Prenatal cocaine alters dopamine and sigma receptor binding in nucleus accumbens and striatum in dams and adolescent offspring.

Maternal cocaine abuse is a societal problem with serious impact on both mother and child. Few studies exist that study the mother/offspring dyad of neurological effects of maternal cocaine abuse. The present study was designed to study alterations in D2, D3 and sigma receptor density in nucleus accumbens and striatum of dams and male and female offspring following gestational cocaine. Long-Evans female rats were implanted with an intravenous (i.v.) access port prior to breeding and were administered saline or 3.0 mg/kg of cocaine from gestational day (GD) GD8-20 (1 injection/day-GD8-14, 2 injections/day-GD15-20). Offspring were raised by maternal dams and allowed to mature until postnatal days 31-35, at which time dams and offspring were sacrificed for assay of radioligand binding. In dams, decreased D2 (24.6%) and D3 (36.9%) binding was observed in striatum. Female offspring displayed no differences in receptor binding in either region. Male offspring displayed decreased D2 receptor binding (27.1%) in nucleus accumbens and increased D3 (75.2% and 33.5%) and sigma receptor binding (73.4% and 53.1%) in accumbens and striatum, respectively. Collectively, these data clearly demonstrate that male offspring exhibit significant alterations in D2, D3 and sigma receptor binding. These results suggest that dams and offspring display long-lasting alterations (5 weeks) in dopamine receptor binding. These alterations in dopamine and sigma receptor binding in offspring following prenatal cocaine and rearing by maternal dams are sex specific and could have profound effects on the development of behavior.

Endogenous glutamate release from frontal cortex of adult and aged rats.

Glutamate (GLU) is a major excitatory neurotransmitter in the frontal cortex. Alterations in GLU neurotransmission are present in a number of neurodegenerative diseases, however, little is known about the normal aging process of GLU utilizing neurons. GLU release, uptake and content were examined in the frontal cortex of adult (6 months old) and aged (24 months old) male, Fisher 344 rats. These markers were used to assess the functional integrity of intrinsic and extrinsic GLU utilizing pathways innervating the frontal cortex. Basal- and potassium- (56 mM) evoked GLU release from brain slices of aged rats were not significantly different from that of adults. Kainic acid (1.0 mM) failed to significantly augment basal or potassium-stimulated GLU release in the frontal cortex of either aged or adult rats. Uptake of [3H] GLU into brain slices was also unaltered as a function of age. In contrast, GLU content was decreased 17% in the frontal cortex of aged rats when compared to the adults. These results suggest that the functional integrity of GLU utilizing nerve terminals in the frontal cortex is maintained in 24-month-old Fisher 344 rats. The decrease in GLU content may reflect a generalized neuronal loss or a defect in neuronal and/or glial GLU metabolism in the metabolic compartment.

L-type calcium channels in the hippocampus and cerebellum of Alzheimer's disease brain tissue.

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.

Long-term treatment of male F344 rats with deprenyl: assessment of effects on longevity, behavior, and brain function.

L-Deprenyl (selegiline) was chronically administered to male Fischer 344 rats via their drinking water beginning at 54 weeks of age (estimated daily dose: 0.5 mg/kg/day). Beginning at 84 weeks of age, the rats were behaviorally evaluated using a sensorimotor battery, a motor-learning task, and the Morris water maze. At 118 weeks of age, cerebellar noradrenergic function was evaluated in the surviving rats using in vivo electrochemistry. The rats were then sacrificed to measure brain monoamine oxidase activity and perform quantitative autoradiography to evaluate the effect of chronic deprenyl treatment on beta-adrenergic receptors in the cerebellum, alpha 2-adrenergic receptors several brain regions, and D1 and D2 dopamine receptors in the striatum. Deprenyl treatment reduced brain monoamine oxidase B activity by 85%, but had no effect on brain monoamine oxidase A. A clear effect of chronic deprenyl treatment upon longevity was not observed. Several measures of CNS function were altered in the deprenyl-treated animals: 1) spatial learning in the Morris water maze was improved; 2) electrochemical signals recorded following local application of NE were reduced, and the responsiveness to the reuptake blocker nomifensine was enhanced, in the cerebellum; 3) beta-adrenergic receptor binding affinity was increased in the cerebellum; 4) alpha 2-adrenergic receptor density was increased in the inferior colliculus; and 5) striatal D1 dopamine receptor density was reduced but binding affinity was enhanced. In contrast, chronic deprenyl treatment did not cause changes in: 1) sensorimotor function, as evaluated by balance beam, inclined screen, or wire hang tasks; 2) motor learning; 3) alpha 2-adrenergic receptor density in any region examined except for the inferior colliculus, or binding affinity in any region examined; or 4) striatal D2 dopamine receptor number or affinity. Thus, long-term oral administration of deprenyl extended the functional life span of rats with respect to cognitive, but not motor, performance.

Basic fibroblast growth factor stimulates the growth and inhibits casein accumulation in mouse mammary epithelial cells in vitro.

The effects of basic fibroblast growth factor (bFGF) on the growth and differentiation of mouse mammary epithelial cells in serum-free collagen gel culture were examined. Epithelial cells obtained from virgin or midpregnant mice grew when bFGF was added to medium containing either insulin at a concentration greater than or equal to 1 microgram/ml or somatomedin-C (Sm-C) at 150 ng/ml. This growth-promoting effect is of the same magnitude as, and additive with, the growth-promoting effect of epidermal growth factor (EGF) or mammogenic hormones. The sensitivity of the cells to EGF or mammogenic hormones was not altered by exposure to bFGF. The progeny cells resulting from growth stimulation by bFGF are capable of accumulating casein upon subsequent stimulation by prolactin (PRL), but accumulate less casein than cells grown in response to EGF. bFGF also appears to reduce casein accumulation if it is added to the cultures at the same time as PRL.

Selective opioid agonist and antagonist competition for [3H]-naloxone binding in amphibian spinal cord.

Opioids elicit antinociception in mammals through three distinct types of receptors designated as mu, kappa and delta. However, it is not clear what type of opioid receptor mediates antinociception in non-mammalian vertebrates. Radioligand binding techniques were employed to characterize the site(s) of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid antagonist that has not been characterized in Rana pipiens. Using the non-selective opioid antagonist, [3H]-naloxone, opioid binding sites were characterized in amphibian spinal cord. Competitive binding assays were done using selective opioid agonists and highly-selective opioid antagonists. Naloxone bound to a single-site with an affinity of 11.3 nM and 18.7 nM for kinetic and saturation studies, respectively. A B(max) value of 2725 fmol/mg protein in spinal cord was observed. The competition constants (K(i)) of unlabeled mu, kappa and delta ranged from 2.58 nM to 84 microM. The highly-selective opioid antagonists yielded similar K(i) values ranging from 5.37 to 31.1 nM. These studies are the first to examine opioid binding in amphibian spinal cord. In conjunction with previous behavioral data, these results suggest that non-mammalian vertebrates express a unique opioid receptor which mediates the action of selective mu, kappa and delta opioid agonists.

Excitatory amino acid involvement in retinal degeneration.

Amino acid analysis using high-performance liquid chromatography demonstrated high levels of the excitatory amino acids, aspartate and glutamate, in the retinas of congenitally blind chicks at the time of photoreceptor degeneration. Concentrations of aspartate were about 2 times higher in blind chicks than in retinas of age-matched sighted chicks that were carriers for the genetic defect. Glutamate levels were similar in blind chicks and carriers at 1 day of age, but doubled and tripled sighted chick values at 1 week and 2 weeks of age in blind chick retinas. Light microscopic immunocytochemistry using antibodies that recognize aspartate and glutamate revealed increased levels of these two amino acids specifically in the photoreceptor layer of blind chicks. This report is the first to demonstrate high endogenous levels of excitatory amino acids associated with a hereditary degeneration of photoreceptor cells.

Dopamine D3 receptor density elevation in aged Fischer-344 x Brown-Norway (F1) rats.

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.

Selective opioid receptor agonist and antagonist displacement of [3H]naloxone binding in amphibian brain.

Opioid receptor ligands have been shown to elicit antinociception in mammals through three distinct types of receptors designated as mu, delta and kappa. These opioid receptors have been characterized and cloned in several mammalian species. Radioligand binding techniques were employed to characterize the sites of opioid action in the amphibian, Rana pipiens. Naloxone is a general opioid receptor antagonist which has not been characterized in R. pipiens. Kinetic analyses of [3H]naloxone in the amphibian yielded a K(D) of 6.84 nM while the experimentally derived K(D) value from saturation experiments was found to be 7.11 nM. Density data were also determined from saturation analyses which yielded a B(max) of 2170 fmol/mg. Additionally, K(i) values were calculated in competition studies for various unlabelled mu-, delta- and kappa-opioid receptor ligands to isolate their site of action. Highly selective antagonists for mu-, delta- and kappa-opioid receptors yielded nearly identical K(i) values against [3H]naloxone.

Pharmacological characterization of [3H]idazoxan, [3H]RX821002 and p-[125I]iodoclonidine binding to alpha 2-adrenoceptors in rat cerebral cortical membranes.

Binding characteristics of alpha 2-adrenoceptors in rat cerebral cortical membranes were compared using the antagonist radioligands [3H]idazoxan, [3H]2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline ([3H]RX821002), and the partial agonist radioligand [125I]2-[2,6-(dichloro-4-iodophenyl)imino]imidazoline ([125I]iodoclonidine). With [3H]RX821002 and alpha 2-adrenoceptor subtype-selective competitors, both alpha 2A/D- and alpha 2C-adrenoceptor subtypes were detected, suggesting rat cortical membranes contain approximately 90% alpha 2A/D-adrenoceptor subtype and 10% alpha 2C-adrenoceptor subtype. Only alpha 2A/D-adrenoceptors were detected with [3H]idazoxan and [125I]iodoclonidine. All three radioligands bound to a single high affinity site (Kd = 0.3-1.6 nM). However, the densities of sites labeled by [3H]idazoxan and [125I]iodoclonidine were 50% greater than the density labeled by [3H]RX821002, likely representing non-adrenoceptor binding sites. The density of [125I]iodoclonidine binding sites in glycylglycine buffer was similar to that labeled by [3H]RX821002. These results suggest that: (1) alpha 2A/D-adrenoceptors are the predominant subtype in rat cerebral cortex, (2) demonstrate that the small number of alpha 2C-adrenoceptors in this tissue can be detected using prazosin to displace [3H]RX821002 binding, and (3) non-adrenoceptor binding with [125I]iodoclonidine can be minimized with the use of glycylglycine buffer.

Kainic acid-induced seizures in aged rats: neurochemical correlates.

This study was conducted to assess the functional integrity of the kainate receptor-mediated seizure response in aged rats. Kainic acid was administered systemically to aged female Long-Evans (LE) rats and aged male F344 rats and the proconvulsant actions of kainic acid was compared to adult controls. The effects of kainic acid on brain regional content of monoamines and amino acids was also determined in the aged female LE and adult control rats. The latency to full clonic-tonic seizures was significantly reduced in aged female LE rats, and the number of seizures was significantly increased above that of the controls. There was increased mortality and a reduction in the latency to exhibit wet dog shakes in the aged F344 rats. Studies were also conducted to evaluate the role of ovarian hormones, route of administration, and dose of kainic acid in mediating the enhanced proconvulsant actions of kainic acid in aged rats. The neurochemical studies suggested that kainic acid significantly enhanced the release of ASP, GLU, and norepinephrine (NE) in the aged rats exhibiting clonic-tonic seizures. The adult rats given the same dose of kainic acid (15 mg/kg, IP) did not exhibit any significant change in brain content of monoamines or amino acids except for a reduction in mediobasal hypothalamic NE. An in vitro study was also conducted using brain slices from adult and aged F344 and it was found that aged rats released significantly more ASP than adults in response to kainic acid. These neurochemical findings were discussed in relation to previous studies of age-related alterations in excitatory amino acids (EAAs) and the role of EAA and NE in modulating limbic seizures. This study has clearly demonstrated that aged rats may be more susceptible to the excitotoxic action of EEAs acting through kainetic receptors.

Characterization of [3H]-diprenorphine binding in Rana pipiens: observations of filter binding enhanced by naltrexone.

Initial studies were undertaken to examine the properties of [3H]-diprenorphine binding to Rana pipiens whole brain tissue using naltrexone for the definition of nonspecific binding. Saturation analysis demonstrated the binding of [3H]-diprenorphine to be saturable with a K(D) value of 0.65 nM and a Bmax value of 287.7 fmol/mg protein. Unlabeled diprenorphine dose-dependently displaced [3H]-diprenorphine from a single noninteractive site in competition studies which yielded a Ki of 0.22 nM. However, control studies in the absence of tissue revealed significant binding of [3H]-diprenorphine to the filter alone. Interestingly, [3H]-diprenorphine in the presence of unlabeled naltrexone as well as with unlabeled naloxone showed significantly greater binding to the filter than did [3H]-diprenorphine alone. Given this observation of increased nonspecific binding, an artificially low Bmax value would be expected. It is our hypothesis that the unlabeled nonspecific drug forms a complex with [3H]-diprenorphine preventing it from being effectively washed through the filter or the unlabeled drug itself is blocking the flow of [3H]-diprenorphine through the filter. The latter is unlikely however as other binding studies done in our lab using the radioligand [3H]-naloxone with unlabeled naltrexone do not show significant binding to the filter.

[3H](+)-7-OH-DPAT and [3H]pramipexole binding in the striatum and nucleus accumbens of Sprague-Dawley and Fischer-344 rats.

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.

A pharmacological analysis of food intake regulation in rats treated neonatally with monosodium L-glutamate (MSG).

Studies were conducted to examine deficits in food intake regulation in MSG-treated rats that result from known or suspected damage to neurotransmitter systems involved in feeding. Male rats were injected with either MSG (4 mg/g) or sodium chloride on postnatal days 2 and 4 (MSG-Lo) or postnatal days 2, 4, 6 and 8 (MSG-Hi). As adults, MSG-treated and control rats (n = 12/group) were examined for deficits in pharmacologically elicited feeding and other measures of food intake regulation. A second group of MSG-treated (n = 9/group) and control rats (n = 12) were used to measure basal blood pressure and nociceptive reactivity in adulthood. Organ weights, body weight and neuropeptide Y (NPY) content in brain regions were determined at the end of the study. MSG-Hi rats consumed significantly less food than controls during the dark part of the light cycle. Both MSG-Hi and MSG-Lo groups ate significantly less food than controls after a 48-hour fast. MSG-Hi and MSG-Lo rats consumed significantly less food than controls in response to 1.0 mg/kg morphine. MSG-Hi rats consumed significantly less food than controls during the dark phase and significantly more food than controls during the light phase in response to naloxone (1.0 mg/kg). MSG-Lo ate significantly more than controls in response to 0.1 mg/kg guanfacine. MSG-Hi and MSG-Lo showed a significant attenuation in diazepam-stimulated feeding when compared to controls. Blood pressure was significantly lower in both MSG-Hi and MSG-Lo rats compared to controls. Tail flick latencies were not altered by MSG-treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Age- and dose-dependent effects of neonatal monosodium glutamate (MSG) administration to female rats.

The age- and dose-dependent effects of neonatal MSG were evaluated in pre- and postpubertal female rats. The neurotoxic action of MSG was assessed by examining monoamine content in microdissected regions of the mediobasal hypothalamus. MSG was administered at a dose of 4 mg/g on postnatal days 2 and 4 (MSG-Lo) or on postnatal days 2, 4, 6 and 8 (MSG-Hi). MSG-Hi treatment significantly reduced dopamine (DA) content in the arcuate nucleus (ANH) and lateral median eminence (LME) on postnatal day 21 when compared to NaCl-injected controls. DA content relative to controls was not altered in the ANH or LME postnatal or postnatal day 60 in MSG-Hi, however, norepinephrine (NE) was significantly (p less than 0.05) decreased on both postnatal day 21 and 60 in the LME. MSG-Lo treatment significantly (p less than 0.05) reduced ANH NE content on postnatal day 60 compared to controls. Both MSG-Hi and MSG-Lo treatment increased 5-hydroxyindoleacetic acid content in the preoptic area (POA) on postnatal day 60 relative to the controls. Developmental changes independent of MSG treatment were noted in the hypothalamus. DA and 3,4-dihydroxyphenylacetic acid (DOPAC) content in the POA were 2-3-fold higher on postnatal day 21 compared to postnatal day 60. In contrast, DA content increased with age in the ANH, LME and medial ME. NE content in the ANH increased as a function of age in controls, but not in MSG-treated rats. The effects of MSG treatment on the postnatal development and maturation of neurons in the mediobasal hypothalamus were discussed in relation to the direct neurotoxicity of MSG.

Dose-response cocaine pharmacokinetics and metabolite profile following intravenous administration and arterial sampling in unanesthetized, freely moving male rats.

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.

Central and peripheral actions of alpha 2-adrenergic agonists on renal function in Long-Evans and Brattleboro rats.

The effects of 3 alpha 2-adrenergic receptor agonists on renal function in vasopressin (AVP)-deficient Brattleboro (DI) rats were evaluated. The aim of this study was to determine the relative contribution of central versus peripheral alpha 2-adrenoceptors in mediating diuresis and natriuresis, as well as the role of alpha 2-adrenoceptors in antagonizing the actions of AVP. In addition to the studies of renal function, the effects of AVP deficiency on renal alpha 2-adrenoceptor affinity and number was evalauted along with determination of peripheral catecholamine stores. The centrally acting alpha 2-adrenergic agonists guanabenz and guanfacine significantly increased urine output and sodium excretion in Long-Evans (LE) rats. Guanabenz and guanfacine increased urine output in DI rats but failed to increase sodium excretion. The polar alpha 2-adrenergic agonist, ST-91, increased sodium excretion in both LE and DI rats, however, at a dose of 1.0 mg/kg urine output was significantly decreased in DI rats. The 3 alpha 2-adrenergic agonists increased potassium excretion in LE rats, but at the 1.0-mg/kg dose of guanabenz and ST-91, potassium excretion was significantly inhibited in DI rats. Renal alpha 2-adrenergic receptors and norepinephrine stores were not altered in DI rats. Adrenal NE stores were significantly elevated in DI rats relative to LE rats. The results of this study suggest that in the absence of AVP, centrally acting alpha 2-adrenergic agonists have limited natriuretic action, although peripheral activation of alpha 2-adrenoceptors is sufficient to elicit natriuresis irrespective of the presence of AVP. The chronic deficiency of AVP does not alter renal alpha 2-adrenergic receptor number, but the natriuretic and kaliuretic actions of alpha 2-adrenergic agonists are altered in DI rats.