A TaqMan-based multiplex real-time PCR assay for the simultaneous detection of Wuchereria bancrofti and Brugia malayi.

With the Global Program for the Elimination of Lymphatic Filariasis continuing to make strides towards disease eradication, many locations endemic for the causative parasites of lymphatic filariasis are realizing a substantial decrease in levels of infection and rates of disease transmission. However, with measures of disease continuing to decline, the need for time-saving and economical molecular diagnostic assays capable of detecting low levels of parasite presence is increasing. This need is greatest in locations co-endemic for both Wuchereria bancrofti and Brugia parasites because testing for both causative agents individually results in significant increases in labor and reagent costs. Here we describe a multiplex, TaqMan-based, real-time PCR assay capable of simultaneously detecting W. bancrofti and Brugia malayi DNA extracted from human bloodspots or vector mosquito pools. With comparable sensitivity to established singleplex assays, this assay provides significant cost and labor savings for disease monitoring efforts in co-endemic locations.

Autoradiographic evidence that prolonged withdrawal from intermittent cocaine reduces mu-opioid receptor expression in limbic regions of the rat brain.

Numerous reports support evidence that dopaminergic mesolimbic pathways interact with opioid systems to influence the reinforcing properties of cocaine. Withdrawal from chronic administration of cocaine in rats causes an upregulation of mesocorticolimbic mu-opioid receptors during early stages, but information about prolonged cocaine abstinence is lacking. We addressed this issue by treating rats with cocaine or saline (control) intermittently (1 mg/kg, i.v., every 12 min for 2 h daily) for 10 days followed by a 10- or 20-day withdrawal period. The animals were then decapitated and the brains removed for quantitative in vitro autoradiographic analysis of 14 brain regions with (125)I-DAMGO. A separate group of animals received two consecutive cycles of the 10-day cocaine/10-day withdrawal regimen. Only the group that participated in the two consecutive cycles showed a significant effect of treatment: downregulation of mu-opiate receptors in limbic cortical layer 3 (17% lower than saline-treated controls, P = 0.03), the core of the nucleus accumbens (16% decrease, P = 0.05), and the nucleus of the diagonal band (18% decrease, P = 0.05). The mu-receptor may manifest, as do other neural markers (e.g., dopamine transporter, dopamine efflux), a biphasic temporal pattern with upregulation during early phases of cocaine withdrawal but a downregulation at later times.

Regional binding to corticotropin releasing factor receptors in brain of rats exposed to chronic cocaine and cocaine withdrawal.

Cocaine, as does exposure to other physiological stressors, releases brain corticotropin releasing factor (CRF), and this release habituates during the course of repeated cocaine administration in animals. Due to the many signs of anxiety and responses to stress that are produced by cocaine withdrawal in humans, the present study was designed to assess the effects of chronic cocaine and its withdrawal on regional 125I-Tyr-oCRF binding to the CRF1 receptor in brains of male Lewis rats. Cocaine or saline was intravenously infused for 10 days in a regimen that resembled a self-administration paradigm (1 mg/kg every 12 min for 2 h each day). Tissues were harvested either 15 min after or 10 days after the last cocaine infusion, and the brains were sectioned and prepared for CRF1 receptor autoradiography. Compared with findings in saline controls, there was a 31% lower level of CRF binding sites in the basolateral nucleus of the amygdala immediately after the last cocaine infusion, but not 10 days later. Neuroendocrine and non-neuroendocrine mechanisms associated with CRF1 receptors do not appear to contribute to long-term withdrawal effects.

Neurochemistry of cocaine withdrawal.

Neural circuits comprised of dopamine-containing and dopamine-receptive neurons are altered functionally after the repeated intermittent administration of cocaine and its withdrawal. The characterization of these neuroadaptive changes at various times after repeated exposure to cocaine is important because they may relate to addictive or withdrawal states associated with cocaine abuse. They also may suggest targets leading to the development of medications to treat one or more aspects of cocaine dependence.

Neurochemical changes in cocaine withdrawal.

Cocaine withdrawal in animals causes a transient increase followed by a long-lasting decrease in mesolimbic dopamine transporters, dopamine efflux and the number of dopamine cells firing spontaneously. Other changes in the nucleus accumbens and frontal cortex also suggest alterations in dopamine-receptive neurones and circuits. In humans, brain imaging has provided evidence for some similar, long-lasting changes in dopaminergic neurones and innervated areas. These results suggest a protracted biochemical abstinence syndrome for cocaine. In this review, Michael Kuhar and Nancy Pilotte focus on biochemical changes that occur following withdrawal from repeated cocaine administration. A key question for treatment is whether (some of) these persistent changes underlie withdrawal symptomatology such as anhedonia and relapse.

Cocaine withdrawal reduces dopamine transporter binding in the shell of the nucleus accumbens.

We have previously shown that withdrawal from repeated, intermittent infusions of cocaine in Lewis rats results in a long-lasting reduction in dopamine transporter levels in the nucleus accumbens. The reduction is dose-dependent, requires multiple injections as well as about a 10-day withdrawal period. In this investigation, we show that the decrease (34%) occurs in the shell rather than in the core of the nucleus accumbens, and that a second cycle of cocaine administration and withdrawal has no additional effect. Also, there were no changes in transporter binding in the caudate putamen, the olfactory tubercle or the ventral tegmental area. These results indicate that the limbic portions of the nucleus accumbens are involved in neurochemical adaptations during withdrawal from cocaine.

Withdrawal of repeated intravenous infusions of cocaine persistently reduces binding to dopamine transporters in the nucleus accumbens of Lewis rats.

Male, Lewis rats were administered cocaine or saline i.v. in an intermittent fashion for 5, 10 or 20 days and killed at various times afterwards. Dopamine transporter binding was then measured in dorsal striatum and nucleus accumbens. Transporter binding was not changed in dorsal striatum under any conditions tested. In the nucleus accumbens, however, binding was decreased in animals given cocaine (10 mg/kg total) for 10 days and withdrawn for 10, 30 or 60 days, but not in animals withdrawn for 0, 1, 3 and 6 days. There were no changes in animals given cocaine for 5 days and withdrawn for 10, or in animals given drug for 20 days and withdrawn for 1 day. Animals given only 1/10 of the cocaine dose had no changes in nucleus accumbens after 10 days of administration and 10 days of withdrawal. Scatchard analysis in control animals indicated that there were significant differences in both Kd and Bmax when comparing nucleus accumbens with dorsal striatum. Within the nucleus accumbens, decreases in binding after a cessation of cocaine administration were associated with a change in Bmax and not in Kd. These data indicate that long-lasting changes in the mesolimbic dopaminergic system can occur during the withdrawal period, and may contribute to behavioral effects during this period.

Reduction in dopamine transporter mRNA after cessation of repeated cocaine administration.

Male, Lewis rats were treated intravenously for 2 weeks with saline or cocaine using a dose and injection schedule that is similar to the doses and patterns of cocaine intake in self-administration studies. Ten days after cessation of treatment, dopamine transporter binding levels were decreased in the nucleus accumbens but not in the striatum. In situ hybridization studies revealed decreases in dopamine transporter mRNA that were restricted to cells of the interfascicular and caudal linear nuclei; these dopaminergic cell groups, found in the ventral tegmentum, project to the nucleus accumbens and other limbic areas. Other dopaminergic cell groups in midbrain which project mainly to other areas did not show a decrease in mRNA. These results indicate that gene expression can be altered many days after withdrawal from cocaine, and provide an example of transporter regulation by a change in gene expression.

Withdrawal of repeated cocaine decreases autoradiographic [3H]mazindol-labelling of dopamine transporter in rat nucleus accumbens.

The in vitro autoradiographic distribution of desipramine-insensitive specific [3H]mazindol binding sites (labelling the dopamine transporter) was determined in brain sections from rats receiving repeated i.v. infusions of saline or cocaine (1 mg/kg, every 12 min for 2 h/day), for 10 days. Brains were removed either within 15 min of or 10 days after the last treatment. A marked dorsal-to-ventral gradient in [3H]mazindol binding appeared in the striatum with the dorsal caudate putamen showing the greatest binding and the medial shell of the nucleus accumbens the least. Cocaine-associated changes in [3H]mazindol-labelled dopamine uptake sites occurred only in the nucleus accumbens (57 and 66% decrease in the lateral core and medial shell, respectively), of animals 10 days after the last treatment. Down-regulation of the dopamine transporter in the nucleus accumbens by withdrawal of chronic cocaine may be one of the mechanisms involved in cocaine's long-term abstinence effects.

Chronic cocaine administration and withdrawal of cocaine modify neurotensin binding in rat brain.

Neurotensin (NT) is a peptide colocalized with dopamine (DA) within some mesocorticolimbic DA neurons that are affected by cocaine. We assessed whether chronic treatment with cocaine and withdrawal from cocaine would alter NT binding within these and other areas in the brain. Rats were given infusions repeatedly of isotonic saline or cocaine (1 mg/kg i.v. every 12 min for 2 hr over 10 days) and then were killed within 15 min of the last treatment session ("cocaine" or "saline") or 10 days later ("withdrawal"). Brains were processed for NT receptor autoradiography. Cocaine affected NT binding in the mesocortical regions differently from other areas. Within the mesocorticolimbic system, NT binding in the parabrachial pigmented nucleus of the ventral tegmental area (VTA) was 67% lower in cocaine-treated rats killed immediately after or 10 days after their final infusion than in rats given saline. In contrast to the perikaryal region, significantly more NT binding occurred postsynaptically in the terminal areas of the VTA (prefrontal cortex [PFC] and substantia nigra, pars compacta) 10 days after withdrawal of cocaine than in the saline controls. NT binding in the nucleus accumbens was unaffected by cocaine or its withdrawal. Cocaine also decreased NT binding in non-mesocorticolimbic areas, including the dorsal hypothalamic area and the zona incerta, but binding returned toward control levels 10 days after withdrawal from cocaine. These data suggest that in central areas poor in DA uptake sites such as the PFC, NT may be a critical element in the inactivation of DA. Chronic cocaine treatment and its withdrawal appear to uncouple the normal NT-DA interaction at both the cell bodies and terminals.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple, but not acute, infusions of cocaine alter the release of prolactin in male rats.

Hypothalamic dopamine tonically inhibits the release of prolactin (PRL) from the anterior pituitary gland. Cocaine, in turn, alters dopaminergic transmission. We compared the effects of acute and repeated injections of cocaine on the release of PRL in male rats to assess whether cocaine could affect dopaminergically mediated hormonal responses. We found that the concentration of PRL in plasma was not affected by single i.v. injections of 1, 3 or 10 mg/kg of cocaine. However, in rats infused repeatedly with 1 mg/kg of cocaine for 5 s every 12 min for 2 h over 10 days, the pre-infusion concentrations of PRL increased in a time-dependent manner whereas cocaine uniformly decreased post-infusion levels of PRL. Repeated administration of cocaine may produce long-term changes in either the tuberoinfundibular dopaminergic neurons or the adenohypophysial dopamine D2-receptors, or both. Changes in the peripheral concentration of PRL after multiple injections of cocaine and during cocaine withdrawal may reflect dopaminergic activity in the hypothalamus. In contrast, single injections of cocaine increased adrenocorticotropin (ACTH) in a dose-dependent manner whereas repeated infusions did not increase peripheral concentrations of ACTH or corticosterone. It seems that repeated injections of cocaine do not result in persistent changes in the hypothalamo-pituitary-adrenal axis.

Ovariectomy permits progesterone to increase the binding of [3H]spiperone to the anterior pituitary gland in estrogen-primed rats.

The binding of the dopamine (DA) D2 antagonist spiperone to DA receptors in the anterior pituitary gland was evaluated in intact and ovariectomized (OVX) estrogen-primed rats in which circulating levels of progesterone (P) were varied. Nembutal (PB; 35 mg/kg, ip) was administered at 1130 h to intact proestrous animals to prevent the LH-stimulated release of ovarian P. Two hours later some of these intact rats were QVX to remove the endogenous ovarian steroids. Both intact and OVX rats were given exogenous P. All rats were killed on the following morning, and adenohypophysial binding of [3H]spiperone was evaluated at a saturating concentration. The binding in intact PB-blocked rats that received P was only 52% of that in PB-blocked rats that were OVX and received P. The binding of [3H]spiperone in PB-treated rats that were OVX and given P did not differ statistically from that in normal estrous rats. A parallel experiment was conducted on other rats that had been OVX 7 days before the sc implantation (on day 0) of Silastic capsules containing estradiol (E2). Two days later (day 2), some of these rats were injected with Nembutal at 1130 h. Two hours later, the E2 capsules of some rats were removed to simulate ovariectomy; Silastic capsules containing P were implanted into both E2-bearing and non-E2-bearing rats. All rats were killed the following morning (day 3), and the density of adenohypophysial DA receptors was assessed. Binding densities were greatest in rats bearing both E2 and P capsules and in rats from which E2 capsules were removed and replaced with P capsules. We conclude that treatment of OVX rats with the sequential combination of E2 and P increases the density of DA receptors in the anterior pituitary gland. However, in intact rats an additional ovarian product prevents the P-induced increase in the density of adenohypophysial DA receptors.

The effects of 9-ene-tetrahydrocannabinol on hormone release and immune function.

We investigated effects of 9-ene-tetrahydrocannabinol (THC) on endocrine and immunological function. Seventeen male volunteers entered into a double blind, randomized study to receive oral THC (10 mg t.i.d. for 3 days and on the morning of the fourth day) or placebo, after at least 2 weeks of abstinence. Plasma prolactin, ACTH, cortisol, luteinizing hormone and testosterone were not altered during or after THC, compared with baseline concentrations. Tests of lymphocyte function showed no differences compared to baseline between THC and placebo groups. As the relatively low dosing regimen of THC (10 mg t.i.d.) resulted in no alterations, another group of 6 men were administered higher doses of THC by inhalation (18 mg/marijuana cigarette) following the same dosing regimen. No endocrine or immunological alterations were observed. When the subjects were grouped according to their history of THC use prior to admission, heavy THC users had lower prolactin concentrations than light users. No differences were observed in concentrations of other hormones or in tests of immune function.

Gender-specific action of thyrotropin-releasing hormone in the mammalian spinal cord.

Thyrotropin-releasing hormone (TRH) potentiated the monosynaptic reflex in isolated spinal cords obtained from 7- to 9-day-old rats. A concentration-dependent increase in the monosynaptic reflex was observed in spinal cords obtained from male but not from female or castrated male rats. In contrast, the magnitude of potentiation in cords from ovariectomized control female rats and in ovariectomized female rats treated with testosterone approached that seen in intact males. The results provide evidence that gender plays a prominent role in the variability of response both of humans with amyotrophic lateral sclerosis and of animal tissues to TRH. Furthermore, exposure to androgen during the neonatal period may determine the responsiveness of motoneurons to TRH. Thus the use of TRH in the treatment of amyotrophic lateral sclerosis may be more effective in males than in females.

Experimental spinal cord injury: effects of trauma or ischemia on TRH and muscarinic receptors.

Traumatic spinal cord injury in rats and ischemic spinal cord injury in rabbits were associated with time-dependent, localized decreases in thyrotropin-releasing hormone (TRH) and muscarinic receptor binding. Changes were not evident in the first 24 to 48 hours, consistent with the hypothesis that secondary alterations in spinal cord may occur relatively late after injury. TRH receptor binding, but not muscarinic receptor binding, recovered by 3 weeks following trauma. Since TRH and acetylcholine may serve as excitatory neurotransmitters within the spinal cord, such changes could contribute to the functional neurologic impairment that follows injury.

Ovarian steroids modulate the release of dopamine into hypophysial portal blood and the density of anterior pituitary [3H]spiperone-binding sites in ovariectomized rats.

The concentrations of dopamine (DA) in pituitary stalk plasma and the number of DA D-2 receptor sites in the anterior pituitary gland were evaluated in ovariectomized (OVX) rats treated for 48-78 h with estradiol (E2) or vehicle and for 2-30 h with progesterone (P) or vehicle. Only the combined administration of E2 for 72-78 h and P for 24-30 h altered the release of DA into pituitary stalk blood and the number of dopaminergic binding sites in the anterior pituitary gland. The DA concentrations in pituitary stalk plasma 72 h after initiation of treatment were 1.9 +/- 0.3 (mean +/- SEM), 2.6 +/- 0.2, and 2.4 +/- 0.3 ng/ml in OVX rats treated with vehicle, E2, and P alone, respectively, and were increased significantly to 4.3 +/- 0.4 ng/ml in E2- and P-treated rats. The binding of [3H] spiperone to DA D-2 receptors was measured by a single point assay in homogenates of the individual anterior pituitary glands obtained from the same rats. Anterior pituitary tissue was incubated with 0.4 nM [3H]spiperone in the presence or absence of 10(-6) M (+)butaclamol. Binding was similar in all animals except those treated with E2 and P, in which a significant increase occurred. Three days after initiation of treatment, binding was equivalent to 49.8 +/- 5.8, 51.3 +/- 5.1, and 55.4 +/- 4.9 fmol/mg protein in OVX rats treated with vehicle, E2, and P, respectively. In contrast, a significant increase in binding to 83.7 +/- 4.6 fmol/mg protein was observed in rats given E2 and P. No differences in either parameter were apparent 48 h after starting the steroid/vehicle treatment, nor were there any differences between morning and afternoon values in any single group. Scatchard analyses were made on the binding kinetics of DA receptors in anterior pituitary glands obtained from additional rats, and 1.5- to 2-fold increases in the density of sites were observed in animals that had received both E2 and P, while the apparent affinity of the receptors was unchanged. We conclude that treatment with the combination of E2 and P is required to increase the release of DA into hypophysial portal blood and that it also increases the density of DA receptors within the anterior pituitary gland.

Characterization and autoradiographic localization of TRH receptors in sections of rat brain.

Receptors for thyrotropin-releasing hormone (TRH) in rat brain have been localized autoradiographically by exposing tritium-sensitive film to sections labeled with [3H]3-Me-His2-TRH. Greatest grain density was found over certain nuclei of the amygdala, with considerable density over several other forebrain areas. Properties of TRH receptor binding in frozen sections closely resembled those of receptors in fresh membrane fragments.