G.Jean Kant

Comparison of stress response in male and female rats: pituitary cyclic AMP and plasma prolactin, growth hormone and corticosterone.

Three potent stressors (forced running, immobilization, and footshock) were found to increase levels of cyclic AMP in the pituitaries of both female and male rats. The pituitary cyclic AMP response in females was generally similar to that observed in males. The tested stressors elevated both plasma corticosterone and prolactin and decreased plasma growth hormone. Plasma corticosterone rose more rapidly in females than in males following stress. Control growth hormone levels were higher in male rats. There was no clear cause and effect relationship between elevations of pituitary cyclic AMP and changes in plasma levels of prolactin, corticosterone, and growth hormone.

Locus coeruleus lesions and learning in the rat

Abstract The performance of rats with bilateral lesions of the nucleus locus coeruleus (LC) was compared with that of sham-lesioned rats using 3 avoidance and 2 appetitive learning paradigms. LC lesions which significantly reduced cortical norepinephrine produced no deficits in learning one-way active or passive shock avoidance responses, nor in acquisition or extinction of a conditioned taste aversion, nor in acquisition of a bar press response for food reinforcement, but did produce impairments in running for food in an L-shaped runway. The runway deficit could not be related to motor difficulties or differences in activity between the two groups. Although locus coeruleus lesions interfere with performance in a runway task they do not produce a general impairment in learning.

Non-cholinergic neurotoxic effects of AF64A in the substantia nigra *

Ethylcholine aziridinium ion (AF64A), a putative specific cholinergic neurotoxin, was unilaterally injected into the substantia nigra of male rats. One week following injection, rats that were challenged with apomorphine exhibited ipsilateral circling behavior. After two weeks, rats were sacrificed and corpus striatum and combined n. accumbens plus o. tubercle were assayed for dopamine. Striatal dopamine levels were depleted by 50%. Histological examination showed that AF64A caused extensive damage at the injection site. These data indicate that injection of AF64A into the substantia nigra can result in damage to dopaminergic neurons.

Effects of chronic stress on sleep in rats

The present study was conducted to determine the effects of chronic stress on sleep using a rodent paradigm of around-the-clock signalled intermittent foot shock in which some rats can pull a chain to avoid/escape shock while another group of rats is yoked to the first group. We measured sleep using telemetry; four-channel EEG was collected 24 h/day in rats during 2 prestress days; days 1, 2, 3, 7, and 14 during chronic stress; and 3 poststress days. States of REM sleep, non-REM (NREM) sleep, and waking were scored for each 15-s period of the EEG recordings. During the prestress period, rats slept (REM plus NREM) 55% of available time during the light hours and 34% of the dark hours with the remainder represented by waking. On the first day of stress, total sleep and, especially REM sleep, decreased markedly. By the second day of stress, only REM sleep in the controllable stress group (but not the uncontrollable stress group) was still significantly decreased compared to prestress levels, and REM sleep returned to baseline levels by day 7 of stress. The recovery of sleep quantity was accomplished by increased sleep during the dark hours, resulting in a long-lasting disruption of normal circadian sleep patterning.

Effects of controllable vs. uncontrollable chronic stress on stress-responsive plasma hormones

We have previously reported effects of chronic stress on circadian rhythms of temperature, eating, and locomotor activity. These studies were conducted using an around-the-clock signalled intermittent footshock paradigm in which some rats have control over shock termination while other rats are yoked to the rats with control. Although this paradigm is stressful, as suggested by decreases in food intake and disrupted circadian rhythms, rats tolerate the paradigm well, continuing to eat, drink, gain weight, and groom. In the present studies, rats were sacrificed following 3 or 14 days of stress, and plasma was collected for hormonal assays. After 3 days of stress, plasma corticosterone and prolactin levels were elevated in both stress groups compared to controls; yoked rats had higher levels of corticosterone than rats in the group with control over shock termination, while prolactin levels in both stressed groups were similar. ACTH levels were similar in stressed and control rats. After 14 days of stress, ACTH and corticosterone levels in both stress groups were similar to control levels. Prolactin levels were elevated in the yoked experimental group compared to levels in control or controllable stress groups. These data support previous studies suggesting that control over stressors attenuates the effects of stress on physiology and demonstrate that two hormones with diverse biological effects are elevated by chronic stress.

Effects of controllable vs. uncontrollable stress on circadian temperature rhythms

The effects of sustained stress on body temperature were investigated in rats implanted with mini-transmitters that permitted remote measurement of body temperature. Temperature was first monitored during control conditions. Following the control period, rats were either shaped to avoid/escape signalled around-the-clock intermittent footshock (controllable stress) or yoked to the controlling rats such that the controlling rat and the yoked rat received shock of the same duration, but only the controlling rat could terminate shock by pulling a ceiling chain. Under control conditions, rats demonstrated regular rhythms in body temperature which averaged 1 degree higher during the 12-h dark cycle than the light cycle. Stress disrupted the rhythm and markedly decreased the night-day difference in temperature, especially in the yoked rats in which almost no difference between light and dark cycle temperature was seen. The disruption was most marked for the first days of stress. A regular temperature rhythm was reestablished following about 5 days of stress although the stress condition continued. Leverpressing for food was also affected by the stress conditions with both stress groups leverpressing less than controls and the uncontrollable stress group pressing less than the controllable stress group. These data offer additional evidence of the increased pathophysiological effects of uncontrollable as compared to controllable stress.

Effects of MK-801 on learning and memory as assessed using a novel water maze

The effects of the NMDA receptor antagonist MK-801 [(+)-10,11-dihydro-5-methyl-5H-dibenzo [a,d]-cyclohepten-5,10 imine hydrogen maleate] on learning and memory were assessed using a water maze. The maze was a traditional type of maze with alleys and choices between various paths, but set inside a pool of water to a height of 25 cm. Different mazes could be configured by altering the arrangement of open vs. closed doors. Both the time required to reach an out-of-the-water exit platform and the errors made during the swim from start to finish were recorded. Learning was assessed during the first 10 to 20 trials in a new maze configuration, while memory was tested after the maze was well learned. Three experiments, some with several phases, were performed. These experiments compared the effects of 0.1 mg/kg of either (+)-MK-801, or (-)-MK-801 vs. saline on learning new maze configurations as well as swimming well-learned mazes. Neither of the MK-801 isomers impaired performance of a previously learned maze. (+)-MK-801 clearly slowed learning of new mazes as measured by both maze completion time and errors committed, while (-)-MK-801 had a significant but smaller effect on learning. Rats given (+)- or (-)-MK-801 (0.1 mg/kg) for 16 days while learning one maze and then challenged to learn a new maze without drug administration performed no differently on the new maze than controls, suggesting that the acute effect of MK-801 on learning is not long lasting.

Release of endogenous norepinephrine and dopamine from rat brain regions in vitro.

Abstract Using radioenzymatic assay procedures, we have measured picomolar amounts of endogenous norepinephrine (NE) and dopamine (DA) released in vitro . The release of NE and DA in response to KCl stimulation was examined in 6 brain regions: cortex, hippocampus, hypothalamus, striatum, combined accumbens-olfactory tubercle, and substantia nigra. NE release was detectable in all regions except striatum. Amounts of NE released by 55mM KCl (expressed as % control) were: cortex (313%), hippocampus (227%), hypothalamus (225%), accumbens-tubercle (278%), s. nigra (155%). KCl stimulated release of DA was detected in 3 regions: striatum (414%), accumbenstubercle (282%), and hypothalamus (312%). DA was measurable in filtrates from the s. nigra but levels in control and KCl stimulated samples were equal. Release of NE and DA was also measured in 12 brain regions after incubation of tissue in vitro with 10−4M d-amphetamine sulfate. d-Amphetamine stimulated NE outflow when compared to controls in all regions examined. DA outflow was markedly increased in most regions, especially striatum (287%), hypothalamus (387%) and accumbens-tubercle (670%). d-Amphetamine doubled endogenous DA outflow from the s. nigra.

The combined effects of pyridostigmine and chronic stress on brain cortical and blood acetylcholinesterase, corticosterone, prolactin and alternation performance in rats

Thousands of soldiers who served in the Gulf War have symptoms that have been collectively termed Gulf War Illness (GWI). It has been suggested that a combination of operational stress and pyridostigmine, a drug given as a pretreatment to protect soldiers against the effects of exposure to nerve agents, might have had unexpected adverse health effects causing these symptoms. Our laboratory has previously modeled operational stress in rats using a paradigm of around-the-clock intermittent signalled footshock. In the present studies, this model was used to investigate the potential synergistic effects of chronic stress and pyridostigmine on physiology and behavior. Seventy-two rats were trained to perform an alternation lever pressing task to earn their entire daily food intake. The rats were then implanted with osmotic minipumps containing vehicle, pyridostigmine (25 mg/ml pyridostigmine bromide) or physostigmine (20 mg/ml eserine hemisulfate). The pumps delivered 1 microl/h, which resulted in a cumulative dosing of approximately 1.5 mg/kg/day of pyridostigmine or 1.2 mg/kg/day of physostigmine, equimolar doses of the two drugs. The rats were then returned to their home cages where performance continued to be measured 24 h/day. After 4 days, 24 of the 72 rats were trained to escape signalled footshock (avoidance-escape group) and 24 other rats (yoked-stressed group) were each paired to a rat in the avoidance-escape group. The remaining 24 rats were not subjected to footshock (unstressed group). Shock trials were intermittently presented in the home cage 24 h/day for 3 days, while alternation performance continued to be measured. Since only 12 test cages were available, each condition was repeated to achieve a final n of six rats per group. Pyridostigmine and physostigmine each decreased blood acetylcholinesterase levels by approximately 50%. Physostigmine also decreased brain cortical acetylcholinesterase levels by approximately 50%, while pyridostigmine had no effect on cortical acetylcholinesterase activity. Alternation performance was impaired on the first day of stress and then recovered. Neither pyridostigmine nor physostigmine affected performance in the absence of stress or increased the effects of stress alone. Corticosterone was significantly increased in the yoked stress group compared to unstressed controls. These data suggest that pyridostigmine does not exacerbate the effects of stress on performance or levels of stress hormones. Furthermore, these data do not suggest that stress enables pyridostigmine to cross the blood brain barrier.

Increase in dopamine in cerebral cortex and other regions of rat brain after microwave fixation: possible diffusion artifact.

Exposure to high-intensity microwave irradiation at 2450 MHz has shown promise as a sacrifice technique which inactivates brain enzymes, reduces or eliminates postmortem change in certain heat-stable substrates and leaves the brain in a condition suitable for regional dissection 1-3. Although microwave inactivation of enzymes is not a prerequisite to the measurement of norepinephrine (NE) or dopamine (DA) in whole brain 24, we desired to establish a method for measuring these amines as well as GABA, glutamic acid, cyclic AMP and cyclic G M P in the same brain regional samples3,4,13. Male Walter Reed strain rats were placed into a plexiglass holder and inserted into a hole in the short-circuiting end plate o f a WR 430 waveguide exposure chamber in such a manner that the longitudinal axis of the head was perpendicular to the microwave E field. The power source and waveguide were modified to achieve greater uniformity and efficiency of inactivation 5,14. Animals were exposed for 5.5 sec with 2.5 kW forward power (1-3 ~ reflected). The power source was a Varian PPS-2.5 modified with electronic control for precise timing and leveling of output power. Frequency was verified at 2440 J20 MHz with a spectrum analyzer. Prior to exposure, each animal was impedance-matched over the range 2420-2460 MHz to a low power (10 mW/sq.cm) signal from a sweep generator, using a double-stub tuner. During every exposure, the number of actual pulses (120/sec) as well as both forward and reflected power were monitored. Norepinephrine and dopamine were assayed using the enzymatic-isotopic method of Coyle and Henry s with slight modifications. Because microwave fixation precludes perfusion, brains to be examined histologically were immersed in phosphate-buffered 10~ formalin. Comparable paraffin sections from both decapitated and microwave-irradiated brains were paired and stained simultaneously in the same jar for the same duration. Thickness of section varied with the type of stain: Luxol Fast Blue (20/~m), or Bielschowsky (8/~m).