Gene expression profiling in the amygdala: an approach to examine the molecular substrates of mammalian behavior.

The molecular substrates of behavior have been difficult to assess because of the large number of messenger RNAs (mRNAs) expressed in a given brain region, the heterogeneous composition of the CNS, and the complexity of mammalian behavior. To gain insight into the molecular components of behavior requires an understanding of the anatomy associated with a specific behavior and the ability to examine multiple gene expression in discrete brain regions. Neuroanatomical and behavioral studies have demonstrated that the amygdaloid complex is an essential component of the neural pathways mediating behaviors, such as fear, anxiety, learning, and memory. The amygdala is composed of several interconnected subnuclei and it is the modulation of information, as it flows through these subnuclei, that underlies amygdala function. To examine the molecular components of the amygdala, we have combined the antisense RNA (aRNA) amplification procedure with microarray technology. This experimental approach permits the simultaneous detection and quantification of numerous mRNAs in fixed tissue sections. Our initial experiment examines region-specific gene expression in naïve mice in order to map the molecular relationship between the subregions of the amygdala. This report provides a general overview of the techniques used to examine regional gene expression, suggests future experiments, and describes a theoretical framework for examining the molecular analysis of behavior.

Traumatic brain injury alters the molecular fingerprint of TUNEL-positive cortical neurons In vivo: A single-cell analysis.

The cerebral cortex is selectively vulnerable to cell death after traumatic brain injury (TBI). We hypothesized that the ratio of mRNAs encoding proteins important for cell survival and/or cell death is altered in individual damaged neurons after injury that may contribute to the cell's fate. To investigate this possibility, we used amplified antisense mRNA (aRNA) amplification to examine the relative abundance of 31 selected candidate mRNAs in individual cortical neurons with fragmented DNA at 12 or 24 hr after lateral fluid percussion brain injury in anesthetized rats. Only pyramidal neurons characterized by nuclear terminal deoxynucleotidyl transferase-mediated biotinylated dUTP nick end labeling (TUNEL) reactivity with little cytoplasmic staining were analyzed. For controls, non-TUNEL-positive neurons from the cortex of sham-injured animals were obtained and subjected to aRNA amplification. At 12 hr after injury, injured neurons exhibited a decrease in the relative abundance of specific mRNAs including those encoding for endogenous neuroprotective proteins. By 24 hr after injury, many of the mRNAs altered at 12 hr after injury had returned to baseline (sham-injured) levels except for increases in caspase-2 and bax mRNAs. These data suggest that TBI induces a temporal and selective alteration in the gene expression profiles or "molecular fingerprints" of TUNEL-positive neurons in the cerebral cortex. These patterns of gene expression may provide information about the molecular basis of cell death in this region after TBI and may suggest multiple avenues for therapeutic intervention.

Positive and negative modulation of the GABA(A) receptor and outcome after traumatic brain injury in rats.

Glutamate-mediated excitotoxicity has been shown to contribute to cellular dysfunction following traumatic brain injury (TBI). Increasing inhibitory function through stimulation of gamma-aminobutyric acid (GABA(A)) receptors may attenuate excitotoxic effects and improve outcome. The present experiment examined the effects of diazepam, a positive modulator at the GABA(A) receptor, on survival and cognitive performance in traumatically brain-injured animals. In experiment 1, 15 min prior to central fluid percussion brain injury, rats (n=8 per group) were injected (i.p.) with saline or diazepam (5 mg/kg or 10 mg/kg). Additional rats (n=8) were surgically prepared but not injured (sham-injury). Rats pre-treated with the 5 mg/kg dose of diazepam had significantly lower mortality (0%) than injured, saline-treated rats (53%). Also, diazepam-treated (5 mg/kg) rats had significantly shorter latencies to reach the goal platform in the Morris water maze test performed 11-15 days post-injury. In experiment 2, at 15 min post-injury, rats were given either saline (n=5) or 5 mg/kg diazepam (n=6). Rats treated with diazepam did not differ in mortality from injured rats treated with vehicle. However, rats treated with diazepam at 15 min post-injury had significantly shorter latencies to reach the goal platform in the Morris water maze than injured, vehicle-treated rats. In experiment 3, the post-injury administration of bicuculline (1.5 mg/kg, n=8), a GABA(A) antagonist, increased Morris water maze goal latencies compared to injured animals treated with saline (n=8). These results suggest that enhancing inhibitory function during the acute post-injury period produces beneficial effects on both survival and outcome following experimental TBI.

Single-cell molecular biology: implications for the diagnosis and treatment of neurological disease.

The normal functioning of the central nervous system (CNS) requires complex interactions among numerous biological components. The pathophysiology of perturbations in this system is as complex as that of neurological disease. Many methods exist to examine the biological output of dysfunctional cells from a diseased system (e.g., immunohistochemical analysis, electrophysiology, and microdialysis), with one goal being to understand the mechanisms of cell death. This understanding may allow the design of therapeutic strategies to prevent cell death and ensuing behavioral abnormalities. Analysis of messenger RNA (mRNA) levels for various genes in CNS tissue may enhance understanding of neurological disease, since cells differ in the complement and abundance of genes they express. One popular method for detecting changes in gene expression is the Northern blot technique, in which total RNA from a sample is extracted and the RNA molecules are separated by size on a denaturing gel and transferred or "blotted" onto nylon membranes that are then probed with radiolabeled DNA for subsequent autoradiograpic detection of gene expression.

Working memory deficits following traumatic brain injury in the rat.

This study was designed to examine working memory following fluid-percussion traumatic brain injury (TBI) using the Morris water maze (MWM). Rats were injured (n = 9) at a moderate level of central fluid percussion injury (2.1 atm) or were prepared for injury but did not receive a fluid pulse (sham injury) (n = 10). On days 11-15 postinjury, working memory was assessed using the MWM. Each animal received 8 pairs of trials per day. For each pair of trials, animals were randomly assigned to one of four possible starting points and one of four possible escape platform positions. On the first trial of each pair, rats were placed in the maze facing the wall and were given 120 sec to locate the hidden escape platform. After remaining on the goal platform for 10 sec, they were placed back into the maze for the second trial of the pair. The platform position and the start position remained unchanged on this trial. After the second trial, the animal was given a 4 min intertrial rest. Between pairs of trials, both the start position and the goal location were changed. Analyses of the latency to reach the goal platform indicated that sham-injured animals performed significantly better on the second trial than on the first trial of each pair. However, injured animals did not significantly differ between first and second trial goal latencies on any day. These results indicate that injured animals have a profound and enduring deficit in spatial working memory function on days 11-15 after TBI.

Exposure to environmental complexity promotes recovery of cognitive function after traumatic brain injury.

This study was designed to determine whether exposure to a complex environment after traumatic brain injury (TBI) would promote the recovery of cognitive function. Rats were injured at a moderate level of fluid percussion injury (2.1 atm) or were prepared for injury but were not injured (sham injury). Immediately after the injury or sham injury, the injured/complex (n = 8) and the sham/complex (n = 7) groups were placed into a complex environment. The complex environment was a 89 x 89-cm enclosure with different types of bedding and objects that provided motor, olfactory, tactile, and visual stimulation. The injured/standard (n = 8) and the sham/standard (n = 8) groups were returned to the animal vivarium where they were housed individually in standard wire mesh cages (24 x 20 x 18 cm). On days 11-15 (postinjury), performance in the Morris water maze was assessed. Analysis of the latency to reach the goal platform indicated that injured animals recuperating in the complex environment performed significantly better than injured animals recovering in the standard environment (p < 0.01). In fact, injured animals in the complex environment performed as well as both sham-injured groups. The improved performance of injured rats recovering in the enriched environment occurred in the absence of environmentally induced alterations in brain weight. These results indicate that exposure to environmental complexity enhances recovery of cognitive function after TBI.

The effect of postinjury kindled seizures on cognitive performance of traumatically brain-injured rats.

The purpose of this experiment was to examine the consequences of postinjury seizures on cognitive performance after experimental traumatic brain injury (TBI). Rats either were injured at a moderate (2.1 atm) level of central fluid percussion TBI (n = 16) or were surgically prepared but did not receive a fluid pulse (sham-injured control, n = 16). Beginning 24 h after TBI, injured animals were injected (ip) once daily (Days 1-24 postinjury) with either saline (n = 8) or 25 mg/kg pentylenetetrazol (PTZ) (n = 8). Sham-injured rats were injected with an equal volume of saline (n = 8) or PTZ (n = 8). In both injured and sham-injured animals, daily injections of PTZ resulted in an increase in the severity of behavioral seizures over days. On Days 25-29 after injury or sham injury, all animals were tested in the Morris water maze (MWM). Analysis of maze performance indicated that in sham-injured animals PTZ-produced seizures had a detrimental effect on performance. In injured animals, however, PTZ-treated animals exhibited significantly faster acquisition and better terminal performance in the MWM than did untreated injured animals. These results show that posttraumatic kindled seizures do not exacerbate behavioral deficits after TBI and may, in fact, improve recovery following injury. The findings of this experiment are consistent with the hypothesis that post-TBI neuronal depression may contribute to behavioral morbidity following injury.

Chronic postinjury administration of MDL 26,479 (Suritozole), a negative modulator at the GABAA receptor, and cognitive impairment in rats following traumatic brain injury.

The present experiment examined the efficacy of postinjury administration of MDL 26,479 (Suritozole), a negative modulator at the gamma-aminobutyric acidA (GABAA) receptor that enhances cholinergic function, in attenuating spatial memory deficits after traumatic brain injury in the rat. Two experiments were performed. In the delayed-dosing experiment, rats received a moderate level (2.1 atm) of fluid-percussion brain injury and were tested in the Morris water maze 11 to 15 days following injury. These rats were injected with either 5 mg/kg (eight rats) or 10 mg/kg (eight rats) of MDL 26,479 60 minutes before each water maze test. Additional rats were injured and treated with saline (eight rats) or were surgically prepared but not injured (eight rats). In the second experiment, an early postinjury dosing procedure was followed. Rats were injured in the same manner but drug treatment began 24 hours after injury and continued daily through Day 15. Results indicated that the rats in the delayed chronic dosing regimen did not differ from the injured, saline-treated rats in their latency to reach the goal platform (p > 0.05). However, those treated chronically beginning 24 hours after injury had significantly shorter latencies than the injured, saline-treated rats (p < 0.05). These results suggest that administration of agents that enhance cholinergic function may be an appropriate strategy for promoting cognitive recovery when given after traumatic brain injury. Furthermore, prolonged treatment may be necessary to elicit beneficial effects.

Impaired gustatory neophobia following traumatic brain injury in rats.

To investigate the function of the amygdala following traumatic brain injury (TBI), rats were tested on a gustatory neophobia task that is sensitive to amygdala and hippocampal damage. Rats were either injured at a moderate level of fluid percussion injury (2.1 atm) or surgically prepared but not injured (sham-injury). Seven days after injury (n = 8) or sham injury (n = 9), rats were habituated to the testing chamber without food items present for 30 min. All rats were then food deprived. Twenty-four hours later, rats were placed in the testing chamber for 30 min and allowed to eat freely from four dishes of different foods: rat chow, raisins, potatoes, and cookies. Results showed that injured and sham-injured rats did not differ in their ability to find hidden food, suggesting that TBI does not produce an enduring impairment of olfaction. There was also no difference in the total amount of food eaten between injured and sham groups (p > 0.05). The percentage of each type of food consumed did differ between the two groups with sham controls consuming more familiar food (rat chow) compared to the unfamiliar foods (p < 0.01). The injured animals distributed their eating evenly among the four foods with no particular preference for any one food (p < 0.05). This pattern of eating behavior in injured animals is similar to animals that have lesions to both the hippocampus and amygdala (Sutherland and McDonald, 1990). Therefore, the results of this experiment suggest that, in addition to the hippocampus, the amygdala may also contribute to the behavioral changes observed following TBI.

Traumatic brain injury enhances the amnesic effect of an NMDA antagonist in rats.

The authors have examined the effect of experimental traumatic brain injury on the amnesia produced by the N-methyl-D-aspartate (NMDA) antagonist MK-801. Rats were either subjected to a moderate level of fluid-percussion injury or prepared for injury but not injured ("sham injury"). Nine days following injury or sham injury, the rats were injected either with saline (sham/saline group, nine rats; injured/saline group, nine rats) or with 0.1 mg/kg of MK-801 (sham/MK-801 group, nine rats; injured/MK-801 group, eight rats) 30 minutes before being trained on a passive-avoidance task. Twenty-four hours later, the rats were tested for retention of the passive-avoidance task. Results revealed that the low dose of MK-801 did not significantly affect retention of the passive-avoidance task in the sham-injured group. In injured animals, administration of MK-801 produced a profound amnesia in contrast to the sham-injured animals treated with MK-801 and the injured animals treated with saline. To further investigate this enhanced sensitivity to the amnesic effects of MK-801 exhibited by the injured animals, nine injured and eight sham-injured rats were injected with 0.3 mg/kg of MK-801 15 minutes before injury. Results indicated that the animals treated with MK-801 before injury did not significantly differ from the sham-injured animals in retention of the passive-avoidance task. In addition, test results in the animals treated with MK-801 before injury and reinjected with MK-801 before passive-avoidance testing did not differ from those in untreated injured animals reinjected with saline before passive-avoidance testing. These findings indicate that MK-801 treatment before injury prevented the enhanced sensitivity to MK-801-induced amnesia that follows traumatic brain injury.

The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury.

The purpose of the present experiment was to examine the effectiveness of a modified rotarod test in detecting motor deficits following mild and moderate central fluid percussion brain injury. In addition, this investigation compared the performance of the rotarod task with two other commonly used measures of motor function after brain injury (beam-balance and beam-walking latencies). Rats were either injured with a mild (n = 14) or moderate (n = 8) level of fluid percussion injury or were surgically prepared but not injured (n = 8). All rats were assessed on all tasks for 5 days following their respective treatments. Results revealed that both the mild and moderate injury levels produced significant deficits in the ability of the animals to perform the rotarod task. Performance on the beam-balance and beam-walking tasks were not significantly impaired at the mild injury level. It was only at the moderate injury level that the beam-balance and beam-walking tasks detected deficits in motor performance. This result demonstrated that the rotarod task was a sensitive index of injury-induced motor dysfunction following even mild fluid percussion injury. A power analysis of the three tasks indicated that statistically significant group differences could be obtained with the rotarod task with much smaller sample sizes than with the beam-balance and beam-walking tasks. Performance on the rotarod, beam-walk, and beam-balance tasks were compared and evaluated by a multivariate stepdown analysis (multiple analysis of variance followed by univariate analyses of covariance). This analysis indicated that the rotarod task measures aspects of motor impairment that are not assessed by either the beam-balance or beam-walking latency. These findings suggest that compared to the beam-balance and beam-walking tasks, the rotarod task is a more sensitive and efficient index for assessing motor impairment produced by brain injury.

Cognitive impairment following traumatic brain injury: the effect of pre- and post-injury administration of scopolamine and MK-801.

In order to examine the effectiveness of pre- and post-injury administration of muscarinic cholinergic and NMDA antagonists in reducing cognitive deficits following traumatic brain injury (TBI), rats were injected with either scopolamine (1 mg/kg) or MK-801 (0.3 mg/kg) 15 min prior to or 15 min after fluid percussion TBI. Cognitive performance was assessed with the Morris water maze procedure on days 11-15 after TBI or sham injury. When scopolamine and MK-801 were injected 15 min before injury, Morris water maze deficits were significantly reduced (P < 0.01 and P < 0.05, respectively). When scopolamine and MK-801 were injected 15 min after TBI, neither drug was effective in attenuating Morris water maze deficits. Consistent with other research, these results suggest that the cognitive deficits produced by TBI are the consequence of a brief period of excessive excitation of cholinergic and NMDA receptor systems. The results of this experiment also suggest that the temporal therapeutic window for the treatment of cognitive dysfunction with receptor antagonist intervention appears to be quite brief (< 15 min) in the rat.

Selective cognitive impairment following traumatic brain injury in rats.

Impairment of cognitive abilities is a frequent and significant sequelae of traumatic brain injury (TBI). The purpose of this experiment was to examine the generality of the cognitive deficits observed after TBI. The performance of three tasks was evaluated. Two of the tasks (passive avoidance and a constant-start version of the Morris water maze) were chosen because they do not depend on hippocampal processing. The third task examined was the standard version of the Morris water maze which is known to rely on hippocampal processing. Rats were either injured at a moderate level (2.1 atm) of fluid percussion brain injury or surgically prepared but not injured (sham-injured control group). Nine days after fluid percussion injury, injured (n = 9) and sham-injured rats (n = 8) were trained on the one-trial passive avoidance task with retention assessed 24 h later. On days 11-15 following injury, injured (n = 9) and sham-injured (n = 8) rats were trained on a constant-start version of the Morris water maze that has the animals begin the maze from a fixed start position on each trial. Additional injured (n = 8) and sham-injured (n = 8) animals were trained on days 11-15 after injury on the standard (i.e. using variable start positions) version of the Morris water maze. The results of this experiment revealed that performance of the passive avoidance and the constant-start version of the Morris water maze were not impaired by fluid percussion TBI.(ABSTRACT TRUNCATED AT 250 WORDS)