Brain Research, 532 (1990) 95-100 Elsevier
95
BRES 15995
Effects of changes in peripheral and cerebral glucose metabolism on locomotor activity, learning and memory in adult male rats Gtinter Mayer, Roger Nitsch and Siegfried Hoyer Department of Pathochemistry and General Neurochemistry, University of Heidelberg, Heidelberg (F.R. G.) (Accepted 8 May 1990) Key words: Hyperglycemia; Cerebral glucose metabolism; Streptozotocin; Locomotor activity; Passive avoidance learning
Interactions of glucose and cognitive function have been reported both in the presence of elevated arterial blood glucose levels and with decreased cerebral glucose metabolism. In order to test the peripheral vs. central effects of this phenomenon, we induced irreversible hyperglycemia and depression of cerebral glucose metabolism in separate designs by means of either intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of streptozotocin (STZ), which is known to damage insulin-producing cells. Behavioral functions, such as locomotor activity, learning, and memory, were investigated under these different conditions. IP treatment with STZ decreased locomotor activity and increased initial step-through latencies on the passive avoidance test. No effects of elevated arterial blood glucose levels on retention of passive avoidance learning checked at 24 h and 144 h after training were observed. I.c.v. treatment of STZ increased the rate of locomotor activity and impaired retention in the passive avoidance test at 24 h, without further forgetfulness at 144 h. This finding may indicate disturbed acquisition and/or consolidation of memory, which may remain impaired but at a constant level, without further deterioration. Enhanced motor activity and impaired acquisition of passive avoidance learning without further impairment have also been reported as a characteristical behavioral pattern after disruption of the cholinergic system. It is therefore postulated that the observed behavioral abnormalities consequent on an impairment of cerebral glucose metabolism may be suggestive of cholinergic dysfunction.
INTRODUCTION Recent findings suggest that disturbances in cerebral glucose and related metabolism may play an important role in brain aging and the genesis of disorders causing dementia lz'18'28'29'3°'52. Glucose is known to be the major fuel for biological energy, and thus for metabolic activity in the central nervous system, in normal conditions 5~'55. A close coupling between cerebral glucose metabolism and neuronal function has been demonstrated in a variety of physiological conditions 54. Brain glucose concentrations below physiological levels can influence the rate of acetylcholine synthesis by modifying the central acetyl-coenzyme A (CoA) concentration, which is derived from glucose by way of glycolysis and the pyruvate oxidation system 4'8'19. This means, for example, that acetylcholine synthesis is significantly decreased in severe hypoglycemia ~9. Abnormalities in the central cholinergic system have been shown to affect behavior, learning, and memory 13'22'56'59. Impairment of passive avoidance learning has been found after the induction of depressed regulation of cerebral glucose metabolism by streptozotocin (STZ) by
the intracerebroventricular (i.c.v.) route 37. It has been suggested that this cognitive impairment might be related to the metabolic action of the impaired cerebral regulation in utilization of glucose, interfering with the synthesis of a critical pool of acetylcholine 42. On the other hand, improved m e m o r y performance has been found in rat and human with peripheral posttraining administration of glucose 16"23"24'39. Glucose also enhances m e m o r y after i.c.v, administration 32. The biochemical mechanisms of the positive effects on performance are still unknown. It has been suggested that glucose may regulate the storage of new information by increasing neuronal glucose availability or via peripheral contributions to glucose by still unknown interactions with the body's glucose regulatory system 21'23. Thus, these findings point to interactions between both peripheral and central glucose metabolism and brain function as determined by learning and memory. The present study was performed to investigate the effect of disturbed glucose regulation after intraperitoneal (i.p.) administration of STZ (STZ-i.p.), producing chronically elevated arterial blood glucose levels 31'4°, and after i.c.v, administration of STZ (STZi.c.v.), generating cerebral hypometabolism of glucose
Correspondence: G. Mayer, Department of Pathochemistry and General Neurochemistry, University of Heidelberg, Im Neuenheimer Feld 220-221, D-6900 Heidelberg, F.R.G. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishe.rs B.V. (Biomedical Division)
96 l a s t i n g f o r s e v e r a l w e e k s 41, o n l o c o m o t o r activity, l e a r n -
LOCOMOTOR ACTIVITY
ing, a n d m e m o r y in a d u l t rats, m e a s u r e d as c l o s e d field
~ CONTROL ~ STZ-IP EZ] STZ-ICV
activity and passive avoidance behavior. 200
*
MATERIALS AND METHODS
Animals
.~_ ~
//A //£
One-year-old (adult) male Wistar rats weighing between 390 and 520 g (breeder: Zentralinstitut ffir Versuchstierzucht, Hannover, F.R.G.) were used in the present study. They were housed in individual cages in a temperature-controlled animal room with a reversed 12:12 h light:dark cycle (lights on at 19.30 h). Experiments were conducted during the dark portion of the cycle. Food pellets (Altromin standard, No. 1320) and water were freely available throughout the experiment. The animals were divided randomly into 4 groups. (1) In one group (n = 18) each animal received a single unilateral (left side) i.c.v, injection of STZ (Sigma, Munich, E R . G . ) in a subdiabetic dose (1.5 mg/kg body weight), sufficient to bring about a significant decrease (by approximately 46%) of several weeks' duration 42 in the cerebral metabolic rate of glucose utilization while the arterial blood glucose concentration (BG) remains unchanged (BG = 9.1 _+ 0.95 mmol/1), as determined by means of commercial test kits (Boehringer Mannheim, ER.G.). (2) The controls for this group (n = 18) received i.c.v, injections of artificial cerebral spinal fluid (CSF) (BG = 9.3 _+ 0.97 mmol/l). (3) In a third group (n = 6), a single i.p. injection of STZ (55 mg/kg body weight) was given to induce insulin-dependent diabetes mellitus (type I) with plasma glucose levels 3 times those of normal animals (BG = 25.3 + 1.56 mmol/1). (4) The corresponding controls (n = 5) received i.p. injections of saline only (BG = 9.7 + 0.84 mmol/l).
placed in the illuminated starting box again and the step-through latency established. The test was concluded when the animal entered the dark compartment or after 5 min if it failed to do so. In order to improve the reliability and validity of the footshock avoidance test, the plates were moistened before each footshock with water, significantly reducing the wide interindividual variability in paw skin resistance of the rats.
Behavioral testing
Statistics
Two behavioral tests were used: (1) The closed-field activity center, to study the rats' locomotor activity, and (2) a single-trial step-through passive avoidance test to investigate learning and memory. (1) Spontaneous locomotor activity was assessed at day 11 after STZ administration. Each animal was observed over a period of 300 s in a square closed-field arena (80 x 80 x 40 cm) equipped with a row of 12 infrared light-sensitive photocells placed 5 cm above the wooden floor of the pen. The 3 photocells on each wall of the square were spaced 20 cm apart, and the last photocell in each row was spaced 20 cm from the next wall. Interruptions of photocell beams were recorded by means of a microcomputer, allowing a record of all horizontal activity as measured by the total number of interruptions of the 12 photocell beams. The closed-field apparatus was housed together with the other behavioral apparatus in a darkened, light- and sound-attenuated and ventilated testing room. During behavioral testing, only one animal and the tester were in the testing room at any time. (2) Single-trial passive avoidance learning. STZ was administered 13 days before the training. The step-through passive avoidance behavior was evaluated by using the light-dark box test. The light-dark box consisted of a 55 × 18 x 40 cm apparatus made of wood. It was divided into two equal compartments by a wooden wall with an 8 x 8 cm opening in it. The opening was controlled by a guillotine door between the two compartments. The light compartment was illuminated by a 60-W lamp fixed 40 cm above its floor in the center. The interior of the dark chamber was painted black and had a ceiling. The floor of each compartment consisted of an individual stainless steel plate. To ensure electrical separation, there was a 5-mm gap between the two plates in the light-dark box at the opening between the two chambers. Each plate had a separate connection to the polar output of our laboratory transformer, which delivered constant-current footshock (AC, 1 mA, 1 s) through the metal floor plates. In each test, single electric shock was applied while the animal was approaching the dark part of the apparatus through the opening. At 24 h and 144h after the shock the rat was
~
100.
~ ~
I
//~
+
//~ //~
so-
///t //~ //~
95
BRES 15995
Effects of changes in peripheral and cerebral glucose metabolism on locomotor activity, learning and memory in adult male rats Gtinter Mayer, Roger Nitsch and Siegfried Hoyer Department of Pathochemistry and General Neurochemistry, University of Heidelberg, Heidelberg (F.R. G.) (Accepted 8 May 1990) Key words: Hyperglycemia; Cerebral glucose metabolism; Streptozotocin; Locomotor activity; Passive avoidance learning
Interactions of glucose and cognitive function have been reported both in the presence of elevated arterial blood glucose levels and with decreased cerebral glucose metabolism. In order to test the peripheral vs. central effects of this phenomenon, we induced irreversible hyperglycemia and depression of cerebral glucose metabolism in separate designs by means of either intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of streptozotocin (STZ), which is known to damage insulin-producing cells. Behavioral functions, such as locomotor activity, learning, and memory, were investigated under these different conditions. IP treatment with STZ decreased locomotor activity and increased initial step-through latencies on the passive avoidance test. No effects of elevated arterial blood glucose levels on retention of passive avoidance learning checked at 24 h and 144 h after training were observed. I.c.v. treatment of STZ increased the rate of locomotor activity and impaired retention in the passive avoidance test at 24 h, without further forgetfulness at 144 h. This finding may indicate disturbed acquisition and/or consolidation of memory, which may remain impaired but at a constant level, without further deterioration. Enhanced motor activity and impaired acquisition of passive avoidance learning without further impairment have also been reported as a characteristical behavioral pattern after disruption of the cholinergic system. It is therefore postulated that the observed behavioral abnormalities consequent on an impairment of cerebral glucose metabolism may be suggestive of cholinergic dysfunction.
INTRODUCTION Recent findings suggest that disturbances in cerebral glucose and related metabolism may play an important role in brain aging and the genesis of disorders causing dementia lz'18'28'29'3°'52. Glucose is known to be the major fuel for biological energy, and thus for metabolic activity in the central nervous system, in normal conditions 5~'55. A close coupling between cerebral glucose metabolism and neuronal function has been demonstrated in a variety of physiological conditions 54. Brain glucose concentrations below physiological levels can influence the rate of acetylcholine synthesis by modifying the central acetyl-coenzyme A (CoA) concentration, which is derived from glucose by way of glycolysis and the pyruvate oxidation system 4'8'19. This means, for example, that acetylcholine synthesis is significantly decreased in severe hypoglycemia ~9. Abnormalities in the central cholinergic system have been shown to affect behavior, learning, and memory 13'22'56'59. Impairment of passive avoidance learning has been found after the induction of depressed regulation of cerebral glucose metabolism by streptozotocin (STZ) by
the intracerebroventricular (i.c.v.) route 37. It has been suggested that this cognitive impairment might be related to the metabolic action of the impaired cerebral regulation in utilization of glucose, interfering with the synthesis of a critical pool of acetylcholine 42. On the other hand, improved m e m o r y performance has been found in rat and human with peripheral posttraining administration of glucose 16"23"24'39. Glucose also enhances m e m o r y after i.c.v, administration 32. The biochemical mechanisms of the positive effects on performance are still unknown. It has been suggested that glucose may regulate the storage of new information by increasing neuronal glucose availability or via peripheral contributions to glucose by still unknown interactions with the body's glucose regulatory system 21'23. Thus, these findings point to interactions between both peripheral and central glucose metabolism and brain function as determined by learning and memory. The present study was performed to investigate the effect of disturbed glucose regulation after intraperitoneal (i.p.) administration of STZ (STZ-i.p.), producing chronically elevated arterial blood glucose levels 31'4°, and after i.c.v, administration of STZ (STZi.c.v.), generating cerebral hypometabolism of glucose
Correspondence: G. Mayer, Department of Pathochemistry and General Neurochemistry, University of Heidelberg, Im Neuenheimer Feld 220-221, D-6900 Heidelberg, F.R.G. 0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishe.rs B.V. (Biomedical Division)
96 l a s t i n g f o r s e v e r a l w e e k s 41, o n l o c o m o t o r activity, l e a r n -
LOCOMOTOR ACTIVITY
ing, a n d m e m o r y in a d u l t rats, m e a s u r e d as c l o s e d field
~ CONTROL ~ STZ-IP EZ] STZ-ICV
activity and passive avoidance behavior. 200
*
MATERIALS AND METHODS
Animals
.~_ ~
//A //£
One-year-old (adult) male Wistar rats weighing between 390 and 520 g (breeder: Zentralinstitut ffir Versuchstierzucht, Hannover, F.R.G.) were used in the present study. They were housed in individual cages in a temperature-controlled animal room with a reversed 12:12 h light:dark cycle (lights on at 19.30 h). Experiments were conducted during the dark portion of the cycle. Food pellets (Altromin standard, No. 1320) and water were freely available throughout the experiment. The animals were divided randomly into 4 groups. (1) In one group (n = 18) each animal received a single unilateral (left side) i.c.v, injection of STZ (Sigma, Munich, E R . G . ) in a subdiabetic dose (1.5 mg/kg body weight), sufficient to bring about a significant decrease (by approximately 46%) of several weeks' duration 42 in the cerebral metabolic rate of glucose utilization while the arterial blood glucose concentration (BG) remains unchanged (BG = 9.1 _+ 0.95 mmol/1), as determined by means of commercial test kits (Boehringer Mannheim, ER.G.). (2) The controls for this group (n = 18) received i.c.v, injections of artificial cerebral spinal fluid (CSF) (BG = 9.3 _+ 0.97 mmol/l). (3) In a third group (n = 6), a single i.p. injection of STZ (55 mg/kg body weight) was given to induce insulin-dependent diabetes mellitus (type I) with plasma glucose levels 3 times those of normal animals (BG = 25.3 + 1.56 mmol/1). (4) The corresponding controls (n = 5) received i.p. injections of saline only (BG = 9.7 + 0.84 mmol/l).
placed in the illuminated starting box again and the step-through latency established. The test was concluded when the animal entered the dark compartment or after 5 min if it failed to do so. In order to improve the reliability and validity of the footshock avoidance test, the plates were moistened before each footshock with water, significantly reducing the wide interindividual variability in paw skin resistance of the rats.
Behavioral testing
Statistics
Two behavioral tests were used: (1) The closed-field activity center, to study the rats' locomotor activity, and (2) a single-trial step-through passive avoidance test to investigate learning and memory. (1) Spontaneous locomotor activity was assessed at day 11 after STZ administration. Each animal was observed over a period of 300 s in a square closed-field arena (80 x 80 x 40 cm) equipped with a row of 12 infrared light-sensitive photocells placed 5 cm above the wooden floor of the pen. The 3 photocells on each wall of the square were spaced 20 cm apart, and the last photocell in each row was spaced 20 cm from the next wall. Interruptions of photocell beams were recorded by means of a microcomputer, allowing a record of all horizontal activity as measured by the total number of interruptions of the 12 photocell beams. The closed-field apparatus was housed together with the other behavioral apparatus in a darkened, light- and sound-attenuated and ventilated testing room. During behavioral testing, only one animal and the tester were in the testing room at any time. (2) Single-trial passive avoidance learning. STZ was administered 13 days before the training. The step-through passive avoidance behavior was evaluated by using the light-dark box test. The light-dark box consisted of a 55 × 18 x 40 cm apparatus made of wood. It was divided into two equal compartments by a wooden wall with an 8 x 8 cm opening in it. The opening was controlled by a guillotine door between the two compartments. The light compartment was illuminated by a 60-W lamp fixed 40 cm above its floor in the center. The interior of the dark chamber was painted black and had a ceiling. The floor of each compartment consisted of an individual stainless steel plate. To ensure electrical separation, there was a 5-mm gap between the two plates in the light-dark box at the opening between the two chambers. Each plate had a separate connection to the polar output of our laboratory transformer, which delivered constant-current footshock (AC, 1 mA, 1 s) through the metal floor plates. In each test, single electric shock was applied while the animal was approaching the dark part of the apparatus through the opening. At 24 h and 144h after the shock the rat was
~
100.
~ ~
I
//~
+
//~ //~
so-
///t //~ //~
