High performance liquid chromatography combined with either single quad or triple quad mass spectral detectors (LC/MS) was used to measure the brain distribution of receptor occupancy tracers targeting dopamine D2, serotonin 5-HT2A and neurokinin NK-1 receptors using the ligands raclopride, MDL-100907 and GR205171, respectively. All three non-radiolabeled tracer molecules were easily detectable in discrete rat brain areas after intravenous doses of 3, 3 and 30 μg/kg, respectively. These levels showed a differential brain distribution caused by differences in receptor density, as demonstrated by the observation that pretreatment with compounds that occupy these receptors reduced this differential distribution in a dose-dependent manner. Intravenous, subcutaneous and oral dose–occupancy curves were generated for haloperidol at the dopamine D2 receptor as were oral curves for the antipsychotic drugs olanzapine and clozapine. In vivo dose–occupancy curves were also generated for orally administered clozapine, olanzapine and haloperidol at the cortical 5-HT2A binding site. In vivo occupancy at the striatal neurokinin NK-1 binding site by various doses of orally administered MK-869 was also measured. Our results demonstrate the utility of LC/MS to quantify tracer distribution in preclinical brain receptor occupancy studies.

BACKGROUND:The binding of [3H]DAMGO to mu-opioid sites was measured in the CNS of selectively bred high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) rats to test the hypothesis that high alcohol preference is associated with higher densities of mu-opioid receptors.METHODS:Adult, alcohol-naïve male HAD and LAD rats from replicate line 1 were decapitated and their brains frozen in isopentane. Brain sections were incubated with 5 nM [3H]DAMGO, and nonspecific binding was determined in the presence of unlabeled DAMGO. Films were exposed for 60 days, then analyzed using quantitative autoradiography.RESULTS:The densities of [3H]DAMGO binding sites were measured within subregions of neocortex, limbic system, basal ganglia, diencephalon, and brainstem. LAD rats had significantly higher [3H]DAMGO binding (10-30%) than HAD rats within the anterior dorsal hippocampus (CA2), posterior hippocampus (dorsal CA1, and ventral CA1, CA3, and dentate gyrus), thalamus (medial dorsal, lateral, medial dorsal, central, ventral lateral, ventral medial, and ventral medial geniculate nuclei), habenula, and amygdala. No significant interline differences were found in the prefrontal, cingulate, frontal, parietal, temporal, occipital or entorhinal cortices, olfactory tubercle, nucleus accumbens, lateral septum, ventral tegmental area, hypothalamus, caudate-putamen, substantia nigra, claustrum, central gray, or superior colliculus.CONCLUSIONS:The present findings with the HAD and LAD lines do not support the hypothesis that high alcohol preference is associated with higher densities of CNS mu-opioid receptors. Instead, the present results, in combination with previously published findings, suggest that the mu-opioid system may play a complex role in regulating high-alcohol-drinking behavior.

Parkinson's disease is a chronic neurodegenerative disorder characterized by the loss of dopamine neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunction. We now report that minocycline, a semisynthetic tetracycline, recently shown to have neuroprotective effects in animal models of stroke/ischemic injury and Huntington's disease, prevents nigrostriatal dopaminergic neurodegeneration in the 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Minocycline treatment also blocked dopamine depletion in the striatum as well as in the nucleus accumbens after MPTP administration. The neuroprotective effect of minocycline is associated with marked reductions in inducible NO synthase (iNOS) and caspase 1 expression. In vitro studies using primary cultures of mesencephalic and cerebellar granule neurons (CGN) and/or glia demonstrate that minocycline inhibits both 1-methyl-4-phenylpyridinium (MPP+)-mediated iNOS expression and NO-induced neurotoxicity, but MPP+-induced neurotoxicity is inhibited only in the presence of glia. Further, minocycline also inhibits NO-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) in CGN and the p38 MAPK inhibitor, SB203580, blocks NO toxicity of CGN. Our results suggest that minocycline blocks MPTP neurotoxicity in vivo by indirectly inhibiting MPTP/MPP+-induced glial iNOS expression and/or directly inhibiting NO-induced neurotoxicity, most likely by inhibiting the phosphorylation of p38 MAPK. Thus, NO appears to play an important role in MPTP neurotoxicity. Neuroprotective tetracyclines may be effective in preventing or slowing the progression of Parkinson's and other neurodegenerative diseases.

Persistent endocrine-disrupting compounds (EDCs) in bodies of water are a concern for human health and constitute an environmental issue, even if present in trace amounts. Conventional treatment systems do not entirely remove EDCs from discharge effluent. Due to the ultra-trace level of EDCs which affect human health and pose an environmental issue, developing new approaches and techniques to remove these micropollutants from the discharged effluent is vital. This review discusses the most common methods of eliminating EDCs through preliminary, primary, secondary and tertiary treatments. The adsorption process is favoured for EDC removal, as it is an economical and straightforward option. The NABC aspects, which are the need, approach, benefits and challenges, were analysed based on existing circumstances, highlighting biochar as a green and renewable adsorbent for the removal of organic contaminants. From the environmental point of view, the effectiveness of this method, which uses natural fibre from the kenaf plant as a porous and economical biochar material with a selected lignocellulosic biomass, provides insights into the advantages of biochar-derived adsorbents. Essentially, the improvement of the natural fibre as an adsorbent is a focus, using carbonisation, activation, and the physiochemical process to enhance the adsorption ability of the material for pollutants in bodies of water. This output will complement sustainable water management approaches presented in previous studies for combating the emerging pollutant crisis via novel green and environmentally safe options.

The densities of subtypes of serotonin (5-HT) and dopamine (DA) receptors were determined in the CNS of male alcohol-naive HAD and LAD lines of rats. Autoradiographic studies were undertaken to measure the densities of (a) 5-HT1A sites labelled with 2 nM [3H]8-OH DPAT, (b) 5-HT2A sites labelled with 2 nM [3H] ketanserin, (c) D1 sites labelled with 1 nM [3H]SCH23390, and (d) D2 sites labelled with 20 nM [3H]sulpiride. Membrane binding, using tissue combined from the olfactory bulb, olfactory tubercle, and nucleus accumbens, was carried out to determine Kd and Bmax values for the binding of 0.25–8.0 nM [3H]7-OH DPAT to D3 sites. Among the 14 regions measured for densities of 5-HT1A sites, no interline differences were found in the cerebral cortical regions or in the septal nuclei; however, within the hippocampus, 15–20% lower binding of [3H]8-OH DPAT was observed in the posterior dorsal CA3 and dentate gyrus of the HAD line. There were no interline differences in any of the 10 regions examined for [3H]ketanserin binding to 5-HT2A sites, or in the densities of D1 and D2 sites in the mesolimbic and nigrostriatal DA systems, except for a 35% higher density of D2 sites in the substantia nigra pars compacta of the HAD line. There were no interline differences in the Kd or Bmax values for [3H]7-OH DPAT binding to D3 sites. Overall, these results indicate that no marked interline differences are evident in the densities of 5-HT1A, 5-HT2A, D1, D2, and D3 receptors within the mesolimbic system that could be associated with the disparate alcohol drinking behaviors of the HAD and LAD rats.

The densities of subtypes of serotonin (5-HT) and dopamine (DA) receptors were determined in the CNS of alcohol-naive alcohol-preferring P and -nonpreferring NP lines of rats. Autoradiography studies were undertaken to measure the densities of 5-HT1B sites labelled with 100 pM [125I](-)-iodocyanopindolol, 5-HT3 sites labelled with 2 nM [3H]LY 278584, and D1 sites labelled with 1 nM[3H]SCH 23390. Membrane binding, using tissue combined from the olfactory bulb, olfactory tubercle, and nucleus accumbens, was carried out to determine Kd and B max values for the binding of 0.25-8.0 nM[3H]7-OH DPAT to D3 sites. Among the 48 regions measured for differences in 5-HT1B recognition sites, statistically significant differences (p < 0.05) were found only in the cingulate and retrosplenial cortices, in the lateral and medial septum, and in the lateral nucleus of the amygdala, with lower values being found in the P than the NP line. There were no significant differences in the regional CNS densities of D1 or 5-HT3 sites between the P and NP lines. There were also no differences between the rat lines in the Kd or Bmax values for [3H]7-OH DPAT binding to D3 sites. The lower densities of 5-HT1B sites in the CNS of the P compared to the NP rats may be a result of reduced numbers of 5-HT1B presynaptic autoreceptors as well as postsynaptic receptors in the P line. The observation that there are no differences in the amount of radioligand binding to D1, 5-HT3, and D3 sites between the P and NP lines suggests that the disparate alcohol drinking behaviors of these two lines is not associated with an innate alteration in the densities of these receptor subtypes.

The regional densities of benzodiazepine (BDZ) recognition sites coupled to GABAA receptors were studied in ethanol-naive alcohol-preferring (P) and -nonpreferring (NP) lines of rats by using quantitative autoradiography to measure the amount of 2 nM [3H]flunitrazepam (FNZ) binding in the absence and presence of 100 μM GABA. Lower values ( p < 0.025) for [3H]FNZ binding (in the absence of GABA) were observed in the prefrontal cortex, layer 4 of the parietal cortex, and the nucleus accumbens shell of the P relative to the NP line. GABA significantly ( p < 0.025) stimulated [3H]FNZ binding in all 50 central nervous system regions examined in both the P and the NP rats. The largest percent increases (190–220%) were observed in the prefrontal, cingulate, frontal, and parietal cortices; shell and core nucleus accumbens; caudate putamen; dorsal lateral, intermediate lateral, ventral lateral, and medial septal nuclei; and lateral hypothalamus. In several layers of the frontal and parietal cortices, a 25–30% greater net or percent increase ( p < 0.025) in GABA-enhanced [3H]FNZ binding was observed in the P rats compared with the NP rats. In contrast, lower net or percent increases ( p < 0.025) in GABA-enhanced [3H]FNZ binding were found in the entorhinal cortex, the mediodorsal thalamus, and the dorsal CA3 area and middle dentate gyrus of the posterior hippocampus of the P line relative to the NP line. The present findings suggest that there are innate regional differences between P and NP rats in the densities and/or affinities of BDZ recognition sites and in the coupling between the GABAA and BDZ binding sites.

The densities of serotonin1A (5-HT1A) receptors, labeled with [3H]8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), were examined in the CNS of alcohol-naive adult male alcohol-preferring (P) and -nonpreferring (NP) rats using quantitative autoradiography. The densities of sites labeled with 2 nM [3H]8-OH-DPAT were a) 20–30% higher in the medial prefrontal, frontal (layers 1, 2, and layers 3–6), parietal (layers 3–6), and cingulate cortex; b) 35–40% higher in the retrosplenial, occipital (all layers), temporal (all layers) cortex; and c) 15% higher in the entorhinal cortex of the P compared with the NP rat. Within the hippocampus, significant differences between the rat lines were observed only in the posterior portion where the densities of [3H]8-OH-DPAT labeled sites were a) 10–15% higher in the dorsal dentate gyrus, dorsal CA1, and dorsal CA3 regions; and b) 15–25% higher in the anterior ventral hippocampal area and ventral dentate gyrus of the P relative to the NP line. In contrast to the above results, the densities of [3H]8-OH-DPAT labeled sites were 15–20% lower in the dorsal, paradorsal, and median raphe nuclei of the P compared with the NP rat. No differences in [3H]8-OH-DPAT binding between the rat lines were found in several basal ganglia, limbic, and brain stem regions. The data indicate that there are greater numbers of postsynaptic 5-HT1A receptors in certain parts of the cerebral cortex and hippocampus of the P compared with the NP rat. In addition, the lower densities of 5-HT1A cell body autoreceptors in the raphe nuclei suggest that there are fewer 5-HT neurons in the raphe nuclei of the P than of the NP rat.

The densities of dopamine D2 recognition sites labelled with [3H]sulpiride were determined in the caudate-putamen, nucleus accumbens (medial and lateral portions), olfactory tubercle, substantia nigra (pars reticulata and pars compacta), and ventral tegmental area (VTA) of alcohol-naive, selectively bred P (N = 7) and NP (N = 7) rats using quantitative autoradiography. The binding of [3H]sulpiride was 20–25% lower (P < 0.05) in the caudate-putamen, medial and lateral nucleus accumbens, and VTA of the P compared with the NP rats. No significant differences were observed between the P and NP rats in the olfactory tubercle or substantia nigra. [3H]Sulpiride binding, using standard membrane preparations, established with Scatchard analysis that the difference in the densities of D2 recognition sites in the caudate-putamen between the P and NP rats was due to lower Bmax values for the P line. The results indicate that the number of dopamine D2 receptor sites is lower in several central nervous system regions of the P rats compared to NP rats.

The densities of serotonin-2(5-HT2) receptors, labelled with [3H]ketanserin (Ket), were examined in the CNS of alcohol-naive, adult, male alcohol-preferring (P) and nonpreferring (NP) rats using quantitative autoradiography. The densities of binding sites labelled with 2.0 nM [3H]Ket were 15–25% lower (p < 0.05) in layer IV of the medial prefrontal, frontal, cingulate, parietal, and temporal cortices of the P line compared with the NP line. [3H]Ket binding in the P rats was also 40–50% lower (p < 0.05) in the medial and lateral nucleus accumbens, olfactory tubercle, and caudate-putamen, and 20% lower (p < 0.05) in the claustrum, compared with the NP line. No differences in 2.0 nM [3H]Ket binding were observed between the lines in the piriform cortex, hypothalamus, amygdala, hippocampus, ventral tegmental area, substantia nigra, central gray, and interpenduncular nucleus. Scatchard analysis of [3H]Ket binding (0.5–6.0 nM), determined in layer IV of the frontal and parietal cortex and in the lateral nucleus accumbens, indicated lower Bmax values without a change in Kd for the P compared to the NP rats. The results of this study indicate that there are fewer 5-HT2 receptors in certain CNS regions of the P relative to the NP rats.

The high-affinity uptake of [3H]serotonin was studied using spinal cord preparations obtained from normal dogs, dogs made paraplegic by midthoracic transection, and dogs made monoplegic by midthoracic hemisection. Measurements were made at 1, 4, and 8 weeks after surgery. Lumbar spinal cord was removed at the above time periods and a myelin-free synaptosomal fraction was obtained by centrifugation and used for studying high-affinity serotonin uptake. At 1 week, there were no significant differences for Km values (nM) among any of the three groups (138 +/- 20, 128 +/- 22, and 118 +/- 20). The Vmax values (pmol/min/mg of protein) at 1 week for the control group (4.9 +/- 0.6) versus the hemisected group (4.3 +/- 0.7) were not significantly different. The Vmax values at 1 week for the completely transected group (2.3 +/- 0.3; P less than 0.01) were significantly reduced by 53 +/- 7%. At 4 weeks, the Vmax values for the hemisected (1.8 +/- 0.2; P less than 0.05) and transected groups (0.6 +/- 0.2; P less than 0.05) were significantly different as compared to the control group. The mean percent drop in Vmax after hemisection was 56 +/- 4%, close to the predicted value of 50%. The mean percent drop in Vmax after transection was 86 +/- 2%. At 8 weeks the mean Vmax values for the hemisected (1.9 +/- 0.3; P less than 0.05) and transected (0.4 +/- 0.4; P less than 0.05) groups were not significantly different compared to their respective 4-week values. It appears that serotonin uptake can be used to quantitate roughly the degree of spinal cord injury.

The high‐affinity uptakes of [3H]serotonin, [3H]‐glutamate, and γ‐[3H]aminobutyric acid were studied using a myelin‐free crude synaptosomal fraction prepared from the spinal cords of normal dogs and spastic dogs following sham treatment or dorsal bilateral rhizotomy surgery. Compared to sham‐operated controls, rhizotomy surgery of normal dogs produced, after 1 week, a 30% reduction in the Vmax value of [3H]glutamate, but did not alter the uptake of γ‐[3H]aminobutyric acid. This treatment also produced a 60% decrease in the Vmax value of [3H]serotonin. Comparison of the effect of rhizotomy surgery on normal and spastic dogs revealed that the spastic group had 60% higher Vmax values for uptakes of [3H]glutamate and γ‐[3H]aminobutyric acid. Comparison of sham‐operated spastic dogs and rhizotomy‐treated spastic animals showed that there was a 25% decrease in the uptake of both amino acids in the rhizotomy‐treated spastic group. Overall, the data (a) support the hypothesis that glutamate is the neurotransmitter from some of the primary afferents, and (b) suggest that sprouting of interneuronal amino acid transmitter systems may occur in the spinal cords of spastic dogs.

Abstract: The high‐affinity uptake of [3H]serotonin, [3H]glutamate, and [3H]‐γ‐aminobutyric acid (3H]GABA) and the Na+‐independent binding of [3H]glutamate and [3H]GABA were studied using spinal cord preparations obtained from normal mongrel dogs and from dogs made paraplegic by midthoracic spinal cord crush. Lumbosa‐cral regions of the spinal cord were removed either before (1 week) or after (3 to 8 weeks) onset of spasticity. A myelin‐free synaptosomal fraction was obtained by cen‐trifugation and used for studying high‐affinity uptake and for preparing synaptic plasma membranes for Na+‐inde‐pendent binding experiments. For the paraplegic groups, the uptake of 30 nM [3H]serotonin was 66 and 18% of control values after 1 and 3 weeks, respectively. Eadie‐Hofstee analysis of [3H]serotonin uptake showed a 90% reduction in Vmax for the paraplegic group relative to control values, thereby indicating the expected loss of descending serotonergic pathways. The high‐affinity uptakes of 1 μM [3H]glutamate and [3H]GABA were the same in both the control and nonspastic paraplegic groups after 1 week. However, after 3 weeks, the uptakes of [3H]glutamate and [3H]GABA were 60‐70% higher for the spastic group than for the control animals. For both amino acids, Eadie‐Hofstee plots revealed no difference in Km and higher Vmax for the spastic group relative to control values. After 1 and 3 weeks, the Na+ ‐independent binding of 5 nM [3H]glutamate was 40‐85% higher and the binding of 10 nM [3H]GABA was 40‐60% lower for the paraplegic groups relative to the values for the control animals. Scatchard analysis revealed significant changes in Bmax values for both amino acids. Overall, the data indicate an increase in segmental amino acid excitatory influence which occurred when signs of spasticity were evident.