- Email: [email protected]
Reversal of scopolamine induced amnesia in rats by the steroid sulfatase inhibitor estrone-3-O-sulfamate
COGNITIVE BRAIN RESEARCH ELSEVIER
Cognitive Brain Research2 (1995) 251-254
Research report
Reversal of scopolamine induced amnesia in rats by the steroid sulfatase inhibitor estrone-3-0-sulfamate Pui-Kai Li, Michael E. Rhodes, Sharada Jagannathan, Graduate School of Pharmaceutical
Sciences, Duquesne Universi&
David A. Johnson
*
Pittsburgh, PA 15282, USA
Accepted2 May 1995
Abstract The intent of the study was to determine whether altering the metabolism of neurosteroids via blockade of the enzyme, steroid sulfatase, could enhance retention test performance in rats. The steroid sulfatase inhibitor estrone-3-0-sulfamate (EMATE) was administered alone and in combination with the neurosteroid dehydroepiandrosterone sulfate (DHEAS) to rats which were then tested for the reversal of scopolamine induced amnesia. EMATE enhanced the reversal of amnesia by DHEAS as measured by a passive avoidance test. When administered without DHEAS, as a single acute dose, EMATE had no effect. When administered without DHEAS over 10 consecutive days, however, EMATE significantly improved retention. These results suggest that steroid sulfatase inhibition can potentiate the memory enhancing properties of DHEAS. The study also suggests that increasing the levels of endogenous sulfated neurosteroids via the inhibition of steroid sulfatase activity may enhance learning and/or memory function. Keywords:
Memory;
Neurosteroid;
Dehydroepiandrosterone
sulfate; Steroid sulfatase
1. Introduction Neurosteroids are concentrated within and are known to produce effects mediated by the central nervous system (CNS) [6,9,11]. Among the effects associated with neurosteroids is the enhancement of memory [l-3]. The mechanism for this enhancement is not well understood but neurosteroids such as pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS) are known to inhibit the actions mediated by the GABA, receptor and facilitate those mediated by NMDA receptors [6,9]. The unsulfated analogs, pregnenolone and dehydroepiandrosterone, can also enhance memory [l]. However, since the metabolism of pregnenolone and DHEA between the sulfated and unsulfated forms occurs bi-directionally within the CNS, it is uncertain whether the sulfated and unsulfated forms produce memory enhancing effects independently or via metabolism from one analog to the other. Estrone-3-0-sulfamate (EMATE) is a potent irreversible inhibitor of steroid sulfatase [5], the enzyme responsible for the metabolism of PS and DHEAS to their
Corresponding author. [email protected] l
Fax:
(1)
(412)
396-5130;
E-mail:
0926-6410/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SsDrO926-6410(95)00010-0
inhibitor;
Estrone-3-0-sulfamate
unsulfated analogs (Fig. 1). A single dose of EMATE (10 mg/kg) can inhibit > 98% of liver steroid sulfatase activity 24 h following administration. After 11 days of treatment, the sulfatase activity in the whole body (including liver, adrenal gland, ovary, uterus and brain) is inhibited
WI. The intent of this study was to determine whether EMATE, by blocking the conversion of endogenous neurosteroids from the sulfated to their unsulfated analogs, could enhance retention test performance as measured by the reversal of scopolamine induced amnesia in a passive avoidance memory test. An additional goal was to determine whether the retention enhancing effects of peripherally administered DHEAS could be potentiated by inhibition of the metabolism of DHEAS to DHEA via the pre-administration of EMATE.
2. Materials
and methods
Male rats weighing between 100 and 124 g were purchased from Zivic-Miller Laboratories (Zelienople, PA) and housed in hanging wire mesh cages in groups of 3 with water and rat chow available ad libitum. The room in which the animals were housed was controlled for both
252
P.-K. Li et al. /Cognitive Brain Research 2 (1995) 251-254
temperature and humidity with a standard 12 h, light/dark cycle. To assess retention test performance, a Gemini Avoidance System (San Diego Instruments) was utilized with a standard passive avoidance paradigm [13]. Briefly, the avoidance apparatus consisted of a box (53 X 53 X 32 cm) with 2 compartments connected by an opening with a sliding door. The compartment in which the rat was placed was brightly lit, while the other compartment was dark. Before the acquisition trial, the animals were allowed to explore the apparatus and were then removed. During the acquisition trial, animals were placed into the light compartment. When an animal entered the dark compartment, the sliding door closed and a mild foot shock (1 mA, 1 s) was administered. The rat was then removed from the apparatus and returned to its cage. Twenty-four hours later the rat was again placed in the ligh; compartment of the apparatus and the time to crossing (crossover latency) to the dark compartment recorded. If the rat did not enter the compartment within 10 min it was removed from the apparatus. Retention was assessed as an increased crossover latency period before entering the dark compartment during the second trial. Significant differences in crossover latency were determined by statistical analysis utilizing one-way analysis of variance with a Dunnett’s test post hoc. Significant differences between groups were interpreted as differences in retention resulting from the various treatments. Scopolamine and DHEAS were purchased from Sigma Chemical Co. (St. Louis, MO). EMATE was synthesized according to the method of Howarth et al. [5]. Preliminary studies were performed to determine whether the administration of DHEAS and EMATE without scopolamine, would alter the retention test perfor-
mance response to foot-shock compared to saline. In these experiments, animals (five animals per group) were injected intraperitoneal (IP) with a combination of DHEAS (1.0 mg/kg) administered 1 h prior to testing and EMATE (10.0 mg/kg) 4 h prior to testing, or deionized water, 1 h prior to the acquisition trial. The acquisition trials utilized one of two intensities of foot shock; either 0.5 or 1.0 mA, for 1 s duration. Twenty-four hours later, crossover latenties were determined. To assess the effect of DHEAS on scopolamine amnesia, 1 h before the acquisition trial groups of ten animals were injected IP with one of several doses of DHEAS (5, 10, 20, 30, 50, 70 mg/kg) dissolved in deionized water or deionized water alone. Thirty minutes prior to testing the animals were also injected IP with scopolamine (1 mg/kg) a dose which previously had been determined would produce amnesia with this paradigm. Twenty-four hours later, the rats were again placed in the apparatus and crossover latencies recorded. To determine whether EMATE could potentiate the reversal of scopolamine induced amnesia by DHEAS, additional groups of ten rats per group were administered EMATE (10 mg/kg; IP) suspended in corn oil IP 48 h before the acquisition trial. On the day of the acquisition trial animals were administered DHEAS and scopolamine as above, except that the dosage range of DHEAS was reduced (0.03, 0.1, 0.3, 1.0, 5.0, 10.0 mg/kg). Twenty-four hours later, crossover latencies for the groups were determined. In order to assess whether EMATE alone could reverse scopolamine induced amnesia amnesia, 4 groups of rats (10 animals per group) were injected with EMATE (10 mg/kg; IP), either 4 h before the acquisition trial, or daily
Stemld sulhtase CHOLESTEROL SULFATE OR LIFOIDAL
-
-
CHOLESTEROL
P-450, 20~DIHYDRO-PREGNENOLONE
j
+aJ PREGNENOLONE SULFATE
-
OXPWGNT
PROGESTERONE 1 5a_rcd”ctase
3B-HSD
7sHYDROXY-PREGNENOLONE i
oRs DEHYDROEPIANDROSTERONE
SULFATE
SWPREGNANE-3.20-DIONE
DEHYDROEPIANDROSTERONE
““@_y
y
7a-HYDROXY-DEHYDROEPIANDROSTERONE
3!_%HYDROXY-5a-PREGNANEZO-ONE
Fig. 1. Neurosteroid biosynthesis and metabolism Figure is a modification of Fig. 1 found in [2,11].
in the rat brain. Dotted arrows indicate metabolic
conversions
3a-HYDROXY-Sri-PP.EGNANE-20-ONE
not yet formally
demonstrated.
Note:
P.-K. Li et al. /Cognitive Table 1 Crossover
latency (s) for rats not administered
Treated Saline control Foot-shock intensity (mA)
2.53
Brain Research 2 (I 995) 2.51-254
scopolamine
199.4 f 66.6 191.3 * 104.1 0.5
0
475.7 * 95.9 450.6 + 82.5 1.0
EMATE
10 mg/Kg
Treated animals received DHEAS (1 mg/kg) and EMATE (10 mg/kgl IP 1 h before acquisition trial. Each group consisted of 5 animals. There was no significant difference in crossover latency between treated and control animals within each level of foot-shock intensity.
for 3, 10, or 15 days before the acquisition trial. Twentyfour hours following the acquisition trial crossover latenties were determined. 01
3. Results
-5
0
1 C?
5
15
i ‘0
Days
In the preliminary studies, those groups tested without the administration of scopolamine had prolonged crossover latencies indicating memory retention of the foot-shock of the previous day. However, there was no difference in crossover latency between control animals administered deionized water and animals administered the combination of DHEAS (1.0 mg/kg) and EMATE (10 mg/kg) for each level of shock intensity (Table 1). These results indicated that the administration of DHEAS and EMATE did not alter the response of rats to foot-shock compared to control animals. Rats administered deionized water 1 h before and the amnestic agent scopolamine (1 mg/kg; IP) 30 min before the acquisition trial had a crossover latency 24 h later of only 21.1 + 10.9 s which was significantly
350
A
DHEAS
0
DHEAS
+ EMATE *
300
250
200
150
100
50
Fig. 3. The effect of EMATF on scopolamine induced amnesia following pre-administration of 10 mg/kg IP once a day for 1, 3, 10 and 15 days. Each group contained 10 animals, bars indicate standard error of the mean. * indicates significance; P < 0.05.
shorter than the crossover latency for groups administered the acquisition trial without scopolamine. This result indicated that the administration of scopolamine would induce amnesia during the acquisition trial of the study. In the groups of animals administered DHEAS in various doses (5-70 mg/kg) 1 h before the acquisition trial and scopolamine 30 min before, there was a significant (P < 0.05) reversal of scopolamine induced amnesia which followed a bell shaped dose response curve (Fig. 2). The maximum effect occurred with the 20 mg/kg dose of DHEAS resulting in a crossover latency of 299.4 + 83.5 s. For those groups of animals treated with both EMATE and DHEAS, there was a 20 fold decrease in the dose of DHEAS producing the maximal effect (1 mg/kg) with a crossover latency of (194.9 f 54.6 s at 1 mg/kg; Fig. 2). For those groups treated with EMATE alone, a single dose of EMATE, or 3 days of treatment failed to increase crossover latency significantly (65.5 f 24.1 and 142.1 + 61.8, respectively). However, the groups administered EMATE daily for 10 and 15 days before the acquisition trial demonstrated a significant (P < 0.05) reversal of scopolamine induced amnesia with crossover latencies of 263.1 f 69.7 s for the 10 day group and 297.0 + 88.7 s for the 15 day group, respectively (Fig. 3).
0
Dose (mg/Kg) Fig. 2. The effects of several doses of DHEIAS and DHEAS in combination with EMATE (10 mg/kg; IP on scopolamine induced amnesia. Crossover latency is the time delay for a rat to enter the dark compartment of the testing apparatus 24 h following the acquisition trial. Each dose group contained 10 animals, bars indicate standard error of the mean. indicates significance; P < 0.05. l
4. Discussion This study confirms the investigations of Flood et al. [3] in demonstrating the reversal of scopolamine induced amnesia by DHEAS. Moreover, the results of this study are the first to suggest that retention in a memory test can be enhanced by blocking the desulfation of endogenous neu-
254
P.-K. Li et al. / Cognirive Brain Research 2 (1995) 251-254
rosteroids. There are several possible mechanisms by which EMATE can enhance retention. The GABAergic neurons of the nucleus accumbens are known to synapse upon cholinergic neurons of nucleus basalis magnocellularis (NBM) which then project to the hippocampus [14]. Local injection of P-carboline, an inverse agonist of GABA into the NBM has been shown to enhance recognition performance in a memory task in rats [7]. When the neurosteroid PS, a GABA, antagonist, was infused into the NBM there was also a demonstration of enhanced memory [S]. Since DHEAS is also known to antagonize the GABA, receptor, it is possible that in this study the administration of DHEAS may have enhanced retention by disinhibiting cholinergic neurons of the NBM. Furthermore, this mechanism would be consistent with the reversal of the amnestic effect of scopolamine, a cholinergic antagonist. Increased brain concentrations of acetylcholine, however, need to be confirmed by methods which allow for the direct monitoring of cholinergic neurochemistry. Since DHEA and DHEAS are synthesized and released by the adrenal gland as well as in the CNS, it is possible that the potentiating effect of EMATE on DHEAS may be related to steroid sulfatase inhibition in either one or both tissues. That EMATE could significantly enhance the potency of DHEAS in reversing scopolamine induced amnesia suggests that it is the sulfated form of DHEA which is responsible for the memory enhancing properties of this neurosteroid. Additionally, the failure of EMATE alone to enhance crossover latency following a single acute administration, indicated that the enzyme inhibitor did not affect the DHEAS binding site directly, since DHEAS could reverse scopolamine induced amnesia following a single acute administration. Ten days and 15 days of pretreatment with EMATE, however, did have a significantly positive effect for enhancing retention impaired by scopolamine. This positive effect may reflect an accumulation of sulfated neurosteroids over a period of days. In this study, EMATE may have increased the absolute concentrations of endogenous sulfated neurosteroids or changed the ratios between sulfated and unsulfated forms. It is possible, however, that EMATE could also influence other steroid hormones which affect memory such as corticosterone or sex hormones
[4,101. That there was no difference between the control and treatment groups when scopolamine was not administered suggests that DHEAS and EMATE did not alter the perception by the rats of the foot-shock. Rather, this result suggests that the effects of the drugs were related to changes in memory acquisition and or processing, not differences in sensory perception. This conclusion is supported by the investigations by Flood of the memory enhancing properties of DHEAS. In those experiments the neurosteroid was administered immediately following the acquisition trial rather than before [3]. In summary, this study demonstrated that blockade of
the enzyme steroid sulfatase by EMATE could potentiate retention test performance mediated by DHEAS. Additionally, when administered over a period of days EMATE alone could enhance retention. These results suggest that increasing concentrations of sulfated neurosteroids can amplify centrally mediated cholinergic functions including memory.
Acknowledgements The authors wish to thank Drs. James Flood, Robert Purdy and Robert Gibbs for their thoughtful suggestions. This research was supported in part by a faculty development award from Duquesne University.
References [l] Flood, J.F., Morley, J.E. and Roberts, E., Memory-enhancing effects in male mice of pregnenolone and steroids metabolically derived from it, Proc. Natl. Acad. Sci. USA, 89 (1992) 1567-1571. [2] Flood, J.F. and Roberts, E., Dehydroepiandrosterone sulfate improves memory in aging mice, Brain Rex, 448 (1988) 178-181. [3] Flood, J.F., Smith, GE. and Roberts, E., Dehydroepiandrosterone and its sulfate enhance memory retention in mice, Brain Res., 447 (1988) 269-278. [4] Gibbs, R.B., Wu, D., Hersh, L.B. and Pfaff, D.W., Effects of estrogen replacement on relative levels of choline acetyltransferase, t&A, and nerve growth factor messenger RNAs in the basal forebrain and hippocampal formation of adult rats, Exp. Neural., 129 (1994) 70-80. [5] Howarth, N.M., Purohit, N.M.A., Reed, M.J. and Potter, B.V.L., Estrone sulfamates: potent inhibitors of estrone sulfatase with therapeutic potential, J. Med. Chem. 37 (1994) 219-221. [6] Majewska, M.D., Neurosteroids: endogenous bimodal modulators of the GABA-A receptor. mechanism of action and physiological significance, Prog. NeurobioI., 38 (1992) 379-395. [7] Mayo, W., Dellu, F., Cherkaoui, C., Chapouthier, G., Dodd, R.H., Le Moal, M. and Simon, H., Cognitive enhancing properties of P-CMM infused into the nucleus basalis magnocellularis of the rat, Brain Rex, 589 (1992) 109-114. [8] Mayo, W., Dellu, F., Robel, P., Cherkaoui, J., Le Moal, M., Baulieu, E.-E. and Simon, H., Infusion of neurosteroids into the nucleus basalis magnocellularis affects cognitive processes in the rat, Brain Res., 607 (1993) 324-328. [9] Mellon, S.H., Neurosteroids: biochemistry, modes of action, and clinical relevance, J. Clin. Endocrbzol. Metab., 78 (1994) 10031008. [lo] Rivas-Arancibia, S. and Vazquez-Pereyra, F., Hormonal modulation of extinction responses induced by sexual steroid hormones in rats, Life Sci., 54 (1994) PL363-367. [ll] Robel, P and Baulieu, E.-E., Neurosteroids: biosynthesis and function, Trends Endocrinol. Merab., 5 (1994) l-8. [12] Roberts, C., Purohit, N.M.A., Williams, G.J., Potter, B.V.L. and Reed, M.J., Duration and extent of in vivo inhibition of steroid sulphatase activity in rat tissues following single and multiple doses of oesterone-3-0-sulphamate, J. Endocrinol. 140 (Suppl) (1994). [13] Rush, D.K., Scopolamine amnesia of passive avoidance: a deficit of information acquisition, Behav. Neural Biol. 50 (1988) 255-274. 1141 Zaborszky, L, Cullinan, W.E. and Braun, A., Afferents to basal forebrain cholinergic projection neurons: an update. In CT. Napier, P.W. Kalivas and I. Hanin (Eds.), Basal Forebrain: Anatomy and Function, Plenum Press, New York, 1991.
Cognitive Brain Research2 (1995) 251-254
Research report
Reversal of scopolamine induced amnesia in rats by the steroid sulfatase inhibitor estrone-3-0-sulfamate Pui-Kai Li, Michael E. Rhodes, Sharada Jagannathan, Graduate School of Pharmaceutical
Sciences, Duquesne Universi&
David A. Johnson
*
Pittsburgh, PA 15282, USA
Accepted2 May 1995
Abstract The intent of the study was to determine whether altering the metabolism of neurosteroids via blockade of the enzyme, steroid sulfatase, could enhance retention test performance in rats. The steroid sulfatase inhibitor estrone-3-0-sulfamate (EMATE) was administered alone and in combination with the neurosteroid dehydroepiandrosterone sulfate (DHEAS) to rats which were then tested for the reversal of scopolamine induced amnesia. EMATE enhanced the reversal of amnesia by DHEAS as measured by a passive avoidance test. When administered without DHEAS, as a single acute dose, EMATE had no effect. When administered without DHEAS over 10 consecutive days, however, EMATE significantly improved retention. These results suggest that steroid sulfatase inhibition can potentiate the memory enhancing properties of DHEAS. The study also suggests that increasing the levels of endogenous sulfated neurosteroids via the inhibition of steroid sulfatase activity may enhance learning and/or memory function. Keywords:
Memory;
Neurosteroid;
Dehydroepiandrosterone
sulfate; Steroid sulfatase
1. Introduction Neurosteroids are concentrated within and are known to produce effects mediated by the central nervous system (CNS) [6,9,11]. Among the effects associated with neurosteroids is the enhancement of memory [l-3]. The mechanism for this enhancement is not well understood but neurosteroids such as pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS) are known to inhibit the actions mediated by the GABA, receptor and facilitate those mediated by NMDA receptors [6,9]. The unsulfated analogs, pregnenolone and dehydroepiandrosterone, can also enhance memory [l]. However, since the metabolism of pregnenolone and DHEA between the sulfated and unsulfated forms occurs bi-directionally within the CNS, it is uncertain whether the sulfated and unsulfated forms produce memory enhancing effects independently or via metabolism from one analog to the other. Estrone-3-0-sulfamate (EMATE) is a potent irreversible inhibitor of steroid sulfatase [5], the enzyme responsible for the metabolism of PS and DHEAS to their
Corresponding author. [email protected] l
Fax:
(1)
(412)
396-5130;
E-mail:
0926-6410/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SsDrO926-6410(95)00010-0
inhibitor;
Estrone-3-0-sulfamate
unsulfated analogs (Fig. 1). A single dose of EMATE (10 mg/kg) can inhibit > 98% of liver steroid sulfatase activity 24 h following administration. After 11 days of treatment, the sulfatase activity in the whole body (including liver, adrenal gland, ovary, uterus and brain) is inhibited
WI. The intent of this study was to determine whether EMATE, by blocking the conversion of endogenous neurosteroids from the sulfated to their unsulfated analogs, could enhance retention test performance as measured by the reversal of scopolamine induced amnesia in a passive avoidance memory test. An additional goal was to determine whether the retention enhancing effects of peripherally administered DHEAS could be potentiated by inhibition of the metabolism of DHEAS to DHEA via the pre-administration of EMATE.
2. Materials
and methods
Male rats weighing between 100 and 124 g were purchased from Zivic-Miller Laboratories (Zelienople, PA) and housed in hanging wire mesh cages in groups of 3 with water and rat chow available ad libitum. The room in which the animals were housed was controlled for both
252
P.-K. Li et al. /Cognitive Brain Research 2 (1995) 251-254
temperature and humidity with a standard 12 h, light/dark cycle. To assess retention test performance, a Gemini Avoidance System (San Diego Instruments) was utilized with a standard passive avoidance paradigm [13]. Briefly, the avoidance apparatus consisted of a box (53 X 53 X 32 cm) with 2 compartments connected by an opening with a sliding door. The compartment in which the rat was placed was brightly lit, while the other compartment was dark. Before the acquisition trial, the animals were allowed to explore the apparatus and were then removed. During the acquisition trial, animals were placed into the light compartment. When an animal entered the dark compartment, the sliding door closed and a mild foot shock (1 mA, 1 s) was administered. The rat was then removed from the apparatus and returned to its cage. Twenty-four hours later the rat was again placed in the ligh; compartment of the apparatus and the time to crossing (crossover latency) to the dark compartment recorded. If the rat did not enter the compartment within 10 min it was removed from the apparatus. Retention was assessed as an increased crossover latency period before entering the dark compartment during the second trial. Significant differences in crossover latency were determined by statistical analysis utilizing one-way analysis of variance with a Dunnett’s test post hoc. Significant differences between groups were interpreted as differences in retention resulting from the various treatments. Scopolamine and DHEAS were purchased from Sigma Chemical Co. (St. Louis, MO). EMATE was synthesized according to the method of Howarth et al. [5]. Preliminary studies were performed to determine whether the administration of DHEAS and EMATE without scopolamine, would alter the retention test perfor-
mance response to foot-shock compared to saline. In these experiments, animals (five animals per group) were injected intraperitoneal (IP) with a combination of DHEAS (1.0 mg/kg) administered 1 h prior to testing and EMATE (10.0 mg/kg) 4 h prior to testing, or deionized water, 1 h prior to the acquisition trial. The acquisition trials utilized one of two intensities of foot shock; either 0.5 or 1.0 mA, for 1 s duration. Twenty-four hours later, crossover latenties were determined. To assess the effect of DHEAS on scopolamine amnesia, 1 h before the acquisition trial groups of ten animals were injected IP with one of several doses of DHEAS (5, 10, 20, 30, 50, 70 mg/kg) dissolved in deionized water or deionized water alone. Thirty minutes prior to testing the animals were also injected IP with scopolamine (1 mg/kg) a dose which previously had been determined would produce amnesia with this paradigm. Twenty-four hours later, the rats were again placed in the apparatus and crossover latencies recorded. To determine whether EMATE could potentiate the reversal of scopolamine induced amnesia by DHEAS, additional groups of ten rats per group were administered EMATE (10 mg/kg; IP) suspended in corn oil IP 48 h before the acquisition trial. On the day of the acquisition trial animals were administered DHEAS and scopolamine as above, except that the dosage range of DHEAS was reduced (0.03, 0.1, 0.3, 1.0, 5.0, 10.0 mg/kg). Twenty-four hours later, crossover latencies for the groups were determined. In order to assess whether EMATE alone could reverse scopolamine induced amnesia amnesia, 4 groups of rats (10 animals per group) were injected with EMATE (10 mg/kg; IP), either 4 h before the acquisition trial, or daily
Stemld sulhtase CHOLESTEROL SULFATE OR LIFOIDAL
-
-
CHOLESTEROL
P-450, 20~DIHYDRO-PREGNENOLONE
j
+aJ PREGNENOLONE SULFATE
-
OXPWGNT
PROGESTERONE 1 5a_rcd”ctase
3B-HSD
7sHYDROXY-PREGNENOLONE i
oRs DEHYDROEPIANDROSTERONE
SULFATE
SWPREGNANE-3.20-DIONE
DEHYDROEPIANDROSTERONE
““@_y
y
7a-HYDROXY-DEHYDROEPIANDROSTERONE
3!_%HYDROXY-5a-PREGNANEZO-ONE
Fig. 1. Neurosteroid biosynthesis and metabolism Figure is a modification of Fig. 1 found in [2,11].
in the rat brain. Dotted arrows indicate metabolic
conversions
3a-HYDROXY-Sri-PP.EGNANE-20-ONE
not yet formally
demonstrated.
Note:
P.-K. Li et al. /Cognitive Table 1 Crossover
latency (s) for rats not administered
Treated Saline control Foot-shock intensity (mA)
2.53
Brain Research 2 (I 995) 2.51-254
scopolamine
199.4 f 66.6 191.3 * 104.1 0.5
0
475.7 * 95.9 450.6 + 82.5 1.0
EMATE
10 mg/Kg
Treated animals received DHEAS (1 mg/kg) and EMATE (10 mg/kgl IP 1 h before acquisition trial. Each group consisted of 5 animals. There was no significant difference in crossover latency between treated and control animals within each level of foot-shock intensity.
for 3, 10, or 15 days before the acquisition trial. Twentyfour hours following the acquisition trial crossover latenties were determined. 01
3. Results
-5
0
1 C?
5
15
i ‘0
Days
In the preliminary studies, those groups tested without the administration of scopolamine had prolonged crossover latencies indicating memory retention of the foot-shock of the previous day. However, there was no difference in crossover latency between control animals administered deionized water and animals administered the combination of DHEAS (1.0 mg/kg) and EMATE (10 mg/kg) for each level of shock intensity (Table 1). These results indicated that the administration of DHEAS and EMATE did not alter the response of rats to foot-shock compared to control animals. Rats administered deionized water 1 h before and the amnestic agent scopolamine (1 mg/kg; IP) 30 min before the acquisition trial had a crossover latency 24 h later of only 21.1 + 10.9 s which was significantly
350
A
DHEAS
0
DHEAS
+ EMATE *
300
250
200
150
100
50
Fig. 3. The effect of EMATF on scopolamine induced amnesia following pre-administration of 10 mg/kg IP once a day for 1, 3, 10 and 15 days. Each group contained 10 animals, bars indicate standard error of the mean. * indicates significance; P < 0.05.
shorter than the crossover latency for groups administered the acquisition trial without scopolamine. This result indicated that the administration of scopolamine would induce amnesia during the acquisition trial of the study. In the groups of animals administered DHEAS in various doses (5-70 mg/kg) 1 h before the acquisition trial and scopolamine 30 min before, there was a significant (P < 0.05) reversal of scopolamine induced amnesia which followed a bell shaped dose response curve (Fig. 2). The maximum effect occurred with the 20 mg/kg dose of DHEAS resulting in a crossover latency of 299.4 + 83.5 s. For those groups of animals treated with both EMATE and DHEAS, there was a 20 fold decrease in the dose of DHEAS producing the maximal effect (1 mg/kg) with a crossover latency of (194.9 f 54.6 s at 1 mg/kg; Fig. 2). For those groups treated with EMATE alone, a single dose of EMATE, or 3 days of treatment failed to increase crossover latency significantly (65.5 f 24.1 and 142.1 + 61.8, respectively). However, the groups administered EMATE daily for 10 and 15 days before the acquisition trial demonstrated a significant (P < 0.05) reversal of scopolamine induced amnesia with crossover latencies of 263.1 f 69.7 s for the 10 day group and 297.0 + 88.7 s for the 15 day group, respectively (Fig. 3).
0
Dose (mg/Kg) Fig. 2. The effects of several doses of DHEIAS and DHEAS in combination with EMATE (10 mg/kg; IP on scopolamine induced amnesia. Crossover latency is the time delay for a rat to enter the dark compartment of the testing apparatus 24 h following the acquisition trial. Each dose group contained 10 animals, bars indicate standard error of the mean. indicates significance; P < 0.05. l
4. Discussion This study confirms the investigations of Flood et al. [3] in demonstrating the reversal of scopolamine induced amnesia by DHEAS. Moreover, the results of this study are the first to suggest that retention in a memory test can be enhanced by blocking the desulfation of endogenous neu-
254
P.-K. Li et al. / Cognirive Brain Research 2 (1995) 251-254
rosteroids. There are several possible mechanisms by which EMATE can enhance retention. The GABAergic neurons of the nucleus accumbens are known to synapse upon cholinergic neurons of nucleus basalis magnocellularis (NBM) which then project to the hippocampus [14]. Local injection of P-carboline, an inverse agonist of GABA into the NBM has been shown to enhance recognition performance in a memory task in rats [7]. When the neurosteroid PS, a GABA, antagonist, was infused into the NBM there was also a demonstration of enhanced memory [S]. Since DHEAS is also known to antagonize the GABA, receptor, it is possible that in this study the administration of DHEAS may have enhanced retention by disinhibiting cholinergic neurons of the NBM. Furthermore, this mechanism would be consistent with the reversal of the amnestic effect of scopolamine, a cholinergic antagonist. Increased brain concentrations of acetylcholine, however, need to be confirmed by methods which allow for the direct monitoring of cholinergic neurochemistry. Since DHEA and DHEAS are synthesized and released by the adrenal gland as well as in the CNS, it is possible that the potentiating effect of EMATE on DHEAS may be related to steroid sulfatase inhibition in either one or both tissues. That EMATE could significantly enhance the potency of DHEAS in reversing scopolamine induced amnesia suggests that it is the sulfated form of DHEA which is responsible for the memory enhancing properties of this neurosteroid. Additionally, the failure of EMATE alone to enhance crossover latency following a single acute administration, indicated that the enzyme inhibitor did not affect the DHEAS binding site directly, since DHEAS could reverse scopolamine induced amnesia following a single acute administration. Ten days and 15 days of pretreatment with EMATE, however, did have a significantly positive effect for enhancing retention impaired by scopolamine. This positive effect may reflect an accumulation of sulfated neurosteroids over a period of days. In this study, EMATE may have increased the absolute concentrations of endogenous sulfated neurosteroids or changed the ratios between sulfated and unsulfated forms. It is possible, however, that EMATE could also influence other steroid hormones which affect memory such as corticosterone or sex hormones
[4,101. That there was no difference between the control and treatment groups when scopolamine was not administered suggests that DHEAS and EMATE did not alter the perception by the rats of the foot-shock. Rather, this result suggests that the effects of the drugs were related to changes in memory acquisition and or processing, not differences in sensory perception. This conclusion is supported by the investigations by Flood of the memory enhancing properties of DHEAS. In those experiments the neurosteroid was administered immediately following the acquisition trial rather than before [3]. In summary, this study demonstrated that blockade of
the enzyme steroid sulfatase by EMATE could potentiate retention test performance mediated by DHEAS. Additionally, when administered over a period of days EMATE alone could enhance retention. These results suggest that increasing concentrations of sulfated neurosteroids can amplify centrally mediated cholinergic functions including memory.
Acknowledgements The authors wish to thank Drs. James Flood, Robert Purdy and Robert Gibbs for their thoughtful suggestions. This research was supported in part by a faculty development award from Duquesne University.
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