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Background: HIV Associated Dementia (HAD) is a common complication of human immunodeficiency virus (HIV) infection that erodes the quality of life for patients and burdens health care providers. Intravenous drug use is a major route of HIV transmission, and drug use is associated with increased HAD. Specific proteins released as a consequence of HIV infection (e.g., gp120, the HIV envelope protein and Tat, the nuclear transactivating protein) have been implicated in the pathogenesis of HAD. In primary cultures of human fetal brain tissue, subtoxic doses of gp120 and Tat are capable of interacting with a physiologically relevant dose of cocaine, to produce a significant synergistic neurotoxicity. Using this model system, the neuroprotective potential of gonadal steroids was investigated., Results: 17beta-Estradiol (17beta-E2), but not 17alpha-estradiol (17alpha-E2), was protective against this combined neurotoxicity. Progesterone (PROG) afforded limited neuroprotection, as did dihydrotestosterone (DHT). The efficacy of 5alpha-testosterone (T)-mediated neuroprotection was robust, similar to that provided by 17beta-E2. In the presence of the specific estrogen receptor (ER) antagonist, ICI-182,780, T's neuroprotection was completely blocked. Thus, T acts through the ER to provide neuroprotection against HIV proteins and cocaine. Interestingly, cholesterol also demonstrated concentration-dependent neuroprotection, possibly attributable to cholesterol's serving as a steroid hormone precursor in neurons., Conclusion: Collectively, the present data indicate that cocaine has a robust interaction with the HIV proteins gp120 and Tat that produces severe neurotoxicity, and this toxicity can be blocked through pretreatment with ER agonists.

Constantly growing body of evidence suggests that hallmarks of oxidative stress are present in various central nervous system (CNS) disorders. Technological advantages in cell culturing made it possible to use neural cell/tissue cultures as experimental models for investigation of molecular mechanisms which underlie the development of oxidative stress condition, damage and adaptive responses to oxidative insults. This review is focused on the application of cell culture methodology for studies of oxidative stress condition in the brain. The review describes studies of biomarkers of oxidative stress-dependent cell damage and adaptive responses in various kinds of brain cell culture models. It discusses the use of cell/tissue culture models for elucidation of the role and pathogenesis of oxidative stress in neurodegenerative brain disorders, AIDS-associated brain pathology, drug abuse, and aging. The review underscores the importance of cell/tissue-based studies for testing of new antioxidants and development of therapeutic strategies for amelioration of oxidative damage in the CNS. The impact of new advances in gene and protein expression analysis on the cell/tissue culture-based research of oxidative stress in the CNS is also discussed.

Neprilysin is a zinc metalloendopeptidase that regulates the activity of a number of physiological peptides through hydrolytic inactivation. Most recently, evidence has accumulated that neprilysin is involved in the clearance of amyloid beta peptides in the brain. Previous studies have shown that the neprilysin gene responds to progesterone, androgen, and glucocorticoids. We now show that estrogen regulates neprilysin activity in rat brain. Ovariectomy leads to a 30% decrease in neprilysin activity at 45 or 85 days, but not 21 days, post surgery. Estrogen replacement restores neprilysin levels back to control values. These changes in neprilysin activity suggest that in women estrogen is required to maintain basal levels of neprilysin.

Cocaine use during pregnancy is associated with neurobehavioral problems in school-aged children that implicate alterations in attentional processes, potentially due to impairments in the noradrenergic system. We analyzed locus coeruleus (LC) neurite outgrowth characteristics following the administration of a physiologically relevant dose of cocaine (3.0 mg/kg) issued during critical phases of gestation (gestational day (GD)8-14, GD15-21, GD8-21). Results showed that cocaine inhibits LC neurite outgrowth and development, as evidenced by a decrease in total neurite length, a decrease in neurite length per cell, and a decrease in the percentage of cells with neurites. Morphological differences between cultures treated with and without cocaine were also evident. Further, the specific gestational exposure period effects were also dependent upon sex of the fetus. Finally, a discriminant function analysis suggested that the pattern and magnitude of alterations that defined the GD8-14 exposure were significantly different from that of the GD15-21 or GD8-21 exposures. Collectively, these data demonstrate a direct, disruptive effect of cocaine on noradrenergic neurons and may provide a neurobiological basis for changes in attentional function seen in offspring exposed to cocaine in utero.

Attentional dysfunction is a persistent behavioral abnormality that is emerging as one of the cardinal features in the investigations of the teratogenic effects of cocaine in humans and rodents. The present study sought to extend this work by using a dose-response design with an alternate strain of rat. Virgin Long-Evans female rats, implanted with an IV access port prior to breeding were administered saline, 0.5, 1.0, or 3.0 mg/kg of cocaine HCl from gestational day (GD) GD8-21 (1x per day-GD8-14, 2x per day-GD15-21). Cocaine had no significant effect on maternal or litter parameters. At 14-15 days of age, 1 male and 1 female from each litter were tested to evaluate the heart rate orienting response (HR-OR). Following 20 min for acclimation, pups were presented an olfactory stimulus for 20s per trial, across four trials, and with an intertrial interval of 2 min. The initial baseline HR was not significantly different across the treatment groups, although cocaine did alter the stability of the QRS complex duration. The magnitude of the HR-OR averaged across trials increased as a linear function of dosage of cocaine. A more complex (quadratic) interaction between cocaine dose and sex of the offspring was also noted. When examined across trials, the controls failed to display any significant within-session variation in the HR-OR; in contrast all of the prenatal cocaine treated groups displayed either sensitization (low and high dose) or habituation of the response (middle dose). Analysis of the peak HR-OR confirmed that the controls were indeed displaying the response on at least one trial of the session, albeit not consistently on any specific trial. The more vigorous HR-OR of the prenatal cocaine groups, relative to vehicle controls, most likely reflects an alteration in development of the neural basis of response; as previously shown, the most vigorous response to the olfactory stimulus is seen early (12 days of age) and progressively decreases across the preweaning period. In sum, prenatal exposure to cocaine, at least when administered by the IV route, provides reproducible alterations in attentional processes, as indexed by the noradrenergically-mediated HR-OR. The documentation of a linear dose-response function suggests that there is likely no threshold for the drug-induced alteration. Moreover, the sex of the animal also appears to play some role in the nature of the expression of the altered HR-OR.

The present study examined the sex-dependent expression of behavioral sensitization as well as changes of dopamine (DA) transporters and D1, D2, and D3 receptors following repeated intravenous nicotine administration. Male and female Sprague-Dawley rats were implanted with indwelling jugular catheters, equipped with subcutaneous intravenous injection ports. Rats were habituated to activity chambers for 3 days and were subsequently administered 15-s bolus injections of intravenous nicotine (50 microg/kg/ml) 1/day for 21 days. Animals were placed in activity chambers for 60 min immediately after the 1st and 21st nicotine injection. Observational time sampling was also performed. Brains were subsequently removed and frozen for autoradiographic DA transporter/DA receptor analysis on the afternoon females were in proestrus. With one exception, no robust sex differences were observed for locomotor activity or any rearing measures either during baseline or after initial nicotine injection. Females exhibited markedly more behavioral sensitization of locomotor activity, rearing, duration of rearing, and incidence of observed rearing. There were no sex differences in the number of D1 or D2 receptors. Females exhibited an increased number of DA transporters and decreased D3 receptors in the NAcc, relative to males. Multiple regression analyses suggest that D3 receptors and DA transporters in various striatal and NAcc subregions differentially predicted nicotine-induced behaviors for males and females. Collectively, these findings demonstrate that repeated intravenous nicotine produces sex differences in the expression of behavioral sensitization, and suggest that nicotine-induced changes of DA transporters and D3 receptors are partly responsible for increased behavioral sensitization in female rats.

Adult Long-Evans rats, exposed prenatally to 1 of 4 doses of cocaine (0.0,0.5,1.0, or 3.0 mg/kg iv), were tested on a 3-choice visual attention task with an olfactory distractor presented unpredictably on one third of the trials. The performance of all 3 cocaine-exposed groups was significantly more disrupted than that of controls by the presentation of distractors. Results demonstrate that prenatal cocaine exposure increases susceptibility to distractors, using a task specifically designed to measure this function. In addition, the present study revealed that individuals exposed to cocaine in utero exhibit greater performance disruption after an error than controls, in certain types of tasks. Both areas of dysfunction, impaired selective attention and impaired arousal regulation, have important functional consequences in humans, possibly affecting the school performance and social development of cocaine-exposed children.

The present study was designed to assess working memory in adult rats exposed to intravenous cocaine in utero, as part of an examination of various cognitive and affective functions. The study included four groups: a saline control and three groups exposed to ascending doses of cocaine from gestational days 8 to 21 (0.5, 1.0, or 3.0 mg/kg). This exposure regimen (route of administration and dose) has been shown to accurately reproduce the pharmacokinetic profile and physiological effects of human recreational cocaine use. This report describes the results of a series of automated alternation tasks, in which the animals were rewarded for alternating their responses between two response ports on successive trials. In the final task, the delay between trials varied randomly between 0, 20, 40, and 80 s, thereby varying the retention interval. Although performance declined dramatically as the retention interval increased, the rate of this decline did not differ across treatment groups. These results suggest that prenatal cocaine exposure, at doses that model recreational use, does not produce lasting changes in explicit memory or working memory. However, subtle, sex-specific effects of prenatal cocaine exposure were seen on measures that indicate impairments in sustained attention and "readiness", as well as altered reactivity to task-related stressors such as waiting for long and unpredictable delays.

The emerging profile for the effects of prenatal cocaine exposure presents two prominent features in the exposed offspring: cognitive/attention deficits and an age-associated trend toward motor/tone abnormalities up to 2 years of age. One candidate mechanism underlying these clinical features is long-lasting alterations to dopamine (DA) neuron function. However, the impact of prenatal cocaine exposure on DA release in dopaminergic terminal fields in vivo in mature offspring is poorly understood. Long-Evans female rats were implanted with an i.v. access port, bred, and given saline or cocaine-HCl (3 mg/kg/ml) for gestational days (GD) 8-14 (1x/day), GD 15-21 (2x/day), or GD 8-21 (1x/day-GD 8-14, 2x/day-GD 15-21). Using in vivo high-speed chronoamperometric recordings, potassium-stimulated DA release was measured in striatum of anesthetized male offspring 90-150 days after birth. There was a trend toward increased potassium-evoked DA signal amplitudes in offspring exposed to cocaine at any time period examined. In offspring exposed to cocaine during GD 8-21 and GD 15-21, but not at GD 8-14, there were significant decreases in the clearance capacity of the potassium-evoked DA signal compared with control offspring. The time required to clear 80% of the evoked DA signal (T(80)) in striatum for DA was significantly prolonged (approximately 150% of control) and this effect was further increased in the mean-evoked DA concentration range for these two groups. We also measured total dopamine transporter (DAT) and tyrosine hydroxylase protein levels in these offspring by blot immunolabeling and found a small, but significant, decrease in DAT protein in striatum from offspring exposed at GD 8-21 and GD 15-21. Collectively, these data demonstrate that prenatal cocaine exposure during dopamine neuron neurogenesis has long-lasting effects on DA neuron function lasting into early adulthood which may be related in part to steady state DAT protein levels. These molecular events may be associated with established cognitive deficits and perhaps the trends seen in altered motor behavior.

Although correlations have been reported between maternal cocaine use and impaired attention in exposed children, interpretation of these findings is complicated by the many risk factors that differentiate cocaine-exposed children from SES-matched controls. For this reason, the present dose-response study (0, 0.5, 1.0, or 3.0 mg/kg cocaine HCl) was designed to explore the effect of prenatal cocaine exposure on visual attention in a rodent model, using an intravenous injection protocol that closely mimics the pharmacokinetic profile and physiological effects of human recreational cocaine use. In adulthood, animals were tested on an attention task in which the duration, location, and onset time of a brief visual cue varied randomly between trials. The 3.0 mg/kg exposed males committed significantly more omission errors than control males during the final 1/3 of each testing session, specifically on trials that followed an error, which implicates impaired sustained attention and increased reactivity to committing an error. During the final 1/3 of each testing session, the 0.5 and 1.0 mg/kg exposed females took longer to enter the testing alcove at trial onset, and failed to enter the alcove more frequently than control females. Because these effects were not seen in other tasks of similar duration and reinforcement density, these findings suggest an impairment of sustained attention. This inference is supported by the finding that the increase in omission errors in the final block of trials in each daily session (relative to earlier in the session) was significantly greater for the 1.0 mg/kg females than for controls, a trend also seen for the 0.5 mg/kg group. Unlike the cocaine-exposed males, who remain engaged in the task when attention is waning, the cocaine-exposed females appear to opt for another strategy; namely, refusing to participate when their ability to sustain attention is surpassed.

HIV-1 transactivating protein Tat is neurotoxic and is believed to play a role in the development of AIDS-associated dementia complex. Neurotoxicity of Tat may be associated with oxidative stress. In this study we examined temporal progression of histopathological changes induced by a single microinjection of Tat 1-72 into the rat striatum. Degenerating neural cells, detected by Fluoro-Jade B staining and increased protein oxidation, determined by protein carbonyl immunostaining, were observed in the striatum as soon as 2 h following the microinjection. Further progression of neuronal degeneration was associated with pronounced infiltration of the area surrounding Tat 1-72 injection site by OX-42 positive macrophages/microglia, which was evident at the 24 h time point. Signs of reactive astrocytosis were found in the striatum of Tat 1-72 injected animals as late as 7 days following the single microinjection. Increased GFAP immunoreactivity and changes in the morphology of astrocytes coincided with a second phase of increased protein carbonyl formation, but not with neuronal degeneration. Control polypeptide, nontoxic Tat delta 31-61, did not cause any cell death, inflammatory reaction or oxidative damage. Results of our study support the hypothesis that oxidative stress may be an early step in the mechanism of Tat neurotoxicity.

The present study was designed to examine the role of cue location and number in spatial navigation of the preweanling Fischer-344N rat in the Morris water maze using a protocol consistent with the pups' response repertoire. The proximal (visible platform) versus distal (hidden platform) cue strategy was used, and spatial cues within the extramaze environment were configured such that the arrangement presented either a double cue or null cull condition relative to the platform location. All pups' performance improved with training; however, probe trial performance, defined by quadrant time and platform crossings, revealed distal-double cue pups demonstrated spatial navigational ability superior to the remaining groups. This experimental dissociation suggests that a pup's ability to spatially navigate a hidden platform is dependent on not only its response repertoire and task parameters but also its visual acuity, as determined by the number of extramaze cues and the location of these cues within the testing environment. The hidden versus visible platform dissociation may not be a satisfactory strategy for the control of potential sensorimotor deficits.

In certain populations around the world, the HIV pandemic is being driven by drug-abusing populations. Mounting evidence suggests that these patient populations have accelerated and more severe neurocognitive dysfunction compared with non-drug-abusing HIV-infected populations. Because most drugs of abuse are central nervous system stimulants, it stands to reason that these drugs may synergize with neurotoxic substances released during the course of HIV infection. Clinical and laboratory evidence suggests that the dopaminergic systems are most vulnerable to such combined neurotoxicity. Identifying common mechanisms of neuronal injury is critical to developing therapeutic strategies for drug-abusing HIV-infected populations. This article reviews 1) the current evidence for neurodegeneration in the setting of combined HIV infection and use of methamphetamine, cocaine, heroin or alcohol; 2) the proposed underlying mechanisms involved in this combined neurotoxicity; and 3) future directions for research. This article also suggests therapeutic approaches based on our current understanding of the neuropathogenesis of dementia due to HIV infection and drugs of abuse.

Rats exposed to cocaine prenatally were administered a series of 3-choice visual attention tasks, with the most pronounced deficits seen in a task in which the onset time, location, and duration of a visual cue varied unpredictably between trials. The cocaine-exposed rats were less accurate than controls but did not differ in the rate of premature responses or omission errors. The pattern of errors, coupled with response latency data, implicated deficits in the ability to rapidly engage attention and maintain a high level of alertness to the task. The cocaine-exposed rats also exhibited a blunted reaction to an error on the previous trial, possibly reflecting an alteration in emotional regulation and/or error monitoring. Implications for underlying neuropathology are discussed.

The present study was designed to test whether prenatal cocaine (COC) exposure alters sensitivity to the attentional effects of idazoxan (IDZ), an alpha-2 adrenergic antagonist that increases coeruleocortical NE activity. The task assessed subjects' ability to selectively attend to an unpredictable light cue and disregard olfactory distractors. IDZ increased commission errors specifically under conditions of distraction, an effect that was similar in the COC and control groups. In contrast, COC animals were significantly more sensitive than controls to the effects of IDZ on omission errors and nontrials. The pattern of effects suggests that the differential treatment response to IDZ on these latter measures resulted from an alteration in norepinephrine (NE)-modulated dopamine release in the COC animals, reflecting lasting changes in dopaminergic and/or noradrenergic systems as a result of the early cocaine exposure. Based on the behavioral measures that showed a differential response to IDZ in the COC animals, it seems likely that these changes may contribute to the alterations in sustained attention and arousal regulation that have been reported in both animals and humans exposed to cocaine in utero.

The present study was designed to examine retention of spatial information in the immature, Fischer-344N rat using a savings paradigm. Following training to locate a hidden platform using extramaze cues, preweanling animals (17 days of age) were immediately probed by testing in the tank with the platform removed. One week later, pups (26 days of age) were given an additional four training trials immediately followed by a second probe test to examine the animals' memory for the location of the platform. Animals that received initial training at 17 days of age demonstrated significant savings of information when tested after the retention interval. These data replicate our recent report of spatial navigation capabilities in the preweanling rat, and extend those findings by demonstrating that preweanling spatial navigation performance permits more rapid and accurate navigation following a 7-day retention interval after a "reinstatement" insufficient to produce accurate navigation in maturation controls., (Copyright 2002 John Wiley & Sons, Inc.)

Oxidative stress has been hypothesized to play a role in the pathogenesis of different neurodegenerative disorders, including HIV-related dementia. Tat, a nonstructural protein of HIV, is implicated in potentiation of neuronal apoptosis by mechanisms involving the disruption of calcium homeostasis and oxidative stress. The injection of Tat caused an increase of protein carbonyl formation in the rat striatum. Increased oxidative modification of proteins occurred early after Tat injection and preceded Tat-mediated astrogliosis. Immunostaining of brain sections demonstrated that an area of prominent protein carbonyl immunoreactivity surrounded an injection site in the striatum of Tat-injected rats. Intense protein carbonyl immunoreactivity was localized in cell bodies. Our study suggests that increased protein oxidation may be an important part of the mechanism of Tat neurotoxicity.

Cocaine use during pregnancy is affiliated with neurobehavioral abnormalities in offspring that are associated with problems of attention. Given the putative role of the noradrenergic system in attentional processes, impairments in the noradrenergic system may underlie specific attentionally sensitive, neurobehavioral alterations. Recent data using a clinically relevant intravenous (iv) route of administration show that the norepinephrine cell bodies of the locus coeruleus (LC) are a primary target for in utero cocaine exposure. Cell survival and neurite outgrowth of LC neurons were studied using two paradigms: (1) in vitro, using a physiologically relevant concentration of cocaine, and (2) in vivo, using a clinically relevant intravenous rat model. Fetal cocaine exposure significantly decreased neuronal survival (in vitro: P=.0001, n=24; in vivo: P=.0337, n=30), reduced neurite initiation (in vitro: P=.001, n=24; in vivo: P=.0169, n=30), decreased the number of neurites elaborated (in vivo: P=.0031, n=30), and reduced total neurite length (in vivo: P=.0237, n=30). The results of this novel approach toward an understanding of noradrenergic neurons as they respond to cocaine during development suggest that cocaine may affect behavior by negatively regulating neuronal pathfinding and synaptic connectivity.

Sensitivity to the attentional effects of SKF81297, a selective full agonist at dopamine D(1) receptors, was assessed in adult rats exposed to cocaine prenatally (via intravenous injections) and controls. The task assessed the ability of the subjects to monitor an unpredictable light cue of either 300 or 700 msec duration and to maintain performance when presented with olfactory distractors. SKF81297 decreased nose pokes before cue presentation and increased latencies and response biases (the tendency to respond to the same port used on the previous trial), suggesting an effect of SKF81297 on the dopamine (DA) systems responsible for response initiation and selection. The cocaine-exposed (COC) and control animals did not differ in sensitivity to the effects of SKF81297 on these measures. In contrast, the COC animals were significantly more sensitive than were controls to the impairing effect of SKF81297 on omission errors, a measure of sustained attention. This pattern of results provides evidence that prenatal cocaine exposure produces lasting changes in the DA system(s) subserving sustained attention but does not alter the DA system(s) underlying response selection and initiation. These findings also provide support for the role of D(1) receptor activation in attentional functioning.

HIV-associated dementia complex is a serious disabling disease characterized by cognitive, behavioral and motor dysfunction. Basal ganglia involvement in HIV-1 infection may be responsible for some of the psychomotor symptoms associated with HIV dementia. The objectives of the present study were to determine: (1) whether gp120 and Tat produce striatal toxicity, and (2) whether gp120 and Tat show synergistic toxicity in the striatum. In these studies, the recombinant proteins gp120, Tat, or saline (0.9%) were stereotaxically injected in the striatum of adult male rats. The striatal sections were evaluated for area of tissue loss (Cresyl-violet stained sections) and the number of GFAP immunoreactive cells 7 days after the injections. Doses of gp120 250 ng/microl or higher and Tat 5 microg/microl or higher produced a significant area of tissue loss and significantly increased the number of GFAP reactive cells. We found no toxicity in animals treated with immunoabsorbed gp120 or Tat. Combined gp120 (100 ng/microl)+Tat (1 microg/microl) injections into the rat striatum significantly increased the area of tissue loss and altered morphology and increased number of GFAP reactive cells, as compared to controls. Thus, the present results suggest the involvement of gp120 and Tat in striatal toxicity and provide a model for further studies to fully characterize their role in HIV-1 toxicity and to develop therapeutic strategies for HIV-1 associated dementia complex.

This study assessed the effects of prenatal cocaine exposure on cognitive functioning, using an intravenous (IV) rodent model that closely mimics the pharmacokinetics seen in humans after smoking or IV injection and that avoids maternal stress and undernutrition. Cocaine-exposed males were significantly impaired on a 3-choice, but not 2-choice, olfactory serial reversal learning task. Both male and female cocaine-exposed rats were significantly impaired on extradimensional shift tasks that required shifting from olfactory to spatial cues; however, they showed no impairment when required to shift from spatial to olfactory cues. In-depth analyses of discrete learning phases implicated deficient selective attention as the basis of impairment in both tasks. These data provide clear evidence that prenatal cocaine exposure produces long-lasting cognitive dysfunction, but they also underscore the specificity of the impairment.

The intravenous route of administration, accessed via a subcutaneous vascular access port, has been recently suggested as an animal model for studying the developmental effects of maternal cocaine abuse in the pregnant and/or group-housed rat. The present study (1) assessed the cardiovascular effects of intravenous (IV) cocaine, delivered via bolus injection, in chronically catheterized near-term pregnant rats, and (2) compared the IV cardiovascular responses to those following cocaine delivered via the commonly employed subcutaneous (SC) and intragastric (IG) routes of administration. Pregnant gestation day 15 (GD15) young adult female Sprague-Dawley rats (n = 21) were anesthetized and catheters surgically implanted into the carotid artery, jugular vein, fundus of the stomach, and a subcutaneous pouch. On GD17-19, heart rate (HR) and mean arterial pressure (MAP) were assessed, using a within-subjects design, prior and subsequent to IV (3 mg/kg), IG (60 mg/kg), and SC (40 mg/kg) cocaine. An interval of 6 h separated IV and IG cocaine administration and an interval of 18 h separated IG and SC cocaine administration. The peak responses of HR (23% downward arrow) and MAP (37% upward arrow) following IV cocaine were noted within 0.5 min. In contrast, the peak responses of HR (4% downward arrow, 6% downward arrow) and MAP (2% upward arrow, 15% downward arrow) after IG (23 min) or SC (26 min) cocaine, respectively, were significantly smaller and markedly delayed. No significant change in aortic blood flow velocity was detected following cocaine via any route of administration, although phasic flow velocities (PFV) were differentially sensitive to route of administration (PFV(dias) not PFV(sys)); IV cocaine increased (55% upward arrow) whereas IG or SC cocaine decreased approximately 35% downward arrow) PFV(dias). The pressor effects of an equimolar dose of IV cocaine methiodide (3.9 mg/kg) were indistinguishable from those of IV cocaine (38% upward arrow vs. 37% upward arrow), as were the effects on PFV(dias) (83% upward arrow vs. 55% upward arrow). The lack of an effect of cocaine methiodide on HR was consistent with the bradycardia effect of cocaine attributable to central mediation of the baroreflex. Finally, the pressor effects of IV cocaine paralleled the rapidly peaking arterial plasma levels of cocaine noted within 30 s after the initiation of drug injection. In sum, prominent effects of IV cocaine on maternal cardiovascular physiology are noted; as such, the recent reports of a lack of maternal/fetal toxicity following daily (3-6mg/kg) IV cocaine during GD8-21 are not due to use of an ineffective drug dose. It was equally clear that the SC and IG routes of exposure did not reproduce the cardiovascular component(s) of the expected physiological response to cocaine.

Repeated intermittent administration of stimulants is well known to produce behavioral sensitization in male animals. The present studies explored whether 1) behavioral sensitization occurred with the i.v. route of administration, 2) sensitization was greater in females than in males, 3) sensitization was modulated by gonadectomy, 4) intact adult female rats maintained normal estrous cytology patterns in response to repeated nicotine administration, and 5) the pharmacokinetics of i.v. nicotine dosing. Adult male, female, castrated, and ovariectomized Sprague-Dawley rats (n = 48) were surgically implanted with an intravenous access port. Animals received 50 microg/kg i.v. nicotine once/day for 14 days. Immediately after the initial nicotine injection and the final day 14 nicotine injection, animals were placed in IR photocell activity chambers for 60 min. Observational time sampling of behavior was also simultaneously performed by an observer blind to treatment condition. An increase in behavioral activity of greater than 120% occurred across the 14-day time course of i.v. nicotine injections. The magnitude of the increase, however, varied as a function of component of activity, gender, and gonadectomy. The behavioral observation data further suggested that the females demonstrated an increased sensitivity to repeated nicotine, as evidenced in a more rapid response, for example, grooming. These behavioral observations were associated with peak arterial levels of nicotine (approximately 25 ng/ml) no greater than the average venous levels of nicotine commonly maintained by cigarette smokers. Repeated i.v. nicotine, at a dose of 50 microg/kg, did not interfere with intact female vaginal cytology or body weight; the failure to detect such alterations were not due to inadequate statistical power. Moreover, no nicotine-treated animals displayed persistent vaginal estrous or were acyclic. Collectively, these data suggest that the i.v. nicotine model may be particularly useful in exploring the gender-dependent effects of nicotine.

Repeated intermittent administration of cocaine is well known to produce behavioral sensitization in male animals. The present studies explored whether intact adult female rats maintained normal estrous patterns in response to repeated IV cocaine administration and whether behavioral sensitization occurred with this route of administration. Adult female Sprague-Dawley rats (N = 48) were surgically implanted with an intravenous access port. Animals received 3.0 mg/kg IV cocaine once/day for 14 days. Daily vaginal lavages indicated that female rats continued to cycle normally throughout the experiment. Estimates of statistical power for detecting alterations in estrous cycle length ranged from 0.61-0.95 for small (0.1) to large (0.4) effect sizes. Moreover, no cocaine-treated animals displayed persistent vaginal estrus or were acyclic and cocaine treatment did not decrease body weight. Immediately after the cocaine injection, animals were placed in IR photocell activity chambers for 60 min. Female rats displayed a significant 75% increase in locomotor activity across the 14-day time course of IV cocaine injections. These data indicate that 3.0 mg/kg of IV cocaine does not interfere with normal estrous cyclicity, and that behavioral sensitization occurs in female rats following repeated daily IV cocaine dosing. Collectively, these data suggest that the IV cocaine-dosing model may be particularly useful in exploring the gender-dependent effects of cocaine using intact female rats.

There is growing evidence that the selective neuronal cell death observed in Alzheimer's Disease (AD) is the result of dysregulation of intracellular calcium (Ca2+) homeostasis. In the present study, L-type voltage sensitive calcium channels (L-VSCCs) were examined in the cerebellum and hippocampus of AD (n = 6; postmortem interval less than 5 h) and age-matched control (n = 6) tissue by homogenate binding techniques and quantitative in vitro receptor autoradiography using [3H]isradipine (PN200-110). Saturation analyses of the cerebellum revealed unaltered [3H]isradipine binding parameters (Kd and Bmax) between AD and control subjects. Analysis of AD and control hippocampus demonstrated significant differences as [3H]isradipine binding increased (62%) in AD, whereas hippocampal cell density decreased (29%) in AD, relative to control subjects. Moreover, AD differentially affected L-VSCC in area CA1 and dentate gyrus. The dentate gyrus had greatly increased binding (77%) with little cell loss (16%) in AD brains, whereas area CA1 had increased binding (40%) with significant cell loss (42%) in AD brains, relative to controls. The results of the present study suggest that hippocampal area CA1 may experience greater cell loss in response to increased L-VSCCs in AD relative to other brain regions.

The binding of the D2-like agonists, (+)-7-hydroxy-N,N-di-n-[3H]propyl-2-aminotetralin (7-OH-DPAT) and [3H]pramipexole (2-amino-4,5,6-tetrahydro-6-propylaminobenzthiazole; MIRAPEX) were determined in membranes from adult male Sprague-Dawley and Fischer-344 rats. Saturation analysis, which optimized binding to D3 receptors, revealed 3-6 fold differences in Bmax values between the two radioligands with no change in affinity. [3H](+)7-OH-DPAT labeled 41.4+/-4.1 to 61.8+/-3.0 fmol/mg protein in nucleus accumbens and striatal homogenates, yet [3H]pramipexole labeled only 7.0+/-1.2 to 18.9+/-5.3 fmol/mg protein. Regional differences with both radioligands were observed in Fischer-344 rats; the striatum exhibited a 52%-69% greater density of sites in comparison to the nucleus accumbens. These data suggest that D3 receptor density can vary significantly between animal strains depending on the radioligand used, and [3H]pramipexole identifies a different ratio of sites in the striatum and nucleus accumbens compared to [3H](+)7-OH-DPAT.

Nitric oxide modulation of dopamine D2 and D3 receptor binding was examined using [125I]epidepride (D2) and (+)7-hydroxy-N,N-di-n-[3H]propyl-2-aminotetralin ([3H](+)-7-OH-DPAT, D3). Nitric oxide, generated by i.c.v. injection of S-nitroso-N-acetyl-penicillamine (SNAP; 5 microg or 10 microg), significantly increased the density of [3H](+)-7-OH-DPAT binding sites (39% and 134%, respectively) in the striatum 24 hours post-injection in the absence of Gpp(NH)p, representing an upregulation of either D3 receptors or high affinity D2 receptors. In the presence of 10 microM Gpp(NH)p, D3 receptor upregulation was maintained in both the 5 microg (increased 35%) and 10 microg SNAP (increased 44%) groups. [3H](+)-7-OH-DPAT binding was reduced in both striatum and nucleus accumbens in the presence of 10 microM Gpp(NH)p compared to binding in the absence of Gpp(NH)p, suggesting an upregulation of D3 receptors. Administration of SNAP did not alter total specific [125I]epidepride binding in either brain region. These data suggest that; 1) D3 receptor density is modified following nitric oxide generation, and 2) the density of high affinity D2 receptors identified by [3H](+)-7-OH-DPAT increases in the striatum, but decreases in the nucleus accumbens.

Despite the wealth of experimental data on cocaine abuse, there are no published dose-response pharmacokinetic studies with bolus i.v. cocaine injection in the male rat. The present study examined the pharmacokinetics of arterial plasma concentrations of cocaine and metabolite profile [benzoylecgonine (BE), ecgonine methyl ester (EME), norcocaine (NC)] following a single i.v. injection of 0.5, 1.0, or 3.0 mg/kg cocaine. Male Sprague-Dawley rats (N = 25) were anesthetized and surgically instrumented with both jugular vein (drug administration) and carotid artery (blood withdrawal) catheters and allowed to recover for at least 24 h. Arterial plasma samples (200 microliters) were obtained at eight time points (0.5, 1.5, 2.5, 10, 20, 30 min) following i.v. bolus injection (15-s injection, 15-s flush) and analyzed by single ion monitoring using GC/MS. Nonlinear regression and noncompartmental pharmacokinetic analysis were employed. Mean +/- SEM peak plasma concentrations of cocaine occurred at 30 s in a dose-response manner (370 +/- 14,755 +/- 119,2553 +/- 898 ng/ml for 0.5, 1.0, and 3.0 mg/kg groups, respectively). T1/2 alpha was < 1 min for all groups, but inversely related to dose. T1/2 beta was independent of dose 13.3 +/- 1.6, 13.0 +/- 1.5, and 12.0 +/- 2.0 min for 0.5, 1.0, and 3.0 mg/kg groups, respectively). MRT (16.0, 15.9, 14.5 min), VdSS (3.3, 3.2, and 2.8 l/kg), and ClTOT (204, 201, and 195 ml/min/kg) also provided little evidence of dose-dependent effects. Although the metabolic profile of i.v. cocaine was similarly ordered for all dose groups (BE > EME > NC), a quantitative shift in metabolite profile was evident as a function of increasing dose. This metabolic shift, perhaps attributable to saturation of plasma and liver esterases, suggests that the recently reported pharmacodynamic effects positively correlated with i.v. cocaine dose are unlikely attributable to NC, a minor but pharmacologically active metabolite. In sum, the i.v. pharmacokinetic profile in rats is distinct from that observed via the SC, IP, and PO routes of administration and offers the potential to provide a reasonable clinically relevant rodent model.

The density of dopamine D3 receptors was determined in young (4-month-old) and aged (37-month-old) Fischer-344 x Brown-Norway (F1) male rats using the putative D3 receptor-preferring agonist, [3H](+)-7.hydroxy-2-(N,N-di-n-propylamino)tetralin ([3H](+)-7-OH-DPAT). In the presence of the non-hydrolyzable GTP analog, 5'-guanylylimidodiphosphate (Gpp(NH)p), the density of dopamine D3 receptors in the striatum and nucleus accumbens was significantly increased (29-102%, respectively) in aged Fischer-344 x Brown-Norway (F1) rats compared to young adults. These findings suggest that dopaminergic activity in aged rats is compromised by increased D3 receptor density, resulting in altered striatal/nucleus accumbens function via presynaptic or postsynaptic modifications.

The dopamine D3 receptor has been implicated as a possible mediator in the reinforcement or abuse of psychostimulants such as cocaine. The present studies examined the effects of repeated (14 day) intravenous cocaine administration (saline vehicle, 0.5, 1.0 and 3.0 mg/kg) on locomotor activity and dopamine D2 and D3 receptor density in the rat striatum and nucleus accumbens. Male Sprague-Dawley rats (n = 40) were implanted with an intravenous access port and allowed to recover for 2 days. An additional group of naive rats was included to control for surgical/injection stress (n = 10). Following 2 days of habituation trials, total, peripheral and central activity (photocell interruptions) data were collected during alternate daily 60-minute test sessions. Repeated cocaine treatment resulted in a significant dose-dependent increase in striatal D3 receptors which was predicted by daily 60-minute central locomotor activity. Conversely, D3 receptors in the nucleus accumbens exhibited a significant dose-dependent reduction which was predicted by the initial 5 minutes of central locomotor activity observed on peak sensitization days (days 6, 8 and 10). Sensitization to the locomotor stimulatory effects of cocaine was dose-dependent, with the time to peak sensitization day following the rank order of 0.5 > 1.0 > 3.0 mg/kg. The density of D2 receptors in the striatum and nucleus accumbens was unchanged by cocaine administration. These data suggest striatal and nucleus accumbens D3 receptor involvement in the expression of cocaine-induced behavioral sensitization. Thus, the D3 receptors in the striatum and nucleus accumbens may be differentially involved in the locomotor stimulation (striatal D3) and reinforcing aspects (nucleus accumbens D3) of repeated cocaine administration.

Receptor binding and gene expression of several members of the IGF gene family were examined in the rat brain following lesion of the hippocampal dentate gyrus granular cells by intradentate colchicine injection. Dentate granular cell loss was accompanied by extensive reactive gliosis in the lesioned hippocampus and damaged overlying cortex, as verified by the increase in GFAP mRNA and BS-1 lectin binding. At 4 days post-lesion, 125I-IGF-2 binding was dramatically increased within the lesioned dentate gyrus and damaged overlying cortex, and corresponded temporally and anatomically with increased IGF-BP2 gene expression following the lesion. Increased IGF-BP3 gene expression was only observed in the overlying cortex at 10 days post-lesion, and corresponded with an increase in 125I-IGF-1 binding at the injured surface of the cortex. Type-2 IGF receptor mRNA expression was reduced to background levels in the lesioned dentate gyrus, suggesting that IGF-BP2 was a major component of the observed increase in 125I-IGF-2 binding. In situ hybridization also revealed a prominent increase in IGF-1 mRNA expression by 4 days post-lesion, which was localized within the lesioned dentate gyrus and damaged cortical areas, and was shown to be expressed by microglia. While no IGF-2 mRNA expression was observed within the CNS, either prior to, or following the lesion, IGF-2 mRNA expression was observed in the choroid plexus, meningeal membranes, and in blood vessel endothelium, providing a potential source for the transport of IGF-2 into the CNS. In the injured CNS, increased IGF-BP2 expression may act to maintain or transport IGF-1 or IGF-2, as well as modulate the local autocrine and paracrine actions of the IGFs. Increased microglial IGF-1 expression following colchicine treatment correlates with the timing of a number of post-traumatic events within the CNS, suggesting that IGF-1 may have a role as a neuroprotectant for surviving neurons and signal for local neuronal sprouting, as well as a role in reactive astrogliosis.

Antibodies directed against choline acetyltransferase and glutamic acid decarboxylase were used in combination with recently developed stereological techniques to quantify changes in cholinergic, GABAergic, and total neuron number (Nissl-stain) within adjacent tissue sections through the horizontal limb/nucleus basalis in young (3 months, n = 6) and aged (27 months, n = 6) Fischer-344 male rats. Unbiased estimates of total neuron number within these regions were produced using a three-dimensional optical probe, the optical disector, in combination with a systematic random sampling scheme. Estimates of cell counts in immunostained tissue sections were conducted throughout the entire horizontal limb/nucleus basalis region. A significant 30% decrease in both cholinergic and total neuron number was detected in the aged animals; GABAergic neuron number remained unchanged. Total neuron number was significantly correlated with both cholinergic (r = 0.94) and glial cell number (r = 0.63), but not with GABAergic cell number. Based on neuron counts within an individual thick tissue section, the cholinergic neurons comprised only 11-15% of all neurons in the nucleus basalis of young and aged animals. Cholinergic neuron loss accounted for only 20% of the total age-related neuron loss within the horizontal limb/nucleus basalis in Fischer-344 male rats. These results indicate that age-related cholinergic neuron loss within the basal forebrain is reflected in reductions in total neuron number; however, GABAergic neurons, many of which project to the cortex, are unaffected by age. The magnitude of the age-related total neuron loss cannot be entirely accounted for by cholinergic cell loss. Therefore, an unidentified non-cholinergic, non-GABAergic component within the basal forebrain is also lost during aging and may contribute to the cognitive deficits previously ascribed to cholinergic dysfunction.

Neutral endopeptidase is a cell surface zinc metal-lopeptidase that regulates the action of a variety of physiologically active peptides. The enzyme exhibits a wide tissue distribution, being most abundant in kidney and lung. Three rat neutral endopeptidase cDNAs with unique 5'-untranslated sequences were isolated. Distribution of the corresponding mRNAs in rat tissues was analyzed by RNase protection assays and by in situ hybridization. In kidney, the type 2b transcript was the major species. In lung and testis, type 1 and type 2b transcripts were expressed in approximately equal amounts, while in brain and spinal cord the type 1 mRNA was the major transcript. These findings were extended by in situ hybridization studies. All three mRNAs were expressed in the proximal tubule of the kidney, with the type 2b transcript giving the strongest signal. In the frontoparietal cortex, expression of the neutral endopeptidase mRNA subtypes was cell- and region-specific. The type 1 transcript was localized to neurons, type 2b mRNA was not detectable, while type 3 mRNA was localized to the oligodendrocytes of the corpus callosum. These results clearly demonstrate that expression of the three neutral endopeptidase mRNAs can be regulated in a cell-specific manner.

Cross-reactions between dopamine D3 and sigma receptor ligands were investigated using (+/-)-7-hydroxy-N,N-di-n-[3H]propyl-2-aminotetralin [(+/-)-7-OH-[3H]-DPAT], a putative D3-selective radioligand, in conjunction with the unlabeled sigma ligands 1,3-di(2-tolyl)guanidine (DTG), carbetapentane, and R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane [R(-)-PPAP]. In transfected CCL1.3 mouse fibroblasts expressing the human D3 receptor, neither DTG nor carbetapentane (0.1 microM) displaced (+/-)-7-OH-[3H]DPAT binding. R(-)-PPAP (0.1 microM) displaced 39.6 +/- 1.0% of total (+/-)-7-OH-[3H]DPAT binding. In striatal and nucleus accumbens homogenates, (+/-)-7-OH-[3H]DPAT labeled a single site (15-20 fmol/mg of protein) with high (1 nM) affinity. Competition analysis with carbetapentane defined both high- and low-affinity sites in striatal (35 and 65%, respectively) and nucleus accumbens (59 and 41%, respectively) tissue, yet R(-)-PPAP identified two sites in equal proportion. Carbetapentane and R(-)-PPAP (0.1 microM) displaced approximately 20-50% of total (+/-)-7-OH-[3H]DPAT binding in striatum, nucleus accumbens, and olfactory tubercle in autoradiographic studies, with the nucleus accumbens shell subregion exhibiting the greatest displacement. To determine directly (+)-7-OH-[3H]DPAT binding to sigma receptors, saturation analysis was performed in the cerebellum while masking D3 receptors with 1 microM dopamine. Under these conditions (+)-7-OH-[3H]DPAT labeled sigma receptors with an affinity of 24 nM. These results suggest that (a) (+/-)-7-OH-[3H]DPAT binds D3 receptors with high affinity in rat brain and (b) a significant proportion of (+/-)-7-OH-[3H]DPAT binding consists of sigma 1 sites and the percentages of these sites differ among the subregions of the striatum and nucleus accumbens.

A novel dopamine receptor mRNA transcript has been identified and classified as the D3 receptor subtype. We have examined the binding of the D2/D3-selective compound [125I]-Iodosulpride using unlabeled D3-selective 7-OH-DPAT to determine the distribution of D2 and D3 dopamine receptor subtypes in the rat basal forebrain. Use of [125I]-labeled ligands has significant advantages over [3H]-labeled compounds for autoradiographic studies, especially for evaluating small brain areas containing low receptor densities. [125I]-Iodosulpride identified two sites with high affinity (< 1 nM) in the presence of (-)sulpiride (1 microM; D2+3) or 7-OH-DPAT (10 nM; D3), with a greater density of D2 receptors (twofold) compared to D3 receptors in the striatum. The density of D2 and D3 receptor subtypes displayed a 1:1 ratio in the nucleus accumbens. [125I]-Iodosulpride with 7-OH-DPAT displayed D2 sites, predominately in the striatum. Digital subtraction autoradiography showed the highest levels of D3 binding in the islands of Calleja as well as in the core and shell regions of the nucleus accumbens. In sum, the advantages in using [125I]-Iodosulpride to label the dopamine receptor subtypes are high specific activity, affinity, and lack of quenching in autoradiographic analyses. Moreover, masking D3 receptors with 7-OH-DPAT permitted indirect determination of D3 receptor density and localization using the [125I]-labeled ligand, without the potential confound of 7-OH-DPAT binding to sigma receptors. The colocalization of the D2 dopamine receptors with D3 receptors suggests that unique interactions may exist between the receptor subtypes in the rat basal forebrain region.

The purpose of this study was to determine whether the calcium binding protein calbindin-D28k was present within the cortically projecting basal forebrain neurons of various rodent species not previously examined. Double-label immunocytochemistry was performed using antibodies against calbindin-D28k and choline acetyltransferase (ChAT) to detect the presence of the calcium binding protein within the cholinergic basal forebrain neurons of various species (i.e., humans, rats, mice, gerbils, guinea pigs). Antibodies against calbindin-D28k, ChAT, and glutamic acid decarboxylase (GAD) were also used in combination with a cortically injected retrograde tracer (Fluoro-Gold) to determine whether calbindin-D28k immunoreactive (IR) neurons within the basal forebrain projected to the frontoparietal cortex. The nucleus basalis of rats was examined for the presence of calbindin-D27k IR within the GABAergic basal forebrain neurons. All species examined had cholinergic, GABAergic, and calbindinergic neurons within the basal forebrain; however, only the cholinergic neurons within the human nucleus basalis of Meynert were also immunoreactive for calbindin-D28k. Although all rodent species had both cholinergic and GABAergic basal forebrain neurons that contained the Fluoro-Gold dye, none of the calbindin-D28k IR neurons, detected using monoclonal and polyclonal antibodies, were found to contain the retrograde tracer. These results indicate that the cortically projecting cholinergic and GABAergic basal forebrain neurons within these rodent species do not contain calbindin-D28k. Therefore, age- and disease-related loss of nucleus basalis projection neurons may not be mediated by alterations in calbindin-D28k.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal models for studying the developmental effects of maternal drug abuse are often based on chronic exposure of the pregnant rat. The suitability of animal models, however, has been constrained by the availability of an appropriate route of administration. The commonly employed SC and PO routes of administration fail to mimic the rapidly peaking pharmacokinetic profile observed in humans with licit (e.g., nicotine) and illicit (e.g., cocaine, methamphetamine) drugs abused via inhalation or i.v. injection. The present study provides a method for the routine use of an i.v. administration model in pregnant and/or group-housed rats. Prior to mating, young adult female Sprague-Dawley rats were anesthetized (ketamine/xylazine) for catheterization. A sterile Intracath i.v. catheter (22 ga., Becton/Dickinson) with a Luer-lock injection cap (Medex) was cut to approximately 8 cm and used as a SC dorsally implanted port for chronic i.v. injections. The distal end of the catheter was inserted into the jugular vein and threaded centrally. Catheter patency was maintained by daily flushing with 0.2 ml of heparinized saline. Following 1 week of surgical recovery, the mean (median)number of estrus cycles to impregnation was 2.5(2). The mean (+/- SEM) duration of catheter patency was 36.6 +/- 1.2 days and was in excess of 30 days for all animals (n = 22). Cocaine at a dose of 3 mg/kg (GD8-14 x 1/day, GD15-20 x 2/day) had no significant effect on dam weight gain, gestation length, litter size, sex ratio, or birth weight. In sum, a subcutaneously implanted port provides a procedure for the routine i.v. administration of drugs to pregnant and/or group-housed rats which avoids (a) the use of anesthesia/surgery during pregnancy, (b) the stress (restraint and/or thermal dilation) associated with tail vein injection, (c) the difficulties of mating and single housing associated with tethered i.v. catheters, and (d) in the case of cocaine, precludes the potential confounds of any drug-induced non-i.v. parenteral lesions.

The diverse morphologies of catecholamine axons in the human brain were examined by using tyrosine hydroxylase immunocytochemistry. Human brain tissue was obtained by either rapid autopsy (mean postmortem delay < 1 h) or routine autopsy (mean postmortem delay 5 h). Tissue blocks from the superior frontal cortex (Brodmann area 9), the hippocampal gyrus and the calcarine cortex (Brodmann area 17) were processed for tyrosine hydroxylase immunoreactivity. First, a quantitative method was developed to reliably identify differing morphologies of catecholamine axons in human brain tissue. A total of 625 tyrosine hydroxylase immunoreactive axons were randomly sampled from coded sections and classified into one of six distinct morphological categories. These categories were based upon axonal morphologies which were readily distinguished by trained observers, and moreover, further investigations demonstrated that entire tissue sections could be reliably re-sampled at intervals of up to six months. Second, regional variations in axonal distribution and the effects of increasing postmortem delay in tissue processing on the categories of tyrosine hydroxylase immunoreactive axon morphologies were examined. Postmortem delays of up to 6.5 hours were found to decrease the frequency of fine axons with varicosities (axon type 2) and increase thick-caliber straight axons (axon type 5) in all regions examined. The frequency of other morphological axon types did not change as a function of postmortem delay. In summary, the use of quantitative neuroanatomical measures of the catecholaminergic system in human brain tissue was found to be reliable and valid. It was furthermore demonstrated that postmortem delays affect selected morphological types of catecholamine axons.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the various diverse morphologies of catecholamine axons in the brains of patients with Alzheimer's disease. Alzheimer's disease and aged control brain tissue were obtained by a rapid autopsy protocol (mean postmortem delay < 1 h). Tissue blocks from the superior frontal cortex (Brodmann area 9), the hippocampal gyrus, and the calcarine cortex (Brodmann area 17) were processed for identification of catecholamine axons using tyrosine hydroxylase immunocytochemistry. A total of 1275 tyrosine hydroxylase immunoreactive axons were randomly sampled from coded sections and classified into one of six distinct axon-type categories. The axon classification from patients with Alzheimer's disease significantly differed from those of an age-matched control population in the hippocampus. The Alzheimer's disease brains were decreased in the frequency of very long, thin, tyrosine hydroxylase immunoreactive axons (type 1) and had an increased frequency of shorter, tortuous, axons (type 3). These selective quantitative shifts in hippocampal catecholaminergic axon morphology are consistent with the hypothesis that sympathetic noradrenergic axons invade the hippocampus of patients with Alzheimer's disease. Multivariate modeling of the frequency sampling data found that the axon type classification scheme successfully predicted the presence of Alzheimer's disease. In particular, the use of quantitative neuroanatomical measures of the catecholaminergic system in human brain tissue was found to have errorless predictive ability with respect to late onset (> 75 years) Alzheimer's disease. In summary, the use of quantitative neuroanatomical measures of catecholamine axonal morphologies in Alzheimer's disease brain tissue identified a specific frequency shift which may represent hippocampal sympathetic ingrowth and this unique measure was found to have predictive utility with respect to Alzheimer's disease.

The 3-dimensional (3-D) distribution of cholinergic neurons located throughout the extent of the entire basal forebrain of young (4-5 months old) and aged (24-25 months old) Fischer-344 rats was examined using choline acetyltransferase immunocytochemistry (ChAT-IR). The number and 3-D position of ChAT-IR neurons spanning the basal forebrain were determined using serial sections and analyzed using computerized image analysis. The effects of aging on ChAT-IR neuron number were analyzed as a cohesive unit with respect to the classically-defined magnocellular subregions located within the basal forebrain (i.e., the medial septum, vertical and horizontal limbs of the diagonal band, and the nucleus basalis). Significant effects of age were found on ChAT-IR neuron number but no significant age-related interactions were noted with either the A-P, M-L, or D-V axes. These results suggest that a significant but diffuse age-related loss of ChAT-IR occurs along the entire length of the basal forebrain, and that this loss is not restricted to individual magnocellular nuclei (A-P axis), the M-L axis, or the D-V axis.

Species differences in the distribution of beta-adrenergic receptors in the hippocampal and retrohippocampal regions of rats and guinea pigs were examined using in vitro autoradiographic techniques. beta 1-receptors were found in the hippocampal area CA1 of both species, although guinea pigs had significantly lower receptor densities in comparison to rats. In guinea pigs, beta 2-adrenergic receptors were predominant in hippocampal area CA1. Hippocampal area CA3 had very low levels of beta 1- and beta 2-receptors in both species. The retrohippocampal area was also found to have a distinct topographic distribution of beta-receptors. In rats, the subiculum and parasubiculum (layers II-III) were heavily labeled for beta 1-receptors; in contrast, guinea pigs had few receptors in these regions. beta 2-receptors were particularly prominent in the parasubicular region in rats. The entorhinal cortex laminae was found to contain beta-receptors in both rats and guinea pigs. Immunohistochemical techniques were used to compare the pattern of catecholaminergic innervation with the receptor distribution within each hippocampal subregion. Despite the general lack of beta-receptors in area CA3, abundant catecholamine immunoreactive fibers were observed in CA3 of rat and guinea pig hippocampus. Significant species differences were found in the distribution of hippocampal beta-adrenergic receptor subtypes, and moreover, in both species the distribution of beta-adrenergic receptors did not coincide with the pattern of hippocampal adrenergic innervation.

c-fos is a proto-oncogene that encodes for a nuclear phosphoprotein with DNA binding properties and is presumed to have an important role in the long-term regulation of neuronal function. It is thought to act as a 'third messenger' molecule in signal transduction systems and its expression has been shown to be induced by a variety of exogenous and endogenous stimuli. This study examines the differential expression of the Fos protein in various brain regions after a single electroconvulsive shock (ECS) in 6-, 13-, and 28-month-old B6C3 mice. The animals received an acute electroconvulsive shock (90 V for 0.3 s), without prior anesthesia, through earclip electrodes and exhibited generalized tonic-clonic seizures lasting 20-36 s. Animals were anesthetized and perfused intracardially with 2.5% acrolein, 4% paraformaldehyde at 0.5, 1.0, 2.0 and 4.0 h postshock. The brains were Vibratome-sectioned (30 microns) and examined using a Fos antibody, directed against a conserved region of both mouse and human Fos by standard immunocytochemical methods. Systematic sampling of the total number of Fos immunostained neurons in amygdala, hippocampus and the cerebral cortex showed peak values at the 1-h time point followed by a steady decline thereafter in all age groups. In a second experiment, Fos-like immunoreactivity was compared 1 h after ECS in the hippocampus, amygdala and the cortex in all 3 age groups. There was increased expression of Fos-like immunoreactivity after ECS- compared to non-ECS-treated controls in all age groups.(ABSTRACT TRUNCATED AT 250 WORDS)

This study demonstrated species differences in IGF-1 and IGF-2 receptor binding and localization in the hippocampus of the rat and mouse. Competition binding studies indicated that there were no differences in the relative binding affinities for the type 1 or type 2 receptors between the brains of these animals. These results suggested that the observed species differences were not attributable to alterations in IGF receptor kinetics. Receptor autoradiographic analyses demonstrated that IGF-1 binding differed in both the localization and overall receptor densities observed, with the rat demonstrating more specific localization and greater receptor density in the hippocampus than the mouse. The rat also exhibited a greater density of IGF-2 receptors in the hippocampus than the mouse. Despite differences in IGF receptor populations, both species exhibit similar hippocampal structure and lamination. Therefore, these results demonstrate a disparity in the localization of IGF receptor binding in the rat and mouse, suggesting that IGFs in these species are differentially regulated, with distinct neuromodulatory, neurotrophic, and/or developmental roles in this region of the brain. Previous comparative anatomical studies of the hippocampal formation of rats and mice fail to offer an explanation for the absence or reduction of binding of IGF-1 in the mouse. Although the mouse has a greater cell density in the s. granulosum than the rat, and both species exhibit similar glia and synaptic contact densities in the s. moleculare of the dentate gyrus, the mouse exhibits a complete absence of IGF-1 binding in this region. The lack of anatomical differences in the hippocampal formation of these species suggests that the patterns observed in IGF binding result from alterations in either neurochemical modulation of these neurons or specific neurotrophic requirements of the cells in this region. Differences have been reported on the concentrations and binding of various neurotransmitters in the hippocampus of these species, however these differences do not easily account for the variations observed in IGF binding in this study. IGFs are known to influence acetylcholine neurotransmission in the hippocampus as well as other brain areas in the rat. Recently, a truncated form of IGF-1, in which a tripeptide is cleaved from the N-terminus of the peptide, has been reported in brain. The cleaved tripeptide has been shown to activate glutamate receptors, which may dramatically influence excitatory neurotransmission in this region. Therefore, in addition to the possible neurotrophic actions of the peptide itself, subsequent processing of IGF-1 may be an important aspect of IGF-1 activity in the brain.(ABSTRACT TRUNCATED AT 400 WORDS)

Somatostatin has been reported to decrease with age in many brain regions and these changes generally have been considered to have important implications for the regulation of both neural activity and neuroendocrine regulatory systems. The purpose of this study was to determine whether the age-related changes in somatostatin concentration in cortex and hypothalamus are attributable to alterations in the regulation of somatostatin gene expression. Hypothalamic and cortical tissue were dissected from young (3-4 month), middle-aged (12-14 month), and old (22 month) male Fischer 344 rats. Total RNA was extracted and dilutions blotted to nitrocellulose. Somatostatin cDNA in expression vector pSP65 was used to produce a 32P-labeled antisense probe for hybridization. After washing, blots were autoradiographed and analyzed by densitometry. Dilutions of total RNA were also probed with 32P-labeled oligo d(pT)16 to determine poly A +RNA levels. Data were expressed as relative somatostatin gene expression (somatostatin mRNA/poly A +RNA). Results indicated that in cortex, relative somatostatin gene expression was similar in young, middle-aged, and old animals (0.54 +/- 0.11, 0.60 +/- 0.08, and 0.51 +/- 0.04, respectively). However, somatostatin gene expression in the hypothalamus decreased consistently with age and ratios in old rats were approximately 50% of levels observed in young animals (p less than 0.05). Northern analysis of RNA revealed a single somatostatin transcript of approximately 0.65 kb in all age groups. In situ hybridization analysis of somatostatin mRNA in the hypothalamus indicated that the age-related decrease in somatostatin gene expression is a consequence of decreased expression within specific hypothalamic nuclei rather than a loss of somatostatin-containing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

The long-term consequences of neonatal exposure to triethyl lead, the putative neurotoxic metabolite of the anti-knock gasoline additive tetraethyl lead, were examined with respect to central nervous system (CNS) development. We presently report a series of studies in which exposure of neonatal rats to organic lead produces profound CNS damage in adulthood as indicated by dose-dependent, persistent behavioral hyperreactivity as well as dose-dependent, preferential, and permanent damage to the hippocampus. General morphological parameters of brain development were not altered. Pharmacological probes of neurotransmitter system integrity suggested a functional and dose-dependent relationship between this behavioral hyperreactivity and hippocampal damage via cholinergic, but not dopaminergic, pathways. Furthermore, these alterations were not accompanied by long-term alterations in motor activity and were not attributable to the presence of lead in adult neural tissue. Finally, these behavioral, anatomical, and pharmacological indices of developmental exposure to organic lead were dissociable from any effects of early undernutrition. These data collectively indicate that organolead compounds may pose a potent neurotoxic threat to the developing CNS.

Although alterations in development due to inorganic lead poisoning have been intensely investigated, little is known about the early toxicity of organic lead compounds. Assessment of developmental consequences due to triethyl lead (TEL) intoxication presently included (1) determination of the acute LD50 as 13 +/- 1 mg/kg, and (2) detailed examination of early neurobehavioral sequelae. The offspring of 12 Fischer-344 dams were administered on postpartum day 5 either a sham-injection, 15% ethanol, 3 mg/kg or 6 mg/kg TEL vis SC injection (20 microliters). Small, but significant, weight reductions of 6% and 13% for the 3- and 6- mg/kg TEL-dosed pups, respectively, were observed (days 14-30). Early sensory deficits of TEL pups as indicated by impaired olfactory discrimination on day 7 and decreased incidence of nipple attachment on day 9 were accompanied by the presence of fine whole body tremor (day 10). While these initial effects were transitory in nature, activity evaluations demonstrated persistent hypoactivity in high dose TEL males (days 15, 22, 24, 26, and 29). Passive avoidance acquisition was not affected by TEL treatment (day 18). However, 72 and 144 hr tests of passive avoidance retention (days 21 and 25) suggested alterations in affective behavior, i.e., hypoactivity in high dose TEL males and hyperactivity in low dose TEL females. A reduction in number, but not magnitude, of startle responses also occurred as a function of TEL exposure. The single postnatal day 5 injection of TEL thus produced transitory effects possibly reflecting direct TEL pharmacological activity, as well as apparent long-term effects suggesting potential permanent alterations in behavioral function.

Sympathetic ingrowth is an unusual neural rearrangement in response to damage of the septohippocampal pathway in which peripheral noradrenergic nerves grow into the hippocampal formation. Hippocampal ingrowth has been extensively studied in rats and has been suggested to be regulated by the mossy fibers of the dentate granule cells, hippocampal interneurons, or glial cells. Sympathetic ingrowth was found to occur in both rats and guinea pigs; however, a discrepancy between the species was observed in the topographical distribution of sympathetic ingrowth. Ingrowth fibers were found in the dentate hilus and area CA3 of guinea pigs and rats. However, in the guinea pig fibers extended into area CA1. Quantitative estimates of fiber number confirmed these observations and identified significant differences between the species in the intrahippocampal lamellar distribution of ingrowth fibers. The topographical differences in sympathetic ingrowth could not be explained by differences in the distribution of the mossy fibers (Timms stain), cholinergic septal afferents (anterograde HRP), or in hippocampal interneurons (GAD-immunoreactive neurons). These species differences are challenging to current theories concerning the regulation of sympathetic ingrowth and may provide a useful model for testing further hypotheses about axonal guidance and target selection.