Penny L. Shultz

An analysis of spatial navigation in prenatally protein malnourished rats

Developing rats were either malnourished or adequately nourished during the prenatal period by feeding their dams diets of low (6% casein) or adequate (25% casein) protein content 5 weeks prior to mating and throughout pregnancy. All pups received adequate nutrition from the day of birth onwards. Male offspring were tested in one of two spatial navigation tests in the Morris water tank. In proximal-cue tests (postnatal days 16-20), the position of a platform, which provided a means to escape from swimming, was denoted by an obvious visual cue located directly on the platform. In distal-cue tests (postnatal days 20-27 and adult ages, days 70-71 and days 220-221), the escape platform was submerged below the surface of the water so that the rats were required to use extramaze visual cues to guide them to the platform. Neither proximal-cue nor distal-cue navigation was significantly impaired in the prenatally malnourished rats relative to controls, at any of the ages tested.

Chlordiazepoxide-Induced Spatial Learning Deficits: Dose-Dependent Differences Following Prenatal Malnutrition

The sensitivity of prenatally protein-malnourished rats to the amnestic properties of the benzodiazepine (BZ) receptor agonist, chlordiazepoxide (CDP), was studied in the male offspring of rats provided with a protein-deficient diet (6% casein) for 5 weeks prior to mating and throughout pregnancy. Rats were tested during acquisition of the submerged platform version of the Morris water maze task using three systemic doses of CDP (3.2, 5.6, and 7.5 mg/kg i.p.) at two ages (day 30 and day 90). At 30 days, prenatally malnourished rats showed less sensitivity to the amnestic effect of the 5.6-mg/kg dose when compared with well-nourished controls by displaying shorter swim paths during acquisition and a more selective search of the target quadrant upon removal of the platform (probe trial). At 90 days, prenatally malnourished rats again showed less sensitivity to CDP at a dose of 5.6 mg/kg, but more sensitivity to the 3.2-mg/kg dose (indicated on the probe trial). No obvious relationship was identified between the nutritional group differences in behavioral sensitivity to CDP at 90 days and their BZ receptor density in the hippocampus or medial septum. It can be concluded that prenatal malnutrition alters the amnestic response to CDP in a dose-dependent and developmentally specific manner, thus providing further support for functional changes within the GABAergic system subsequent to malnutrition.

Prenatally protein-malnourished rats are less sensitive to the amnestic effects of medial septal infusions of chlordiazepoxide.

Evidence is mounting that prenatal protein malnutrition affects the physiological properties of the GABAergic neurotransmitter system in rats. To investigate the functional behavioral consequences of these changes, chlordiazepoxide (CDP, a positive modulator of the GABAA receptor) was applied directly to the medial septum and the amnestic response appraised. In adulthood, male offspring of rats provided with a protein‐deficient diet (6% casein) for 5 weeks prior to mating and throughout pregnancy underwent stereotaxic surgery to implant steel cannulae aimed at the medial septum. After recovery, spatial learning performance in the submerged platform version of the Morris water maze task was assessed immediately following a 1 μl infusion of either artificial cerebrospinal fluid (aCSF), or one of three doses of CDP (15, 30 and 60 nmol). Well‐nourished control rats demonstrated a robust amnestic response to intraseptal CDP. During task acquisition, well‐nourished rats administered each of the doses exhibited significantly longer escape latencies than those given aCSF. On the probe trial (platform removed) a lower proportion of time was spent in the target quadrant (all three doses) at a greater average distance from the former platform location (30 and 60 nmol doses). In contrast, prenatally malnourished rats exhibited a muted sensitivity to CDP, most notable at the 30 nmol dose. These findings provide further support for functional changes within the GABAergic system consequent to malnutrition.

Development of spatial navigation following prenatal cocaine and malnutrition in rats: Lack of additive effects

The effects of prenatal cocaine exposure and protein malnutrition on the development of spatial navigation were assessed in rats. Sprague-Dawley dams were fed a low-protein (6% casein), adequate protein (25% casein), or a laboratory chow diet prior to mating and throughout pregnancy. Within each diet group, rats received either cocaine injections (30 mg/kg i.p. two times per week prior to mating and then 30 mg/kg s.c. daily from day 3 to 18 of pregnancy) or saline injections. All litters were fostered on the day of birth to saline-injected mothers fed either the 25% casein diet or the chow diet. Gestation length was decreased by prenatal cocaine exposure whereas litter size was reduced by prenatal malnutrition. On postnatal days 21, 25, 30, or 70, rats were tested for their ability to locate a submerged platform in a Morris water maze. In well-nourished rats, prenatal cocaine increased the mean distance swum during acquisition over days 21-30, a difference that was abolished in rats with prenatal malnutrition. In the absence of drug exposure (saline groups), prenatal malnutrition was itself associated with longer swim paths. Neither prenatal insult affected the accuracy of the spatial navigation at these ages, as determined by their search pattern when the platform was removed. On postnatal day 25, rats raised on the chow diet exhibited superior performance to that of rats raised on the 25% casein diet, but by day 30 these two well-nourished groups were comparable. At day 70, prenatal cocaine impaired spatial performance on the first session, in well-nourished rats only. Thus, these results provide no support for the hypothesis that prenatal cocaine and protein malnutrition combine to produce a greater effect on behavioral development than either insult alone.

Prenatal protein restriction increases sensitization to cocaine-induced stereotypy.

The present study characterized the total amount of stereotyped behavior following acute and repeated administration of cocaine in male and female prenatally protein malnourished rats. Adult offspring of female Sprague-Dawley rats fed either a low (6% casein) or adequate (25% casein) protein diet 5 weeks prior to mating and throughout their pregnancy were studied. Once every 3 days (for a total of six injections), half the rats from each nutritional treatment group (repeated exposure) were injected with cocaine (30 mg/kg, i.p.) and their total amount of stereotypy (rearing, forepaw treading, compulsive sniffing and head bobbing) monitored. The remaining rats received five saline injections followed by a cocaine injection on the last injection day (acute exposure group) and their behavioral response was also measured. Despite being slightly less sensitive to cocaine following their first injection, by the sixth injection, prenatally protein malnourished animals in the repeated-exposure group exhibited significantly greater sensitization to the psychomotor stimulant effects of cocaine than well-nourished controls. In the acute exposure groups, however, prenatally malnourished males, but not females, exhibited significantly more stereotypy than well-nourished subjects following a single cocaine injection. These findings have implications for characterizing addiction potential in the previously malnourished rats, as well as providing additional information regarding factors which can influence sensitization.

Prenatal cocaine but not prenatal malnutrition affects foster mother-pup interactions in rats

The separate and combined effects of prenatal cocaine exposure and malnutrition on mother-pup interactions in rats were assessed daily from postnatal day 2 to day 21. Sprague-Dawley dams were fed a diet of low protein content (6% casein), an isocaloric diet of adequate protein content (25% casein, control), or a laboratory chow diet prior to mating and throughout pregnancy. Within each diet group, rats received either cocaine injections (30 mg/kg IP two times per week prior to mating and then 30 mg/kg SC daily from days 3 to 18 of pregnancy) or saline injections. Litters were fostered on the day of birth to control mothers (i.e., nondrug-exposed dams fed the control or chow diet). Foster mothers fed the 25% casein diet showed increased contact with cocaine-exposed pups compared with nondrug-exposed pups in the second postnatal week but lower levels as the pups approached weaning. Passive nursing was increased in dams caring for prenatally malnourished, cocaine-exposed pups compared with those caring for similar pups with no drug exposure. Chow-fed mothers did not differ in their behavior towards pups with or without prenatal cocaine treatment. Prenatal cocaine and malnutrition independently compromised birth weight and various reflexive milestones but the attainment of physical milestones was affected only by prenatal cocaine. There were no additive effects of the two prenatal insults on any measure of mother-pup interaction or pup development.

The effects of cocaine exposure prior to and during pregnancy in rats fed low or adequate protein diets

Sprague-Dawley rats were fed a diet of low protein content (6% casein), an isocaloric diet of adequate protein content (25% casein), or a laboratory chow diet for 5 weeks prior to mating and throughout pregnancy. Within each diet group, rats received either cocaine (30 mg/kg IP two times per week prior to mating and 30 mg/kg or 40 mg/kg SC daily from days 3 to 18 of pregnancy) or saline injections. Cocaine produced a greater reduction in food intake during pregnancy in the malnourished group compared with the other two diet groups. The effect of cocaine on food intake was minimal in chow-fed rats. Weight gain in pregnancy was reduced by cocaine in a dose-dependent manner, and by malnutrition. Both prenatal cocaine and malnutrition impaired skeletal maturation of the pups, but there was no additive effect of the two insults on this measure. Litter size was significantly reduced by the 40 mg/kg, but not by the 30 mg/kg dose of cocaine across all diet groups. Consequently, the 40 mg/kg dose of cocaine proved to be fetotoxic in this model. Birth weight was significantly reduced by prenatal malnutrition but not by prenatal cocaine. Gestation length was unaffected by either insult. Hence, the ability to detect a diet x drug interaction was dependent upon the variable being measured.

Prenatal protein malnutrition enhances stimulus control by CDP, but not a CDP/THIP combination in rats

In the present study, the effects of prenatal protein malnutrition on stimulus control exerted by the benzodiazepine (BZ), chlordiazepoxide (CDP) and the GABA-A receptor agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) were characterized. The adult, male offspring of female Sprague-Dawley rats fed either low (6% casein) or adequate (25% casein) protein diets 5 weeks prior to mating and throughout pregnancy served as subjects. Subjects were first trained to discriminate CDP (8.0 mg/kg ip) from saline using drug discrimination procedures. Once a criterion level of performance was achieved, generalization tests were performed to lower doses of CDP (4.0, 2.0, 1.0, 0.5 and 0.25 mg/kg) and then to several doses of THIP (10.0, 7.5, 5.6 and 3.2 mg/kg). Lastly, the ability of a single dose of THIP (3.0 mg/kg) to enhance discriminative control by several low doses of CDP (4.0, 2.0, 1.0 and 0.5 mg/kg) was assessed. Although both diet groups acquired the original CDP/saline discrimination at the same rate, malnourished rats exhibited significantly more generalization to low doses of CDP than their well-nourished counterparts. Neither diet group exhibited significant generalization to THIP nor a difference in THIPs ability to enhance the CDP cue. These results suggest that a subjects sensitivity to the stimulus properties of drugs can be selectively modified by prenatal malnutrition.

Effects of diet on sensitization to cocaine-induced stereotypy in female rats

The progressive increase in cocaine-induced stereotyped behavior that accompanies repeated cocaine injections (sensitization) was examined in rats consuming different diets. Adult female Sprague-Dawley rats were fed one of three diets: low protein (6% casein), adequate protein (25% casein), or a standard chow diet. Following 1 week of adaptation to the diets, the rats were injected every 3-4 days with either cocaine (30 mg/kg, IP) or saline, and the total amount of stereotypy was measured over a 90-min interval following each of four injections. Cocaine-induced stereotypy peaked at 40-50 min following each injection, after which it declined for all diet groups. With repeated injections, the total amount of stereotypy increased in all diet groups. By the fourth injection, the low protein diet group (6% casein) exhibited a slower onset and a possibly prolonged duration of cocaine-induced stereotypy when compared with the two adequate protein diet groups (25% casein and chow). Interestingly, the rats in the two purified diet groups (6% casein and 25% casein) exhibited significantly more stereotypy across injections than those in the chow diet group. Weight differences did not explain the differences in stereotypy present among the diet groups. This study concludes that diet significantly alters the pattern of cocaine-induced stereotypy in female rats, especially after repeated exposure.

Effects of an every other day rapid kindling procedure in prenatally protein malnourished rats

Prenatally protein (6/25) rats have been reported to require significantly more stimulations to attain a stage 5 seizure than well-nourished controls (25/25) when using either a traditional or rapid every day, kindling procedure. In the present study, a rapid kindling procedure was utilized where both prenatally malnourished and control rats received every other day perforant path kindling (50 Hz, 10 s train) 12 times a day at 5-min intervals. Using this procedure, stage 5 seizures and a fully state were attained in both nutritional groups at approximately the same rate. It is postulated that it is the every other day component of the present procedure which overcomes seizure-induced inhibition in the 6/25 subjects, thereby allowing them to attain stage 5 seizures at the same rate as controls.