Psychopharmacologia (Berl.) 45, 167-169 (1975) - 9 by Spfinger-Verlag 1975
Effects of d-Amphetamine on the Incorporation of Carbon Atoms of D-Glucose into the Brains of Differentially-Housed Mice* P. A. DeFEUDIS**, R. M. PAOLINO, and F. V. DeFEUDIS** Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, and Department of Pharmacology and Therapeutics, Purdue University, Lafayette, Indiana, U.S.A. Received February 20, 1975
Abstract. Mice were housed either individually ("isolated") or in groups of 2 0 - 25 ("aggregated") for 5 - 9 weeks or for 22 weeks. A decreased incorporation of radioactivity into brain from subcutaneously-administered U-14C-D-glucoseoccurred in "isolated" mice as compared to grouped animals. Amphetamine, administered before labelled glucose, produced
a dose-dependent decrease of radioactivity which was selective to the brains of the "isolated" mice. The data support the correlation between isolation-induced changes in behavior and central metabolic pathways and indicate further that these changes may be altered by administration of psycho-active agents.
Key words: d-Amphetamine - Isolation - Cerebral metabolism - Environment.
Introduction Individual housing of male mice, or other rodents causes pronounced behavioral changes (Valzelli, 1967; Essman, 1971; Bennett and Rosenzweig, 1971; DeFeudis, 1974) which have been correlated with alterations in cerebral carbohydrate metabolism (DeFeudis, 1971a, 1972, 1973; DeFeudis and Black, 1972; DeFeudis and Paolino, 1972). Herein, we report a sensitive method for demonstrating the changes in cerebral metabolism which can be produced by differential housing.
Methods and Results In the first set of experiments, male C-57 black mice, 45 days old at onset of differential housing, were kept for 22 weeks under previously-described conditions (DeFeudis, 1971a). All animals had access to food and water throughout experiments. After the period of differential housing, each animal was placed in a 2000-ml beaker for 1.5 hrs during which injections of d-amphetamine and D-glucose were made. Thirty minutes before sacrifice, all animals were injected subcutaneously with 0.1 gCi/g of U-14C-D-glucose (New England Nuclear Corp. ; 217 mCi/mmole). Each animal was given an intraperitoneal injection of 5, * Supported in part by a grant from the Eli Lilly Co. ** Present address: Departamento de Ciencias Fisiol6gicas, Facultad de Medicina Universidad Aut6noma, Madrid 34, Spain.
10 or 15mg/kg d-amphetamine-SO4 10min after~ the labelled glucose; a control group received an equivalent volume o f 0.154 M NaC1. All mice were decapitated, and their brains (rostral to the inferior colliculi; excluding the cerebellum) were excised rapidly and homogenized in 3.0 ml ice-cold 80~o ethanol solution (v/v). Radioactivity due to 1~C was determined in 0.5 ml aliquots o f homogenates (DeFeudis, 1971a, b). As shown in Table 1, less 14C was incorporated into the brains o f the isolated animals
Table 1. Effects of dramphetamine on the incorporation of carbon atoms of o-glucose into the brains of differentiallyhoused mice Dose of d-amphetamine (mg/kg)
Radioactivity; dpm (x
10-3)/g brain
'~Isolated. . . . mice
Aggregated" mice
Control; no drug 5 10 15
245 234 229 222
282 + 300 + 281 + 284 +
+__10.9 (8) + 13.9 (5) i- 15.2 (5) +_+_16.6 (5)
11.3 (8)* 17.9 (5)* 13.0 (5)* 15.6 (5)*
Mice were housed individually or in groups of 20-25 for 5.5 months. Means _+ standard errors; numbers of mice in parentheses. * Indicates P < 0.05 when these values are compared with corresponding values for "isolated" mice; Student's t-test; two-tailed. NO significant differences were found between animals that received the drug and control animals under either "isolated" or "aggregated" conditions of housing.
168
Psychopharmacologia (Berl.), Vol. 45, Fasc. 2 (1975) Table 2. Effects of d-amphetamine on the incorporation of carbon atoms of D-glucose into the brains and sera of differentially-housed mice
Dose of d-amphetamine (mg/kg)
Control 15 25 50
Radioactivity; dpm (x-3)/g brain, or ml serum "Isolated" mice
"Aggregated" mice
Brain
Serum
Brain
Serum
201 185 147 152
240 169 156 143
217 207 213 199
252 • 10.7 (5) 219 • 6.2 (5)* ++ 194 • 17.4 (5)* 166 • 4.4 (5)*** +
+ 8.2 (15) • 14.8 (10) ___ 6.7 (5)*** • 6.9 (15)***
• 15.2 (5) • 7.1 (5)** • 9.4 (5)** • 7.6 (5)***
• 8.2 (15) • 10.3 (5) • 4.0 (5) + • 4.5 (16)* +
Mice were kept individually or in groups of 20-25 for 5 - 9 weeks. Means + standard errors; numbers of mice in parentheses; *, ** and *** indicate P < 0.05, P < 0.01, and P < 0.00t for comparisons between these values and the control values for "isolated" or "aggregated" mice; + and + + indicate P < 0.05 and P < 0.001 for comparisons between these values and corresponding values for "isolated" mice (Student's t-test; two-tailed).
and d-amphetamine injections appeared to produce a decrease in cerebral 14C incorporation into the brains of the "isolated" animals. However, only the differences between "isolated" and "aggregated" mice were significant. In an effort to assess more fully this decrease in cerebral 14C incorporation into the brains of"isolated" mice, a second set of experiments was performed in which the age o f the animals, the dose range of d-amphetamine, the time following d-amphetamine injection, and the feeding schedule were modified. Male, C-57 black mice, 21 days of age, were kept "isolated" or in groups of 2 0 - 2 5 for 5 - 9 weeks, and fasted for 24 hrs prior to injections. All animals received 0.1 gCi/g o f U-14C-D-glucose (s.c.) 30 min prior to sacrifice and d-amphetamine-SO4 (doses of 15, 25, or 50 mg/kg, i.p.) 1 hr before sacrifice. Animals were decapitated, and brain homogenates were prepared as described above. In addition, radioactivity was determined in 0.05 ml aliquots o f serum, d-Amphetamine caused a selective decrease in ~ C incorporation into the brains o f the "isolated" animals (Table 2). This effect was apparently not related directly to changes in serum levels of radioactivity since injections of d-amphetamine decreased **C incorporation into the sera of both "isolated" and "aggregated" mice, the effect being slightly more pronounced in "isolated" mice (Table 2). Calculations of brain/ sel-um ratios and o f '~~0 equilibrated spaces" indicated that although an increase in cerebral ~4C, relative to serum ~4C, occurred as the injected dose of d-amphetamine was increased, this change was similar for both groups o f mice (Table 3).
Discussion Prior intraperitoneal injections of the psychomotor stimulant, d-amphetamine, decreased selectively the
Table 3. Effects of injections of d-amphetamine on brain/ serum ratios and " ~ equilibrated spaces" of carbon atoms of glucose in differentially-housed mice Dose of d-amphetamine (mg/kg)
dpm/g brain
~ Equilibrated space
dpm/ml serum "Isolated" mice
None 15 25 50
0.82 ___0.05 0.95 + 0.03 1.13 • 0.09 1.15 +_ 0.07
97.4 112.9 134.2 136,6
"Aggregated" mice None 15 25 , 50
0.86 0.88 0.95 0.96
• 0.07 • 0,04 +__0.04 __+0.12
100.4 102.8
111.0 I12.1
These calculations were based on/hatched values from the data o [ dpm/g brain \ presented in Table 2. ~ Equilibrated space = / . ~ : : 7 , . - ~ / "[~/o H 2 0 serums
\u~,,L~,,u ~clu.,/ k~o H--aO--b~]-n)x 100;thisisatheoreticalvaluewhichprovides the ratio between the dpm in brain and serum corrected for differences in total tissue water content. Previously-reported values for %oH20 of serum and ~ H20 of brain of "isolated" and "aggregated" mice were used for estimate s of }o/ equilibrated space (DeFeudis and Marks, 1973). Means • S.E.M. ; 5 mice in all cases.
incorporation o f carbon atoms of injected D-glucose into the brains o f "isolated" mice in a dose-dependent fashion, an effect which apparently reflects an environmentally-induced change in cerebral metabolic capacity. These findings might be contrasted with those which indicated that the incorporation of 14C of injected 1-~4C-pyruvate into the brains of"isolated '~ mice was selectively increased by prior treatment with Li +, an anti-manic agent (DeFeudis and Paolino, 1972). However, both sets of results provide further
P. A. DeFeudis et al. : Environment and d-Amphetamine support for the c o n t e n t i o n that the dramatic changes in emotional behavior caused by p r o l o n g e d isolation o f mice are intimately related to changes in cerebral glucose metabolism, and that the central actions o f psychoactive agents are m o r e p r o n o u n c e d when mice are subjected to individual housing.
References
Bennett, E.L.; Rosenzweig, M. R. : Chemical alterations produced in brain by environment and training. In: Handbook of neurochemistry, Vol. 6, A. Lajtha, ed., pp. ]73-201. New York: Plenum Press ]971 DeFeudis, F. V. : Effects of environmental changes on the incorporation of carbon atoms of D-glucose into mouse brain and other tissues, Life Sci. 10 (Part II), 1187-1194 (1971 a) DeFeudis, F. V. : Studies on the content and metabolism of L-glutamate in peripheral nerve in vitro. Res. Commun. Chem. Path. Pharmacol. 2, 189-205 (1971b) DeFeudis, F. V. : Effects of isolation and aggregation On the incorporation of carbon atoms of D-mannose and Dglucose into mouse brain. Biol. Psychiat. 4, 239-242 (1972)
169 DeFeudis, F. V. : Effects of D-amphetamine on the incorporation of carbon atoms of I>mannose into the brains and sera of differentially-housed mice; Short-term reversibility of these effects. Biol, Psychiat. 7, 3 - 9 (1973) DeEeudis, F. V, : Cerebral biochemical and pharmacological changes in differentially-housed mice. In: Current developments in psychopharmacology, Vol. I, W. B. Essman and L. Valzelli, eds. New York: Spectrum Publ. 1975 DeFeudis, F. V., Black, W. C. : Effects of environment on the incorporation of radioactive carbon atoms of glucose into various structures of mice; evidence for dim-hal rhythmicity, Exp. Neurol. 36, 4 1 - 45 (1972) DeFeudis, F. V., Marks, J. H. : Brain to serum distribution of radioactivity of injected 3H-D-amphetamine in differentially-housed mice, Biol. Psychiat. 6, 8 5 - 8 9 (1973) DeFeudis, F. V., Paolino, R. M. : Effects of environmental changes on the incorporation of carbon atoms ofpyruvate into mouse brain and liver. Experientia (Basel) 28, 309- 310 (1972) Essman, W. B. : Neurochemical changes associated with isolation and environmental stimulation. Biol. Psychiat. 3, 141-147 (1971) Valzelli, L.: Drugs and aggressiveness. In: Advances in pharmacology, Vol. 5, S. Garattini and P. A. Shore, eds., pp. 79-108. New York: Academic Press 1967
Dr. F. V. DeFeudis, Chief, Section of Neurochemistry, Departamento de Ciencias Fisiol6gicas, Facultad de Medicina Universidad Aut6noma, Madrid 34, Spain
Effects of d-Amphetamine on the Incorporation of Carbon Atoms of D-Glucose into the Brains of Differentially-Housed Mice* P. A. DeFEUDIS**, R. M. PAOLINO, and F. V. DeFEUDIS** Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, and Department of Pharmacology and Therapeutics, Purdue University, Lafayette, Indiana, U.S.A. Received February 20, 1975
Abstract. Mice were housed either individually ("isolated") or in groups of 2 0 - 25 ("aggregated") for 5 - 9 weeks or for 22 weeks. A decreased incorporation of radioactivity into brain from subcutaneously-administered U-14C-D-glucoseoccurred in "isolated" mice as compared to grouped animals. Amphetamine, administered before labelled glucose, produced
a dose-dependent decrease of radioactivity which was selective to the brains of the "isolated" mice. The data support the correlation between isolation-induced changes in behavior and central metabolic pathways and indicate further that these changes may be altered by administration of psycho-active agents.
Key words: d-Amphetamine - Isolation - Cerebral metabolism - Environment.
Introduction Individual housing of male mice, or other rodents causes pronounced behavioral changes (Valzelli, 1967; Essman, 1971; Bennett and Rosenzweig, 1971; DeFeudis, 1974) which have been correlated with alterations in cerebral carbohydrate metabolism (DeFeudis, 1971a, 1972, 1973; DeFeudis and Black, 1972; DeFeudis and Paolino, 1972). Herein, we report a sensitive method for demonstrating the changes in cerebral metabolism which can be produced by differential housing.
Methods and Results In the first set of experiments, male C-57 black mice, 45 days old at onset of differential housing, were kept for 22 weeks under previously-described conditions (DeFeudis, 1971a). All animals had access to food and water throughout experiments. After the period of differential housing, each animal was placed in a 2000-ml beaker for 1.5 hrs during which injections of d-amphetamine and D-glucose were made. Thirty minutes before sacrifice, all animals were injected subcutaneously with 0.1 gCi/g of U-14C-D-glucose (New England Nuclear Corp. ; 217 mCi/mmole). Each animal was given an intraperitoneal injection of 5, * Supported in part by a grant from the Eli Lilly Co. ** Present address: Departamento de Ciencias Fisiol6gicas, Facultad de Medicina Universidad Aut6noma, Madrid 34, Spain.
10 or 15mg/kg d-amphetamine-SO4 10min after~ the labelled glucose; a control group received an equivalent volume o f 0.154 M NaC1. All mice were decapitated, and their brains (rostral to the inferior colliculi; excluding the cerebellum) were excised rapidly and homogenized in 3.0 ml ice-cold 80~o ethanol solution (v/v). Radioactivity due to 1~C was determined in 0.5 ml aliquots o f homogenates (DeFeudis, 1971a, b). As shown in Table 1, less 14C was incorporated into the brains o f the isolated animals
Table 1. Effects of dramphetamine on the incorporation of carbon atoms of o-glucose into the brains of differentiallyhoused mice Dose of d-amphetamine (mg/kg)
Radioactivity; dpm (x
10-3)/g brain
'~Isolated. . . . mice
Aggregated" mice
Control; no drug 5 10 15
245 234 229 222
282 + 300 + 281 + 284 +
+__10.9 (8) + 13.9 (5) i- 15.2 (5) +_+_16.6 (5)
11.3 (8)* 17.9 (5)* 13.0 (5)* 15.6 (5)*
Mice were housed individually or in groups of 20-25 for 5.5 months. Means _+ standard errors; numbers of mice in parentheses. * Indicates P < 0.05 when these values are compared with corresponding values for "isolated" mice; Student's t-test; two-tailed. NO significant differences were found between animals that received the drug and control animals under either "isolated" or "aggregated" conditions of housing.
168
Psychopharmacologia (Berl.), Vol. 45, Fasc. 2 (1975) Table 2. Effects of d-amphetamine on the incorporation of carbon atoms of D-glucose into the brains and sera of differentially-housed mice
Dose of d-amphetamine (mg/kg)
Control 15 25 50
Radioactivity; dpm (x-3)/g brain, or ml serum "Isolated" mice
"Aggregated" mice
Brain
Serum
Brain
Serum
201 185 147 152
240 169 156 143
217 207 213 199
252 • 10.7 (5) 219 • 6.2 (5)* ++ 194 • 17.4 (5)* 166 • 4.4 (5)*** +
+ 8.2 (15) • 14.8 (10) ___ 6.7 (5)*** • 6.9 (15)***
• 15.2 (5) • 7.1 (5)** • 9.4 (5)** • 7.6 (5)***
• 8.2 (15) • 10.3 (5) • 4.0 (5) + • 4.5 (16)* +
Mice were kept individually or in groups of 20-25 for 5 - 9 weeks. Means + standard errors; numbers of mice in parentheses; *, ** and *** indicate P < 0.05, P < 0.01, and P < 0.00t for comparisons between these values and the control values for "isolated" or "aggregated" mice; + and + + indicate P < 0.05 and P < 0.001 for comparisons between these values and corresponding values for "isolated" mice (Student's t-test; two-tailed).
and d-amphetamine injections appeared to produce a decrease in cerebral 14C incorporation into the brains of the "isolated" animals. However, only the differences between "isolated" and "aggregated" mice were significant. In an effort to assess more fully this decrease in cerebral 14C incorporation into the brains of"isolated" mice, a second set of experiments was performed in which the age o f the animals, the dose range of d-amphetamine, the time following d-amphetamine injection, and the feeding schedule were modified. Male, C-57 black mice, 21 days of age, were kept "isolated" or in groups of 2 0 - 2 5 for 5 - 9 weeks, and fasted for 24 hrs prior to injections. All animals received 0.1 gCi/g o f U-14C-D-glucose (s.c.) 30 min prior to sacrifice and d-amphetamine-SO4 (doses of 15, 25, or 50 mg/kg, i.p.) 1 hr before sacrifice. Animals were decapitated, and brain homogenates were prepared as described above. In addition, radioactivity was determined in 0.05 ml aliquots o f serum, d-Amphetamine caused a selective decrease in ~ C incorporation into the brains o f the "isolated" animals (Table 2). This effect was apparently not related directly to changes in serum levels of radioactivity since injections of d-amphetamine decreased **C incorporation into the sera of both "isolated" and "aggregated" mice, the effect being slightly more pronounced in "isolated" mice (Table 2). Calculations of brain/ sel-um ratios and o f '~~0 equilibrated spaces" indicated that although an increase in cerebral ~4C, relative to serum ~4C, occurred as the injected dose of d-amphetamine was increased, this change was similar for both groups o f mice (Table 3).
Discussion Prior intraperitoneal injections of the psychomotor stimulant, d-amphetamine, decreased selectively the
Table 3. Effects of injections of d-amphetamine on brain/ serum ratios and " ~ equilibrated spaces" of carbon atoms of glucose in differentially-housed mice Dose of d-amphetamine (mg/kg)
dpm/g brain
~ Equilibrated space
dpm/ml serum "Isolated" mice
None 15 25 50
0.82 ___0.05 0.95 + 0.03 1.13 • 0.09 1.15 +_ 0.07
97.4 112.9 134.2 136,6
"Aggregated" mice None 15 25 , 50
0.86 0.88 0.95 0.96
• 0.07 • 0,04 +__0.04 __+0.12
100.4 102.8
111.0 I12.1
These calculations were based on/hatched values from the data o [ dpm/g brain \ presented in Table 2. ~ Equilibrated space = / . ~ : : 7 , . - ~ / "[~/o H 2 0 serums
\u~,,L~,,u ~clu.,/ k~o H--aO--b~]-n)x 100;thisisatheoreticalvaluewhichprovides the ratio between the dpm in brain and serum corrected for differences in total tissue water content. Previously-reported values for %oH20 of serum and ~ H20 of brain of "isolated" and "aggregated" mice were used for estimate s of }o/ equilibrated space (DeFeudis and Marks, 1973). Means • S.E.M. ; 5 mice in all cases.
incorporation o f carbon atoms of injected D-glucose into the brains o f "isolated" mice in a dose-dependent fashion, an effect which apparently reflects an environmentally-induced change in cerebral metabolic capacity. These findings might be contrasted with those which indicated that the incorporation of 14C of injected 1-~4C-pyruvate into the brains of"isolated '~ mice was selectively increased by prior treatment with Li +, an anti-manic agent (DeFeudis and Paolino, 1972). However, both sets of results provide further
P. A. DeFeudis et al. : Environment and d-Amphetamine support for the c o n t e n t i o n that the dramatic changes in emotional behavior caused by p r o l o n g e d isolation o f mice are intimately related to changes in cerebral glucose metabolism, and that the central actions o f psychoactive agents are m o r e p r o n o u n c e d when mice are subjected to individual housing.
References
Bennett, E.L.; Rosenzweig, M. R. : Chemical alterations produced in brain by environment and training. In: Handbook of neurochemistry, Vol. 6, A. Lajtha, ed., pp. ]73-201. New York: Plenum Press ]971 DeFeudis, F. V. : Effects of environmental changes on the incorporation of carbon atoms of D-glucose into mouse brain and other tissues, Life Sci. 10 (Part II), 1187-1194 (1971 a) DeFeudis, F. V. : Studies on the content and metabolism of L-glutamate in peripheral nerve in vitro. Res. Commun. Chem. Path. Pharmacol. 2, 189-205 (1971b) DeFeudis, F. V. : Effects of isolation and aggregation On the incorporation of carbon atoms of D-mannose and Dglucose into mouse brain. Biol. Psychiat. 4, 239-242 (1972)
169 DeFeudis, F. V. : Effects of D-amphetamine on the incorporation of carbon atoms of I>mannose into the brains and sera of differentially-housed mice; Short-term reversibility of these effects. Biol, Psychiat. 7, 3 - 9 (1973) DeEeudis, F. V, : Cerebral biochemical and pharmacological changes in differentially-housed mice. In: Current developments in psychopharmacology, Vol. I, W. B. Essman and L. Valzelli, eds. New York: Spectrum Publ. 1975 DeFeudis, F. V., Black, W. C. : Effects of environment on the incorporation of radioactive carbon atoms of glucose into various structures of mice; evidence for dim-hal rhythmicity, Exp. Neurol. 36, 4 1 - 45 (1972) DeFeudis, F. V., Marks, J. H. : Brain to serum distribution of radioactivity of injected 3H-D-amphetamine in differentially-housed mice, Biol. Psychiat. 6, 8 5 - 8 9 (1973) DeFeudis, F. V., Paolino, R. M. : Effects of environmental changes on the incorporation of carbon atoms ofpyruvate into mouse brain and liver. Experientia (Basel) 28, 309- 310 (1972) Essman, W. B. : Neurochemical changes associated with isolation and environmental stimulation. Biol. Psychiat. 3, 141-147 (1971) Valzelli, L.: Drugs and aggressiveness. In: Advances in pharmacology, Vol. 5, S. Garattini and P. A. Shore, eds., pp. 79-108. New York: Academic Press 1967
Dr. F. V. DeFeudis, Chief, Section of Neurochemistry, Departamento de Ciencias Fisiol6gicas, Facultad de Medicina Universidad Aut6noma, Madrid 34, Spain
