Intracerebroventricular Administration of ...

Behavioral Neiiroscience 1998, Vol. 112, No. 5. 1199-1208

Copyright 1998 by the American Psychological Association, Inc 0735-7044/98/53.00

Intracerebroventricular Administration of Streptozotocin Causes Long-Term Diminutions in Learning and Memory Abilities and in Cerebral Energy Metabolism in Adult Rats Heinrich Lannert and Siegfried Hoyer

This document is copyrighted by the American Psychological Association or one of its allied publishers. This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.

University of Heidelberg Drastic abnormalities have been demonslrated to occur in cerebral glucose and energy metabolism in sporadic Alzheimer's disease, pointing to a primary disturbance in neuronal insulin and insulin receptor signal transduction and contributing to the causation of dementia. The compound Streptozotocin (STZ) is known to inhibit insulin receptor function. The study was designed to investigate whether intracerebroventricularly (icv) applied STZ would inhibit neuronal insulin receptor function and would induce changes in both behavior and neuronal energy metabolism. Adult rats with icv-mjected STZ developed long-term and progressive deficits in learning, memory, and cognitive behavior, indicated by decreases in working and reference memory in the holeboard task and the passive avoidance paradigm, along with a permanent and ongoing cerebral energy deficit. This animal model may be appropriate for investigations related to sporadic Alzheimer's dementia.

Experimental evidence suggests that the process underlying learning and memory formation starts with a series of molecular events (Rankin, 1994) that are linked to each other by transient effects with time constants of increasing length. Events somehow related to memory formation can be observed at different cellular levels (i.e., at the membrane, in the cytoplasm and/or at the nucleus). Although initial parts of the "learning cascade" may last only for milliseconds, intermediate steps, such as long-term potentiation (Izquierdo & Medina, 1995; Maren & Baudry, 1995; Richter-Levin, Canevari, & Bliss, 1995), continue to be elicited for hours, and products of protein synthesis and/or glycosylation in the endoplasmic reticulum (ER) and Golgi apparatus may persist for days to weeks (Matthics, 1989). A general role in learning and memory function falls to the cerebral energy metabolism dependent on glucose utilization. Glucose is known to be the major fuel for biological energy and, thus, for metabolic activity in the central nervous system in normal conditions (Siesjo, 1978; Sokoloff, 1980). A close coupling between cerebral glucose metabolism and neuronal function has been demonstrated in a variety of physiological conditions (Sokoloff, 1977). Damaged glucose metabolism in the brain caused by a single intracerebroventricular (icv) injection of the diabetogenic drug Streptozotocin (STZ) was shown to lead to an impairment of passive avoidance behavior 3 weeks after icv

Heinrich Lannert and Siegfried Hoyer, Department of Pathochemistry and General Neurochemistry, University of Heidelberg, Heidelberg, Germany. We thank Vcra Schuhmann and Roland Galmbacher for skillful technical assistance.

Correspondence concerning this article should be addressed to Siegfried Hoyer, Department of Pathochemistry and General Neurochemistry, University of Heidelberg, Im Neuenheimer Feld 220/221, 69120 Heidelberg, Germany. Electronic mail may be sent [email protected].

STZ (Blokland & Jolles, 1993; Mayer, Kitsch, & Hoyer, 1989, 1990). In contrast, memory was enhanced after icv administration of glucose (Lee, Graham, & Gold, 1988; Wenk, 1989), demonstrating that glucose metabolism in the brain may well be correlated with learning and memory capacity. Administration of icv STZ in a subdiabetogenic dose did, in fact, result in decreased use of glucose in the brain (Duelli, Schrock, Kuschinsky, & Hoyer, 1994), increased release of lactate from the brain (Nitsch, Mayer, & Hoyer, 1989), and reduced activities of glycolytic enzymes (Plaschke & Hoyer, 1993), indicating a damaged neural glucose metabolism leading to reduced formation of adenosine triphosphate (ATP) and creatine phosphate (CrP; Nitsch & Hoyer, 1991). With respect to neurotransmission, cholinergic deafferentiation (Hellweg, Nitsch, Hock, Jaksch, & Hoyer, 1992) and changes in the concentrations of monoaminergic neurolransmitters were found (Ding, Nitsch, & Hoyer, 1992). So far, short-term effects on behavior, glucose and energy metabolism, and related metabolism have been studied after a single icv STZ challenge. Several neurodegenerative disorders in human beings, such as sporadic Alzheimer's disease in particular, are characterized by a progressive deterioration of both memory and cognitive functions and of cerebral glucose and energy metabolism (Hoyer, 1992; Hoyer, Nitsch, & Oesterreich, 1991). Cerebral glucose use was found to be diminished in all areas of the cerebral cortex but was accentuated in the frontal and parietotemporal cerebral cortices (Mielke, Herholz, Grand, Kessler, & Heiss, 1992). The reduction of the use rate of glucose ranged from 10% (mild) to more than 40% (severe), indicating that severity of dementia may parallel the diminution of glucose use in these cortical areas (Kumar et al., 1991). To our knowledge, so far no animal model is available that corresponds to such abnormalities over a long period of time without improvement. On the basis of observations of short-term effects of a single icv

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STZ administration, we decided to inject STZ icv in triplicate and to study the effect of this on behavior and brain energy metabolism over a period of 80 days (long-term effect). We were interested in investigating the progression of behavioral deterioration during this period and the degree of damage to the neuronal energy pool and the energy metabolism in the cerebral parietotemporal cortex. Additionally, only rats with good behavioral performance (good performers) were used in this investigation. Good performers form a relatively homogeneous experimental group (Steelier, Muller, & Hoyer, 1997) and may be expected to develop more pronounced disturbances in learning, memory, and cognition after neuronal damage. Here we report that triplicate icv STZ injection induced long-term abnormalities in learning, memory, and cognition, with deteriorating tendency and accompanying disturbances in the neuronal energy pool and in energy metabolism.

Method

Rats One-year-old (adult) male Wistar rats weighing between 420 and 540 g (Zentralinstitut fur Versuchstierzucht, Hannover, Germany) were used throughout the study. They were housed in individual cages in a temperature-controlled animal room with a reversed 12:12 hr light-dark cycle (lights on at 1900). Experiments were conducted during the dark period of the cycle. Food pellets from Altromin (standard, no. 1320) were used. Water was freely

available throughout the experiment. However, during the habituation phase, the training phase and 2 days before each test in the retest phase, the rats were deprived of food (5 g/day) to enhance their motivation to perform the test (Ando & Ohashi, 1991). This deprivation of food did not cause arterial hypoglycemia (data not shown). We started our investigations with 30 animals.

Behavioral Testing The Holeboard Test Apparatus. A holeboard of the type devised by Oades and Isaacson (1978) was slightly modified. An open field (70 cm X 70 cm) surrounded by black-tinted Plexiglas walls (40 cm high) contains 16 holes (diameter of 3.5 cm) in a 4 X 4 array. In the middle of one wall, an attached Plexiglas start box is separated from the testing area by a guillotine door, which can be operated from a distance. The holes are 10 cm apart (Figure 1). In our study, each hole supplied with food pellets was covered by a false bottom (a metal cup, 3 cm deep) to mask potential odor cues emanating from the reward in the baited holes. Thus, rats were unable to discriminate between baited and unbaited holes by olfactory stimuli. Habituation phase. The rats were habituated to the holeboard apparatus on 4 consecutive days. The rats were deprived of food during this period, which caused their body weights to fall to 85% of that at the start of this phase. During habituation each hole was baited with a 50-mg food pellet. Each animal was placed in the start box alone, after which the guillotine door was opened. A trial started when the guillotine door was closed and the animal had

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Figure 1. Time course in days (d) of the experimental design for psychometric testing. Upper file of numbers demonstrate the duration of the respective test periods. Lower file of numbers shows the duration of the whole psychometric procedure. P = pause; R = retest.


CEREBRAL DEFICITS AFTER STREPTOZOTOCIN entered the testing area. The trial was finished after 10 min or after all food pellets had been collected. The number of hole visits was recorded by a microcomputer, On the 4th day, the rat had to find at least 15 of 16 pellets; rats with a poorer result were excluded (first criterion for the exclusion of poor performers; 4 animals were excluded) from further experiments and evaluation. Training phase. For the remaining rats (n — 26), a (raining phase starting 3 days after habituation followed for the next 7 days, with a pause on the 6th day (P). The rats were trained to collect pellets from a fixed set of four holes (Al, B3, C2, and D4, marked with a T symbol for the test phase in Figure 2, one trial) within 5 min. Four (rials were executed each day. A rat was placed in the start box, and the trial was initiated by raising the guillotine door between the start box and the holeboard and terminated after the rat had found the four food pellets or 5 min had elapsed, whichever occurred first. The number of hole visits was recorded by a microcomputer, which identified nose pokes by light beam crossings. At the end of a trial, the rat was replaced in the start box and food pellets were placed in appropriate holes of the arena before the next trial. After each trial, visible traces of urine and feces were removed with a dry cloth. The interval between trials was approximately 1 min. Training sessions of four consecutive trials were given each morning. The second criterion for exclusion of poor performers was determined by values for working and reference memory ratio (see definitions in Retest phase). Rats with values less than or equal to 50% for working memory ratio or 40% for reference memory ratio at Day 6 in this training phase were excluded from further testing. In our case, 6 rats were excluded. A pool of 20 good performers was obtained. Retest phase. The 20 good performers were divided randomly into two experimental groups: an STZ-treated group and a control group. After the icv injections (see Surgery) the rats had to collect pellets in four trials from a fixed set but a different set of four holes as compared with the training phase (A4, B2, C3, and Dl, marked with R forretestin Figure 2). This new order of baited holes was in principle the same as in the training phase but was rotated by 90°. This procedure was equivalent to the cone field apparatus described by van der Staay, Krechting, Blokland, and Raaijmakers (1990). In our study, this procedure was performed because of different reasons. At the end of the training phase, the animals reach a higher level of mental capacity that also may include a test wisdom

A B C D

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start hnx Figure 2. The holeboard apparatus. Schematic drawing of testing apparatus. Numbers and letters designate rows and columns as described in text. T = the placement of food in the training phase; R = the retest phase.

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frequently observed in human beings. To avoid this possibility of habituation to the once-learned order of baited holes during the training phase, baited holes A4, B2, C3, and Dl on Days R19, R40, and R80. Furthermore, if icv STZ would cause a fall in learning and memory capacities, the difference between training phase and retest phase might be expected to become more prominent when the task to be performed is slightly different from former tasks. Both mean run time and number of visited holes were recorded by a microcomputer. The latter parameter was identified via nose pokes by light beam crossing. Consumed food was not rebaited within a trial. The number of visited holes also served as a measure for motivation. Mean run time is correlated with the performance level: The more errors the animal makes, the more time is needed to complete the trial (Rapp, Rosenberg, & Gallagher, 1987). The mean visit interval was determined by dividing the time between the first and the last visit of a trial by (number of visits — 1; van der Staay, van Nies, & Raaijmakers, 1990). This variable provides a measure for the speed of visiting the holes. The experimental set-up of the holeboard permits a definition of two distinct memory functions, that is, working memory (WM) and reference memory (RM). Performance of both components can be expressed as ratios of different types of hole visits (van der Staay et al., 1990). 1. WM ratio was defined as number of food rewarded visits divided by number of visits and revisits to the baited set of holes. Thus, this measure represents the percentage of all visits to the baited set of holes that had been reinforced with food (van der Staay, Raaijmakers, & Collin, 1986; van der Staay, Raaijmakers, Sakkee, & van Bezooijen, 1988; van der Staay et al., 1990). 2. KM ratio was defined as number of visits and revisits to the baited set of holes divided by number of visits and revisits to all holes. This measure expresses the number of visits to the baited set of holes as a percentage of the total number of visits to all holes. In the present study, WM ratio and RM ratio were calculated (Beldhuis, Everts, Van der Zee, Luiten, & Bohus, 1992; van der Staay et al., 1990) using block means of four trials.

Single-Trial Passive Avoidance Learning The step-through passive avoidance behavior was evaluated by using the light-dark avoidance box test. The inhibitory apparatus (70 X 45 X 40 cm wooden box) consisted of a light (30 X 45 X 40 cm) and a dark (40 X 45 X 40 cm) compartment. The light compartment was illuminated by a 60-watt 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 consisted of a metal grid connected to a shock scrambler. The two compartments were separated by a guillotine door that could be raised 10 cm. On Day R17, rats were placed in the light compartment and the latent period (initial), that is, the time lapse before each animal entered the dark compartment and had all four paws inside it, was measured. Twenty-four hours later the rats were placed directly in the dark compartment; after 20 s, the guillotine door was opened and a scrambled footshock (AC, 1 mA, 1 s) was delivered. After the rats had left the dark compartment (active avoidance), they stayed in the light compartment (passive avoidance) for a maximum of 300 s. A further step-through latency was measured 24 hr after the shock (Day R19). The rat was again placed in the illuminated starting box and the test was concluded when the animal had all four paws in the dark compartment or after 5 min if it failed to go right into the dark compartment. The test was repeated on Days R40 and R80. To improve the reliability and validity of the footshock avoidance test, we moistened the grid with water before each footshock, significantly reducing the wide interindividual variability in paw skin

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resistance of the rats. The first icv injection of STZ or artificial CSF was given 17 days before the training.


Closed Field Activity Test Spontaneous locomotor activity was assessed on Days R19, R40, and R80 after the first 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 lightsensitive 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 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 sited in a darkened, lightand sound-attenuated, and ventilated testing room together with the other behavioral testing apparatus. During behavioral testing, only one animal and the tester were in the testing room at any time. The sequence of the behavioral tests in the retest phase on Days R19, R40, and R80 was locomotor activity, followed by memory performance on the holeboard, and finally, passive avoidance behavior.

Surgery After habituation and training (exclusion of poor performers), and at the beginning of the retest phase at Day Rl (Figure 1), the animals were divided randomly into two groups. Animals were anesthetized with chloral hydrate (240 mg/kg body weight ip). The head was positioned in a stereotactic frame (Uhl, Asslar, Germany), the skin over the skull was incised sagittally, and bur holes were drilled into the skull 2.0 mm lateral (right and left) and 0.7 mm caudal to bregma (Paxinos & Watson, 1986). Injection cannulas were lowered into the cerebral ventricles under stereotactic guidance (4.0 mm ventral to the brain surface; Paxinos & Watson, 1986). In the first group (n ~ 10), each rat received a bilateral icv injection of STZ (Sigma, Munich, Germany) in a subdiabetogenic dose (0.25 mg STZ dissolved in 2 ul artificial CSF/injection site). The injections were repeated on Day R3 and Day R20. In the second group, which served as the control group, artificial CSF containing 120 mM NaCl, 3 mM KC1, 1.15 mM CaCl2, 0.8 mM MgCl2, 27 mM NaHCO3, and 0.33 mM NaH2PO4, adjusted to pH 7,2 by CO2 insufflation (all chemicals from Merck, Darmstadt, Germany) was injected, the time course being the same as in STZ-treated animals. The cannulas were left in place for a further 2 min before slow removal. The bur holes were closed with bone wax, and the skin incision was sutured.

neutralized to pH 7.2 ± 0.4 with KOH and filtered through a 45-p.m Millipore membrane filter, after which 100 |jl of each filtrate was analyzed and ATP, guanosine triphosphate (GTP), adenosine diphosphate (ADP), and phosphocreatine (CrP) concentrations were measured by high-performance liquid chromatography (HPLC; Harmson et al., 1982). "~P" was defined as available phosphate represented by the sum of ATP and CrP.

Reagents For standards, the nucleotides and CrP were purchased from Sigma, Munich, Germany and only ATP was from Boehringer, Mannheim, Germany. Water was purified with Milli-Ro4/Milli-Q system (Millipore, Beford, MA). Standards were always freshly prepared.

High-Performance Liquid Chromatography A BESTA-HPLC system (HD-2-400) was used to determine energy rich phosphates. The system consisted of two positive displacement pumps, a variable-wavelength UV detector (Shimadzu, SPD-6A) set at 210 nm, a pneumatic sampling device (autosampler Gilson 231) tempered rack (1°C). Data were collected by an intelligent interface series 900 (Nelson Analytical) and chromatograms were calculated by Nelson 2600 chromatography software (Revision 5.0). Buffers were prepared on the day of use and filtered through a 0.45 urn filter (Millipore). Buffer A was 0.01 M H3PO4, adjusted to pH 2.85 with 6 M KOH; buffer B was 0.7 M KH2PO4, pH 4.40. The column (Partisil-SAX, 4.6 X 250 mm, particle size 10 urn; Whatman, Maidstone, Great Britain) was eluted with buffer A at a flow rate of 2.0 ml/min. Five minutes after injection, a gradient started with an increase of 4% B per minute until 100% B. A precolumn, Partisil-SAX, 4.6 X 20 mm, particle size 10 urn (Whatman, Maidstone, Great Britain), was used.

Statistics Data are displayed as mean values ± standard deviations. The statistical comparisons of the WM and RM ratios, of the passive avoidance values and of the locomotor activity values in the two groups, were performed by means of the Mann-Whitney U nonparametric statistic. An alpha level of 0.05 was the criterion of statistical significance. Statistically significant differences in the biochemical data (nucleotides, ATP/ADP ratio and —P) between the groups investigated were also calculated by the Mann-Whitney U test. Statistical advice from the Institute of Medical Biostatistics, University of Heidelberg is gratefully acknowledged.

Biochemical Analysis

Ethics

After behavioral testing, the animals in both groups were killed by in situ freezing of their brains with liquid nitrogen under steady-state conditions of arterial normotension, normoxemia, normocapnia, and normothermia. The brains were chiselled out from the skull and stored at —80° C. Cerebral parielotemporal cortex was prepared at approximately —20° C in a cryostat (Brandau, Darmstadt, Germany). The brain was dissected rostrocaudally into thin slices (approximately 1.5 mm), and areas were identified with reference to a stereotactic atlas (Paxinos & Watson, 1986). After dissection, tissues were weighed and homogenized in 10 vol CHC13 at approximately -25°C with an Ultraturrax. Proteins were precipitated with 1.97% HC1O4 (0.8 M) and homogenates were centrifuged at 10,000 g for 10 min. Supernatants were

The experimental protocol was approved by the review committee for animal experimentation of the Medical Faculty of the University of Heidelberg and by the responsible government agency.

Results Behavior Behavior During the Training Phase The data sampled on Days 1 and 7 of this phase are presented in Table 1 and Figures 3A and 3B. It becomes

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Table 1 Mean Values and Standard Deviations of Mean Run Time, Number of Visited and Revisited Holes, and Mean Intervisit Interval From the Holeboard Task Training


Behavioral parameters from the holeboard Mean run time (in s) Control STZ Number of visited and revisited holes Control STZ Mean intervisit interval (in [s/1 hole]) Control STZ

Retest

Day 1

Day 7

195 ± 31

87 ± 193

20 ± 3

10.2 ± 0.9

Day R19

Day R40

Day R80

94 -+- 16 179 -t- 29"

97 ± 17 232 ± 3]b.c

101 ± 18 251 ±36 b

13 + 2 20 -4- 2b

13 ± 2 19 ± 2b

13 ± 2 18 ± 2 "

8.1 ± 0.5 12.9 ± 0.8b-c

X.4 ±0.6 14.8 ± 0.9b-c

13 ± 2 »

7.3 ±0.4" 7.8 9.4

+ ±

0.5 0.6b

Note. After STZ damage, significant differences compared to control became obvious in all parameters studied over the hole retest period. STZ = streptozotocin. 'During training significance. 'Control versus STZ group. CSTZ group: R19 versus R40 and R40 versus R80.

obvious that mean run time, number of visited and revisited holes, and the mean intervisit interval decreased significantly (Table 1). Both WM and RM were found as in other studies conducted by our group in rats on Day 1 of the training phase. A 6-day training enhanced WM by 100% and RMby93%.

Behavior During the Retest Phase In the control group, mean run time, number of visited and revisited holes, and mean intervisit interval were maintained at the same levels over the whole retest phase and were found not to be different from Day 7 of the training phase.

REFERENCE MEMORY

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Figure 3. (A) Working memory (WM) ratio (number of food rewarded visits/number of visits and revisits to the baited set of holes) of 1 -year-old Wistar rats (good performers). Values are M ± SD. The course of training indicates the improvement of WM performance: Significant increase between Day 1 (Tl) and the last day of training (T7) is marked by a plus sign. Comparison between streptozocin (STZ) applicated group (« = 10) and control group (« = 10) in the retest phase shows significantly (*) decreased WM values at Days R19, R40, and R80. (B) Reference memory (RM) ratio (number of visits and revisits to the baited set of holes/number of visits and revisits to all holes) of 1 -year-old Wistar rats (good performers). Values are M ± SD. The course of training indicates the improvement of RM performance: Significant increase between Day 1 (Tl) and the last day of training (T7) is marked by a plus sign. Comparison between streptozocin (STZ) applicated group (n = 10) and control group (n = 10) in the retest phase shows significantly (*) decreased RM values at Days R40 and R80. d = days; R = retest.

R80


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The same was true for WM and RM whose levels of enhancement were maintained throughout the experimental period. The data after icv STZ are also presented in Table 1 and Figures 3A and 3B. Mean run time of the animals was significantly higher on Days R19, R40, and R80 as compared with the control rats. It increased steadily and showed statistically significant differences between R19 and R40, but significance between R40 and R80 was just barely missed. The number of visited holes also increased significantly as compared with the control group throughout the retest phase. A slight decrease but no significant differences became apparent between the Days R19, R40, and R80. The mean intervisit interval differed significantly from control conditions throughout the retest phase. This parameter increased steadily and was found to be significantly different between R19 and R40, and R40 and R80. It is of note that the mean run time of icv STZ-treated animals that did not succeed in collecting all four food pellets within 5 min was set at 300 s. Accordingly, the number of incomplete trials was 6 on Day R19, 10 on Day R40, and 16 on Day R80. With a failure of a WM ratio the number of food rewarded visits was put either at 4 (completed trial) or 0 (all or nothing law). WM and RM declined drastically and in a progressive manner, WM to 41% (R19), 29% (R40), and 18% (R80), and RM to 72% (R19), 35% (R40), and 18% (R80) of the level for all animals after training at Day T7. There were also statistically significant differences between the different time points tested: For WM, capacity fell by 42% from Day R19 to Day R40, and by 26.5% from Day R40 to Day R80; and for RM, by 66% from Day R19 to Day R40, and by 49% from Day R40 to Day R80 (Figures 3 A and 3B). Passive avoidance behavior. The average initial stepthrough latency on Day R17 did not differ significantly between the control group (15.8±5.2s) and the icv STZ group (19.4 ± 12.1 s; Figure 4). In the STZ group, the latencies of the retention test (passive avoidance) conducted on Day R19 (1 day after the footshock on Day R18, and 1 day before the third injection of STZ) increased nearly six-fold to 107.2 ± 33.2 s, whereas the latency in the control group increased to 279.2 ± 34.5 s. By days R40 and R80, in the retest period, the latencies had increased to the maximum of 300 s in control animals. In contrast, after triplicate icv STZ injection the latencies fell to 37.7 ± 23.3 s (Day R40) and to 39.1 ± 24.3 s (Day R80)— that is, around two thirds of those on Day R19 (Figure 4). Locomotor activity. The spontaneous locomotor activity in the open field did not differ significantly between the STZ group and the control group on Days R19, R40, and R80 (Figure 5). The mean values in the STZ group were 100.0 ± 38.6 counts per 5 min on Day R19,122.1 ± 48.4 counts per 5 min on Day R40, and 89.5 ± 28.0 counts per 5 min on Day R80. In the control group, 83.0 ± 37.41 counts per 5 min were found on Day R19, 92,7 ± 37.4 counts per 5 min on Day R40, and 66.1 ± 36.0 counts per 5 min on Day R80 (Figure 5).

PASSIVE AVOIDANCE BEHAVIOR 300

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d Figure 4. Effects of streptozotoein (STZ) icv pretreatment on retention passive behavior in rats (STZ was administered for the first time 18 days before the training; see time course described in the Method section). STZ treatment did not affect the step through latency (initial test at Day R17). Foot shock was applied at Day R18. Retention tests were conducted at Days R19, R40, and R80. Height of columns shows mean values; bars, SD, and significance significantly decreased in comparison with retention values in the control animals, d = days; PA = passive avoidance.

Energy Metabolism After triplicate icv STZ administration and at the end of all psychometric tests, the concentrations of ATP, GTP, and CrP, and the sum of available P (~ P) were found to be significantly lower than control values in parietotemporal cerebral cortex, whereas ADP increased, though the difference just failed to reach statistical significance. As conseLOCOMOTOR ACTIVITY 200 dCONTROL E3STZ

150

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R19

R40

R80

d Figure 5. Effects on spontaneous locomotor activity after streptozocin (STZ) administration. Height of columns shows mean values; bars indicate SD. The STZ-applicated animals show a nonsignificant slight increase of locomotor activity during the retest phase in comparison with the control animals, d = days; R = retest.

CEREBRAL DEFICITS AFTER STREPTOZOTOCIN

quence, the ATP:ADP ratio, an indicator of ATP turnover, decreased significantly (Table 2).


Discussion As is clearly demonstrated in this study, mental training performed over 6 days significantly increased short-term memory (WM) and long-term memory (RM) and decreased mean run time, the number of visited holes, and mean intervisit interval in all animals. This effect was maintained in control animals throughout the investigation period of 12 weeks. Control animals also completely avoided entering the dark compartment in the passive avoidance task. We thus tentatively conclude that mental training enhances and maintains learning, memory, and cognition capacities longer in well-performing animals. Only the latter animals were used in this study to allow better discrimination between controls and STZ-damaged animals. In contrast to the results of the training phase and the retest phase in control animals, a disturbance in the neuronal insulin signal transduction cascade by a triplicate icv STZ (Ar'Rajab & Ahrfn, 1993; Kadowaki, Kasuga, Akanuma, Ezaki, & Takaku, 1984) induced long-term abnormalities in learning, memory, and cognition abilities. The number of visited holes approached the same level as in Day 1 of the training phase accompanied by an increase in mean run time, which exceeded this value from Day R40 onward. Accordingly, mean intervisit interval also increased together with the number of incomplete trials with the duration of the damage. These results clearly point to a decline of learning and memory function in the STZ group independent from the motivation to search food pellets and to visit holes. The specific errors of different types of hole visits were analyzed with ratios for working memory and reference memory (van der Staay et al., 1990). Both WM and RM deteriorated progressively in a stepwise manner during the same period of time, and no improvement could be observed. Also, the results recorded in the passive avoidance behavior test Table 2 Influence of Triplicate icv Application of Streptozotocin on Tissue Concentrations (nmol/mg Net Weight) of Energy-Rich Phosphate in Cerebral Parietotemporal Cortex Parietotemporal cortex Energy-rich phosphates

Controls __ (rtjMO)__

icv Streptozotocin (« = 10)

^TP ADP GTP CrP

~2.54± 0.29~ 0.41 ± 0.07 0.63 ± 0.07 5.76 ± 0.99

2.15 0.51 0.54 5.17

ATP/ADP ~P

6.29 ± 0.93 1.33 ±0.13

±0.23* ± 0.15 ± 0.04* ± 0.85*

4.58 ± 1.19* 1.16 ±0.12*

Note. Values are given as M ± SD. Statistical analysis was performed by Mann-Whitney analysis (* = significant at a = .05). ATP — adenosine triphosphate; ADP — adenosine diphosphate; GTP = guanosine triphosphate; CrP — phosphocreatine; ~-P = sum of available phosphate.

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reinforce the data recorded in the holeboard test. STZdamaged animals revealed significantly reduced latencies on Day R19 with a further significant fall by Day R40. Passive avoidance was also found to not improve at all during the course of the whole investigation period in rats damaged with icv STZ. It can thus be concluded that the present animal model is appropriate for studies addressing ongoing and long-lasting disturbances in learning, memory, and cognition. On the basis of the data presented above, it is suggested that the impairment of passive avoidance behavior may reflect poorer acquisition and/or retention of memory rather than accelerated forgetting after icv STZ injection. These findings are consistent with deficits found in WM and RM. Otherwise, there seems to be no evidence that icv STZ damage might induce motivational or sensory effects. The number of visited holes, which was regarded as a measure for food-deprived motivation (see Table 1), was not reduced by STZ but was increased as compared with control rats. Sensory (olfactory) stimuli did not vary during the whole experiment (see the Method section); that is, successful food search was guided by memory rather than olfactory sensorial stimuli. Furthermore, there was no evidence that the animals had become blinded by STZ during the experiment which became obvious from the holeboard task (STZ animal entered the holeboard from the startbox with the same short latencies as control animal and started to visit food holes) and the passive avoidance task. It can therefore be concluded that the data arising from different behavioral tests indicate that icv STZ-induced damage of the neuronal insulin receptor caused progressive deteriorations in the mental capacities of learning, memory, and cognition and that these deteriorations were maintained at a low level over a long period of time. A very similar decline in such acquired capacities along with its steady deterioration has become obvious in human beings suffering from neurodegenerative disorders such as sporadic Alzheimer's disease. The results of the open field performed during the retest phase indicated that icv STZ administration did not significantly increase activity levels, although there was a tendency to this. The present results were different from our earlier data (Mayer, Nitsch, & Hoyer, 1990), which were observed 3 weeks after a single icv STZ injection. Several variables could contribute to these differences, for example, intensity of handling and the exclusion of poor learners from this study. The latter animals had a reduced number of NMDA-binding sites in the hippocampus, which was accompanied by enhanced locomotor activity (Stecher et al., 1997). Regardless of the reasons for these differences, the results suggest that the deficit in inhibitory avoidance retention performance observed after icv STZ treatment was not due to increased locomotor activity. This excludes the possibility that activity per se may have contributed to the severe deficits in holeboard and passive avoidance learning after icv STZ treatment. From previous studies by our group, we obtained indirect evidence that both cerebral energy pool and energy turnover were increased by mental training as compared with mental rest (Dutschke, Nitsch, & Hoyer, 1994; Nitsch & Hoyer, 1991). Subsequent studies in which the cerebral energy pool


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was directly compared between mental activity and mental rest over a longer period of time confirmed this result (Hoyer & Haag, 1998). In the present study, the cerebral energy pool and energy turnover of control animals was found to be in the same range as in the previous investigations of mental activity and thus higher than typically observed at mental rest. We therefore suggest that the experimental manipulations performed accounted for the enhancement that was counteracted by icv STZ resulting in an impairment of brain energy metabolism characterized by significant decreases in the cortical tissue concentrations of ATP, GTP and CrP and, thus, in available energy, ~P. Because neither glucose nor lactate concentrations in arterial blood nor the energy state of liver and muscle were altered by icv STZ (data not shown), STZ injected into the cerebral ventricles did not produce diabetes mellitus but damaged brain metabolism exclusively. The disturbances found in this study were of the same quality, albeit slightly more pronounced in quantity, as abnormalities seen 3 weeks after a single icv STZ application (Nitsch & Hoyer, 1991). This indicates that brain energy metabolism can be kept at a lower level than normal over a longer period of time after triplicate icv STZ. The decreases in CrP and ATP, together with the increased ADP concentration, and the resulting reduction in the ATP:ADP ratio, may be indicative of an imbalance between energy production and energy use. This imbalance of energy metabolism may reflect a state of metabolic neuronal stress, which resembles that observed, for example, in carbon monoxide intoxication (MacMillan, 1975) and during cerebral ischemia and the subsequent recovery phase (Hoyer & Betz, 1988; Ljunggren, Schutz, & Siesjo, 1974). The depletion of available energy, in particular ATP and GTP, can be assumed to influence energy-dependent cellular processes that are also involved in learning and memory functions. These processes include folding (Braakman, Helenius, & Helenius, 1992; Gething & Sambrook, 1992) and sorting of proteins (Rothman & Wieland, 1996), vesicular transport of proteins (Rothman, 1996; Rothman & Orel, 1992) and other substances (e.g., neurotransmitters, neurotransmission itself [Kadekaro, Crane, & Sokoloff, 1985]), and the functional organization of the Golgi apparatus for glycosylation of proteins (Hirschberg & Snider, 1987; e.g., receptors and glycosphingolipid-biosynthesis [Lannert, Biinning, Jeckel, & Wieland, 1994; Wiegandt, 1995]), a process that needs energy-activated sugars (Hirschberg & Snider, 1987; Lannert, Gorgas, MeiBner, Wieland, & Jeckel, 1998). In this respect, the energy deficit found in this study may underlie at least the deterioration of behavior, in part, especially because reduced glucose use has been found to correlate with a decline in cognitive function (Wree, 1991). In sporadic Alzheimer's disease, a dramatic decrease in cerebral glucose use has been found (Fukuyama et al., 1994; Hoyer et al., 1991; Mielke et al., 1992), with a consequent reduction in available energy (Hoyer, 1992). These abnormalities were hypothesized to be caused by a dysfunction of the neuronal insulin signal transduction cascade (Craft et al., 1998; Henneberg & Hoyer, 1995; Hoyer, 1998). Indeed, a reduced insulin concentration and a disturbance in insulin receptor function have been found post-mortem in the

Alzheimer brain (Frolich, Blum-Degen, Hoyer, Beckmann, & Riederer, 1997; Frolich et al., 1998) and in cerebrospinal fluid of Alzheimer patients (Craft et al., 1998). Prominent histopathological markers of this neurodegenerative disorder are neuritic placques and neurofibrillary tangles. The former mainly consist of amyloid, which is formed from the amyloidogenic derivative (3A4, a metabolite from amyloid precursors protein (APP) metabolism. Neurofibrillary tangles consist of hyperphosphorylated tau-protein and fragmented microtubuli. Faulty APP metabolism producing more (3A4 can be assumed to occur when both the tyrosine kinase activity and energy availability are reduced (Gabuzda, Busciglio, Chen, Matsudaira, & Yankner, 1994; Petryniak, Wurtman, & Slack, 1996; Slack, Breu, Muchnicki, & Wurtman, 1997; Slack, Breu, Petryniak, Srivastava, & Wurtman, 1997). In addition, hyperphosphorylation of the tau-protein also depends on the availability of ATP. The activities of the tau-protein kinases PK40 and PK36 were found to be highest when ATP was low (Roder & Ingram, 1991); in other words, the lower the ATP level in the neuron, the higher the chance that hyperphosphorylated tau-protein will be formed. The formation of hyperphosphorylated tau-protein was found to be facilitated when insulin could not perform its function (Hong & Lee, 1997). Thus, abnormalities in both insulin signal transduction and energy metabolism can be assumed to contribute considerably to the formation of the abnormal proteins B A4 and hyperphosphorylated tau in sporadic Alzheimer's disease. On the basis of data from Alzheimer's disease pathophysiology and based on our study, we therefore tentatively assume that icv STZ administration, which leads to disturbances in behavior and in glucose and energy metabolism, can be considered as an appropriate animal model for sporadic Alzheimer's disease. Although icv STZ mimics several abnormalities in behavior and oxidative metabolism of this clinical condition, further detailed morphological and molecular biological research is needed to test this model for similarity to sporadic Alzheimer's disease.

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Received December 17, 1997 Re vision received April 2, 1998 Accepted April 3, 1998 •