Pargamon Press
Life Sciences, Vol . 25, pp . 487-496 Printed in the U .S .A .
EFFECTS OF INTRAHIPPOCAMPAL INJECTION OF CHEMICALS ON THE LEVELS OF PLASMA CORTICOSTERONE IN RATS Akira Rawa, Ratsumi Mizuguchi, Yoshio Maeda, Yoshiyasu Taniquchi, Seüchiro Ryu, Syunsaku Yamashita, Takao Ariyama, Takeshi 1(amisaki and Takuya Ranehisn The First Department of Internal Medicine, Ragosh ima University Medical School, Ragoshima 890, Japan . (Received in final form June 20, 1979)
Summary The possibility that the hippocampus can influence the function of the hypothalamo-pituitary-adrenal axis was examined by injecting small quantities of various neurotransmitter substances into this brain structure . Injections of either noradrenaline or 5-hydroxytryptamine into the dorsal hippocampus had no effect on plasma concentrations of corticosterone (B) . ,An injection of carbachol into the dorsal hippocampus elicited a significant rise in B concentrations, while that of hemicholinium into the same brain structure resulted in an inhibition of noise-induced rise of plasma 8 concentration . An injection of carbachol into the dorsal hippocampus counteracted dexamethasone-induced decrease in plasma B concentration, while that of hèmicholinium enhanced it . Although the hippocampus has been implicated in the regulation of ACTH secretion based mainly on results of experiments involving electric stimulation or lesions of this brain structure, some conflicting evidence has been reported . While Endröczi et al . (1) and Rnigge (2) have advocated that the hippocampus exercises an inhibitory influence on hypothalamo-pituitary-adrenocortical (H-P-A) axis, Casady 1~ Taylor (3) reported a facilitatory role of the hippocampua in the regulation of secretion of corticosterone (B) in rats . Lamer et al . (4), in a study of differential hippocampal damage, failed to prove the involvement of the hippocampus in regulation of B secretion in rats . Furthermore the number of papers relating to neurotransmitter specificity in the hippocampus in the regulation of H-P-A activity is still limited . Results of experiments on intrahippocampal application of chemicals (5)(6) do not appear to be . conclusive . In this report the results of our studies on a possible role of the dorsal hippocampus in the regulation of H-P-A activity will be presented with special reference to neurotransmitter specificity . Materials and Methods The animals used were male Wistar rats, weighing 300 g . They were caged individually in rooms provided with automatically controlled 0024-3205/79/060487-09$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
488
Dorsal Hippocampue and Corticoat®tone
Vol . 25, No . 6, 1979
lighting (12 hours of light, from 7 :00 a .m . till 7 :00 p .m . and 12 hours dark daily), and were kept at a constant temperature of 22 + 2°C and a constant humidity of 55 + 58 . The animals were allowed free access to food and water . Pérmanent bilateral cannuli, 0 .7 mm in diameter, were stereotaxically implanted into the dorsal hippocampus . Through them injection cannuli, 0 .3 mm in diameter, were inserted to deliver the chemicals . The stereotaxic coordinates according to Albe-Fessard et al . (7) Kere as follows : AP ; 3 .8 mm, H; 7 .0 mm and L ; 3 .0 mm . Intrahippocampal injection of Chemicals was carried out 14 days after surgery . All the animals were well adapted to the experimental procedures and environment before the experiments . Noradrenaline (NA : 0 .05, 0 .10 or 0 .50 ug ), 5-hydroxytryptamine (5-HT : 0 .1, 1 .0 or 3 .0 ug), carbachol Carb : 0 .1, 0 .4 or 0 .8 ug) or hemicholinium-3 (HC-3 : 0 .5, 1 .0 or 5 .0 ug) dissolved in 0 .5 ul of an 0 .9 $ solution of NaCl (saline) were injected into the dorsal hippocampus in a volume of 0 .5 ul for each site . The chemicals in this volume were delivered with Control animals received an infusion pump in a period of 61 sec . the same volume of vehicle . The animals were decapitated 40 min after injection of the chemicals . Each animal was injected only once . Trunk blood was collected and centrifuged . The samples were kept frozen until assayed fluorometrically for B according to the method of Guillemin et al . (8) . The brains were histologically examined for verification of injection site . Only the animals, in which the site of injection was histologically verified as being the dorsal hippocampus, were used for the assay of B . Observations were made in Carb and HC-3 group animals under the following conditions : 1) basal concentrations 2) exposure to the stressors, 3) dexamethasone . All experiments were performed between 3 :00 p .m . and 5 :00 p .m . except the experiments designed to examine the effects of centrally administered Carb on the circadian rhythm, which were carried out at 8 :00 a .m . and in the afternoon . The following stressors were used : i) auditory stimulation : The animals were exposed to the noise of a buzzer (about 85 phony for 30 min and then killed immediately afterwards ; ü) ether : rats were placed for 1 min in a glass jar saturated with ether vapour and then returned to their home cage for 30 min before decapitation . Dexamethasone (20 ug/rat) was injected intraperitoneally 9 hours and 5 hours prior to the injection into the dorsal hippocampus . In each experiment controls and treated animals were tested and analysed at the same time . The values are expressed in Mean + SD . Student's t test was used for statistical analyses . Results The values of pH and osmolality of the solutions are presented in Table 1 . The results of an injection of either NA or 5-HT into the dorsal hippocampus are summarized in FIG . 1 . Injections of NA in doses of either 0 .05, 0 .10 or 0 .50 ug had no effect on the concentrations of plasma B . The concentration of plasma B in animals in-
Vol . 25, No . 6, 1979
Dorsal Hippocaapus and Corticosterone
489
TABLE I The values of pH and osmolality of the solutions NaCl Carb (0 .4 HC-3 (1 .0 NA (0 .05 5-HT (0 .1
(0 .9 ug / ug / ug /
uq /
$) 0 .5 ul) 0 .5 yl) 0 .5 yl) 0 .5 yl)
P8 6 .27 6 .20 6 .37 8 .50 3 .56
osmolality(m Osm/1) 277 286 287 278 295
jected with 5-HT in doses of either 0 .1, 1 .0 or 3 .0 ug did not differ from those observed in the controls . Results of intrahippocampal injection of Carb (dose-response) are summarised in FIG . 2 . There was a tendency for plasma B to increase after an injection of 0 .1 Wg of Carb, but the difference was not statistically significant . Though the existence of Circadian rhythm in the levels of B was obvious, in both morning and afternoon experiments intrahippocampal injections of 0 .4 Pg of Carb resulted in significant increases of plasma B . intrahippocampal injections of HC-3 caused no change in basal concentrations of plasma B as far as the doses used in the present studies were concerned (FIG . 3) . FIG . 4 shows the effects of intrahippocampal injections of Csrb on
FIG. 1 . The Effects of Injection of Noradrenaline(NA) or 3erotonia(5-HT) into the Dorsal Hippocampus on the Basal Concentration of Plasma Corticosterone (B) in Rats (Dose Response) . The Experiments were carried out at 3 :00 p .m . . Tha Doses of Monoaminea are expressed in ug . Numbers in Parenthesis show the Numbers of Sxperimenta .
FIG . 2 . The Effects of Injection of CarbachollCarb),into the Dorsal Hippocampus on the Basal Concentrations of Plasma Corticosterone (B) in Rats (Dose Reaponsa) . The Experiments ware carried out at either 3s00 p .m . or 8x00 a .m . . The Doses of Carb are expressed in ug . Numbers in Parenthesis show the Numbers of Experiments . * : P < 0 .05 as compared with ** : P < 0 .01 the Controls .
490
Vol . 25, No . 6, 1979
Dorsal Hippocampus and Corticosterone
70
Plasma
20
B
30
(Lg1100m1) "0
Life Sciences, Vol . 25, pp . 487-496 Printed in the U .S .A .
EFFECTS OF INTRAHIPPOCAMPAL INJECTION OF CHEMICALS ON THE LEVELS OF PLASMA CORTICOSTERONE IN RATS Akira Rawa, Ratsumi Mizuguchi, Yoshio Maeda, Yoshiyasu Taniquchi, Seüchiro Ryu, Syunsaku Yamashita, Takao Ariyama, Takeshi 1(amisaki and Takuya Ranehisn The First Department of Internal Medicine, Ragosh ima University Medical School, Ragoshima 890, Japan . (Received in final form June 20, 1979)
Summary The possibility that the hippocampus can influence the function of the hypothalamo-pituitary-adrenal axis was examined by injecting small quantities of various neurotransmitter substances into this brain structure . Injections of either noradrenaline or 5-hydroxytryptamine into the dorsal hippocampus had no effect on plasma concentrations of corticosterone (B) . ,An injection of carbachol into the dorsal hippocampus elicited a significant rise in B concentrations, while that of hemicholinium into the same brain structure resulted in an inhibition of noise-induced rise of plasma 8 concentration . An injection of carbachol into the dorsal hippocampus counteracted dexamethasone-induced decrease in plasma B concentration, while that of hèmicholinium enhanced it . Although the hippocampus has been implicated in the regulation of ACTH secretion based mainly on results of experiments involving electric stimulation or lesions of this brain structure, some conflicting evidence has been reported . While Endröczi et al . (1) and Rnigge (2) have advocated that the hippocampus exercises an inhibitory influence on hypothalamo-pituitary-adrenocortical (H-P-A) axis, Casady 1~ Taylor (3) reported a facilitatory role of the hippocampua in the regulation of secretion of corticosterone (B) in rats . Lamer et al . (4), in a study of differential hippocampal damage, failed to prove the involvement of the hippocampus in regulation of B secretion in rats . Furthermore the number of papers relating to neurotransmitter specificity in the hippocampus in the regulation of H-P-A activity is still limited . Results of experiments on intrahippocampal application of chemicals (5)(6) do not appear to be . conclusive . In this report the results of our studies on a possible role of the dorsal hippocampus in the regulation of H-P-A activity will be presented with special reference to neurotransmitter specificity . Materials and Methods The animals used were male Wistar rats, weighing 300 g . They were caged individually in rooms provided with automatically controlled 0024-3205/79/060487-09$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
488
Dorsal Hippocampue and Corticoat®tone
Vol . 25, No . 6, 1979
lighting (12 hours of light, from 7 :00 a .m . till 7 :00 p .m . and 12 hours dark daily), and were kept at a constant temperature of 22 + 2°C and a constant humidity of 55 + 58 . The animals were allowed free access to food and water . Pérmanent bilateral cannuli, 0 .7 mm in diameter, were stereotaxically implanted into the dorsal hippocampus . Through them injection cannuli, 0 .3 mm in diameter, were inserted to deliver the chemicals . The stereotaxic coordinates according to Albe-Fessard et al . (7) Kere as follows : AP ; 3 .8 mm, H; 7 .0 mm and L ; 3 .0 mm . Intrahippocampal injection of Chemicals was carried out 14 days after surgery . All the animals were well adapted to the experimental procedures and environment before the experiments . Noradrenaline (NA : 0 .05, 0 .10 or 0 .50 ug ), 5-hydroxytryptamine (5-HT : 0 .1, 1 .0 or 3 .0 ug), carbachol Carb : 0 .1, 0 .4 or 0 .8 ug) or hemicholinium-3 (HC-3 : 0 .5, 1 .0 or 5 .0 ug) dissolved in 0 .5 ul of an 0 .9 $ solution of NaCl (saline) were injected into the dorsal hippocampus in a volume of 0 .5 ul for each site . The chemicals in this volume were delivered with Control animals received an infusion pump in a period of 61 sec . the same volume of vehicle . The animals were decapitated 40 min after injection of the chemicals . Each animal was injected only once . Trunk blood was collected and centrifuged . The samples were kept frozen until assayed fluorometrically for B according to the method of Guillemin et al . (8) . The brains were histologically examined for verification of injection site . Only the animals, in which the site of injection was histologically verified as being the dorsal hippocampus, were used for the assay of B . Observations were made in Carb and HC-3 group animals under the following conditions : 1) basal concentrations 2) exposure to the stressors, 3) dexamethasone . All experiments were performed between 3 :00 p .m . and 5 :00 p .m . except the experiments designed to examine the effects of centrally administered Carb on the circadian rhythm, which were carried out at 8 :00 a .m . and in the afternoon . The following stressors were used : i) auditory stimulation : The animals were exposed to the noise of a buzzer (about 85 phony for 30 min and then killed immediately afterwards ; ü) ether : rats were placed for 1 min in a glass jar saturated with ether vapour and then returned to their home cage for 30 min before decapitation . Dexamethasone (20 ug/rat) was injected intraperitoneally 9 hours and 5 hours prior to the injection into the dorsal hippocampus . In each experiment controls and treated animals were tested and analysed at the same time . The values are expressed in Mean + SD . Student's t test was used for statistical analyses . Results The values of pH and osmolality of the solutions are presented in Table 1 . The results of an injection of either NA or 5-HT into the dorsal hippocampus are summarized in FIG . 1 . Injections of NA in doses of either 0 .05, 0 .10 or 0 .50 ug had no effect on the concentrations of plasma B . The concentration of plasma B in animals in-
Vol . 25, No . 6, 1979
Dorsal Hippocaapus and Corticosterone
489
TABLE I The values of pH and osmolality of the solutions NaCl Carb (0 .4 HC-3 (1 .0 NA (0 .05 5-HT (0 .1
(0 .9 ug / ug / ug /
uq /
$) 0 .5 ul) 0 .5 yl) 0 .5 yl) 0 .5 yl)
P8 6 .27 6 .20 6 .37 8 .50 3 .56
osmolality(m Osm/1) 277 286 287 278 295
jected with 5-HT in doses of either 0 .1, 1 .0 or 3 .0 ug did not differ from those observed in the controls . Results of intrahippocampal injection of Carb (dose-response) are summarised in FIG . 2 . There was a tendency for plasma B to increase after an injection of 0 .1 Wg of Carb, but the difference was not statistically significant . Though the existence of Circadian rhythm in the levels of B was obvious, in both morning and afternoon experiments intrahippocampal injections of 0 .4 Pg of Carb resulted in significant increases of plasma B . intrahippocampal injections of HC-3 caused no change in basal concentrations of plasma B as far as the doses used in the present studies were concerned (FIG . 3) . FIG . 4 shows the effects of intrahippocampal injections of Csrb on
FIG. 1 . The Effects of Injection of Noradrenaline(NA) or 3erotonia(5-HT) into the Dorsal Hippocampus on the Basal Concentration of Plasma Corticosterone (B) in Rats (Dose Response) . The Experiments were carried out at 3 :00 p .m . . Tha Doses of Monoaminea are expressed in ug . Numbers in Parenthesis show the Numbers of Sxperimenta .
FIG . 2 . The Effects of Injection of CarbachollCarb),into the Dorsal Hippocampus on the Basal Concentrations of Plasma Corticosterone (B) in Rats (Dose Reaponsa) . The Experiments ware carried out at either 3s00 p .m . or 8x00 a .m . . The Doses of Carb are expressed in ug . Numbers in Parenthesis show the Numbers of Experiments . * : P < 0 .05 as compared with ** : P < 0 .01 the Controls .
490
Vol . 25, No . 6, 1979
Dorsal Hippocampus and Corticosterone
70
Plasma
20
B
30
(Lg1100m1) "0
