable heterogeneity (possibly representing further subtypes) may exist. The theoretical and experimental implications of these findings are discussed. Sprague-Dawley rats Fecal pellet scores
Motility
Dopamine
Dihydroxyphenyl acetic acid
THE spontaneous motor behavior shown by rats in a novel environment is dependent on (amongst other factors) the integrity of the forebraln dopaminergic system (9,21). Depletion of dopamine reduces the spontaneous motor behavior, while enhancement of dopaminergic transmission has the opposite effect (5, 6, 12, 17). Thus, spontaneous motor behavior (baseline motility) can be, and has been, used to study the dopaminergic system in the brains of intact animals (3, 5, 13). The nigrostriatal dopaminergic system is of special relevance in this regard (7,20). A large negative genetic correlation between open field activity and open field defecation has been reported in mice (10,11). In an inbred strain of mice, it has been reported that animals showing lesser activity have higher defecation scores, and animals with greater activity have lower defecation scores (2). Previous researchers have found that 20% of the additive genetic covariance of open field activity and fecal pellet scores is attributable to the involvement of a particular genetic locus. In our laboratory, motility measurements using automated devices showed consistent heterogeneity in male Sprague-Dawley rats obtained from an inbred strain. It was hypothesised that this heterogeneity in motility is accompanied by heterogeneity in other behavioral parameters, such as fecal pellet score, and by heterogeneity in nigrostriatal dopamine content as well. The present investigation was designed to test this hypothesis.
Homovanillic acid
Institute of Mental Health and Neurosciences, Bangalore, India) were used in the experiment. The rats were fed with standard Hindustan Lever chow pellets and water ad lib. They were housed in pairs in Plexiglas cages and reared in a 12/12 hour light-dark cycle, temperature controlled (23 -+ 1°C), soundproof animal house, one week prior to commencing the experiments. Motility monitoring was conducted by use of computerised methods. Taking the median value of the distance travelled in cm, the rats were segregated into two groups. From each group, rats were taken randomly for fecal pellet scoring and neurochemical experiments.
Behavioral Experiments At the start of the behavioral experiments, the animals were acclimatized to the monitoring cage for 30 minutes every day, for three consecutive days. Thereafter, the spontaneous motility of the animals was measured using the Optovarimax Apple II system (Columbus Instruments, International Corp., OH). The measurement consisted of five trials per session for a duration of 21.7 minutes. The monitoring began 5 min after the animal was placed in the Optovarimax monitoring cage. All monitoring was done between 9 a.m. and 1 p.m. to reduce the confounding influence of diurnal variation in motility. Seven parameters of motility were obtained by this procedure. These were: a) total distance travelled, in cm, b) total time spent in resting, in sec, c) time spent in stereotypic movement, in sec, d) time spent in ambulatory behavior, in sec, e) number of bursts of stereotypic movements, f) number of clockwise rotations, and g) number of counter clockwise rotations. The rats were segregated into two groups on the basis of the first parameter, i.e., total distance travelled, in cm. The rats above the median value were
METHOD
Subjects Male Sprague-Dawley rats weighing 180-200 grams from an inbred strain (Central Animal Research Laboratory of the National
705
706
PRADHAN, ARUNASMITHA AND UDAYA
categorised into a high motile group, and those below, as low motile.
TABLE 1 MEAN± SEM MOTILITY SCORES IN LOW AND HIGH MOTILE RATS
Biochemical Analysis The animals were randomly chosen from the high motile group and the low motile group for neurochemical estimations. The rats were sacrificed between 10 a.m. and 11 a.m. every time, to reduce the influence of diurnal variation. The brain was dissected, the corpus striatum separated on ice and homogenized in ice cold 0.1 M PCA with an internal standard (Isoproterenol) at a concentration of 1 ~,g/ml. The homogenates were centrifuged in cold, the supernatant separated and ultrafiltered through an Amicon (MPS) micorpartition system. The clear ultrafiltrate was used for the quantification of dopamine (DA) and its metabolites by HPLC with electrochemical detection (8). An isocratic HPLC system comprised of a HPLC pump (Model 302 Gilson, France) with the pulse dampener, Rheodyne injector with a 100 Ixl loop (Rheodyne Corp.) and an Amperometric detector (LC 4B Bioanalytic System), online solvent filter, prefilters and C-18 guard columns (Supelco) were used. The glassy carbon electrode potentials were set to 0.65 V for DA and DOPAC and 0.9 V for HVA. The compounds were eluted with TEA phosphate buffer system (pH 2.80) containing 0.1 mM EDTA and 50 mM/litre of heptane sulphonic acid as an ion-pairing agent on a 5 cm C-18, 3-~m packing (Supelco) column with a flow rate of 0.8 ml/min, at ambient temperature.
Variable
Low Motile Rats (n = 37)
High Motile Rats (n = 38)
Distance travelled (cm)
1250.24 -4-84.01
3051.34 --+152.32
t
Time resting (sec)
1037.35 -+42.30
819.52 _+38.11
p
