MAP2 IHC detection: a marker of antigenicity in CNS tissues.

Immunohistochemistry (IHC) is used to detect antibody-specific antigens in tissues; the results depend on the ability of the primary antibodies to bind to their antigens. Therefore, results depend on the quality of preservation of the specimen. Many investigators have overcome the deleterious effects of over-fixation on the binding of primary antibodies to specimen antigens using IHC, but if the specimen is under-fixed or fixation is delayed, false negative results could be obtained despite certified laboratory practices. Microtubule-associated protein 2 (MAP2) is an abundant microtubule-associate protein that participates in the outgrowth of neuronal processes and synaptic plasticity; it is localized primarily in cell bodies and dendrites of neurons. MAP2 immunolabeling has been reported to be absent in areas of the entorhinal cortex and hippocampus of Alzheimer's disease brains that were co-localized with the dense-core type of amyloid plaques. It was hypothesized that the lack of MAP2 immunolabeling in these structures was due to the degradation of the MAP2 antigen by the neuronal proteases that were released as the neurons lysed leading to the formation of these plaques. Because MAP2 is sensitive to proteolysis, we hypothesized that changes in MAP2 immunolabeling may be correlated with the degree of fixation of central nervous system (CNS) tissues. We detected normal MAP2 immunolabeling in fixed rat brain tissues, but MAP2 immunolabeling was decreased or lost in unfixed and delayed-fixed rat brain tissues. By contrast, two ubiquitous CNS-specific markers, myelin basic protein and glial fibrillary acidic protein, were unaffected by the degree of fixation in the same tissues. Our observations suggest that preservation of various CNS-specific antigens differs with the degree of fixation and that the lack of MAP2 immunolabeling in the rat brain may indicate inadequate tissue fixation. We recommend applying MAP2 IHC for all CNS tissues as a pre-screen to assess the quality of the tissue preservation and to avoid potentially false negative IHC results.

Seroquel: biochemical profile of a potential atypical antipsychotic.

Seroquel and the atypical antipsychotic clozapine were compared using a number of biochemical measures in rats which are indicative of potential antipsychotic activity and possible extrapyramidal side effect liability. Both in vitro and in vivo, these compounds are low potency D-2 dopamine (DA) receptor antagonists and are relatively more potent 5-HT2 antagonists than typical antipsychotic drugs. Seroquel also exhibited low affinity for D-1 DA receptors in vitro, but D-1 receptor occupancy was not detectable in vivo. Unlike clozapine, Seroquel lacks appreciable activity at either D-1 DA or muscarinic receptors. Following IP administration, both compounds produce similar elevations in DA metabolite concentrations. Following 1 month of daily administration, at doses which produce large increases in striatal DA metabolite concentrations, both Seroquel and clozapine fail, unlike typical antipsychotics, to increase the number of striatal D-2 receptors, but do decrease the number of 5-HT2 receptors in frontal cortex. ICI 204,636 produces a short-lasting increase in plasma prolactin levels, but these increases are much greater than those that are produced by clozapine. One day after 3 weeks of daily administration, tolerance, to the ability of Seroquel to elevate DA metabolite and plasma PRL concentrations is not observed. These biochemical observations are discussed with regard to the atypical profile of Seroquel in behavioral and electrophysiological studies.

Clozapine and haloperidol have differential effects on glutamic acid decarboxylase mRNA in the pallidal nuclei of the rat.

The striatum, and one of its targets, the pallidum (globus pallidus and entopeduncular nucleus) are based ganglia nuclei involved in extrapyramidal movement control. Gamma-aminobutyric acid (GABA)ergic neurons of the pallidum may be important for the expression of the effects of agents which alter striatal neurotransmission. In this study, rats were treated once daily for 28 days with either haloperidol or clozapine, two drugs which respectively, do and do not, induce extrapyramidal movement disorders. In situ hybridization histochemistry was used to quantify the levels of labeling for the messenger ribonucleic acid encoding glutamic acid decarboxylase, the main synthesizing enzyme for GABA in neurons of the striatum, globus pallidus, and entopeduncular nucleus. Neither drug treatment altered levels of labeling in the striatum. Haloperidol treatment increased the level of labeling in the entopeduncular nucleus and clozapine treatment increased labeling in the globus pallidus suggesting that these drugs exert different regulatory effects on pallidal neurons.

Glucose concentrations in brain and blood: regulation by dopamine receptor subtypes.

The stimulation of D1 and D2 dopamine (DA) receptors by selective agonists produced large increases in brain glucose concentrations. D2 receptor stimulation also produced large increases in blood glucose. The D1-induced increases were somewhat variable in normal animals, but were more reliably observed and greatly increased in animals with brain DA depletions. Blockade of D1 receptors prevented these increases. Likewise, centrally acting D2 antagonists, but not the peripheral D2 antagonist domperidone, prevented D2 agonist-induced increases in brain and blood glucose. These observations point to an important role for DA in the regulation of glucose homeostasis.

5-HT2 receptor blockade by ICI 169,369 and other 5-HT2 antagonists modulates the effects of D-2 dopamine receptor blockade.

The effects of D-2 dopamine (DA) receptor blockade were modulated by ICI 169,369, a selective 5-hydroxytryptamine (5-HT)2 receptor antagonist, and by other 5-HT2 antagonists. Specifically, it appears that blockade of 5-HT2 receptors can attenuate the effects of D-2 receptor blockade on rat striatal dopaminergic transmission. Thus, the blockade of D-2 receptors by haloperidol results in a compensatory increase in rat striatal DA metabolism, which is enhanced by ICI 169,369. By itself, ICI 169,369 did not significantly alter DA metabolism. Conversely, several compounds which possess appreciable activity at 5-HT2 sites in ex vivo binding assays, but possess little activity at D-2 sites (i.e., pirenperone, setoperone, fluperlapine and clozapine), all produced large increases in striatal DA metabolism. Therefore, these data suggest that the 5-HT2 component of these compounds, by enhancing DA metabolism, may act to attenuate the blockade of striatal D-2 receptors by these compounds. Consistent with this hypothesis, the chronic blockade of D-2 receptors by haloperidol increases the number of striatal D-2 DA receptors, and these increases are attenuated by the coadministration of ICI 169,369. Likewise, pirenperone and clozapine, at doses which acutely produced elevations in DA metabolism which were similar to those produced by haloperidol, failed to increase the number of D-2 receptors in striatum. Interestingly, 5-HT2 receptor blockade did not appear to potently modulate the effects of D-2 receptor blockade in the olfactory tubercle, a brain region which is innervated by mesolimbic DA-containing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute treatment with clozapine blocks D1 dopamine receptor binding in discrete brain areas of the male rat.

The ability of acute treatment with clozapine in vivo to block D1 receptors in the rat telencephalon and midbrain was investigated using the irreversible inactivation of [125I]SCH 23982 binding sites by N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ; 6 mg/kg, i.p., 24 h). As analyzed by quantitative autoradiography, clozapine (40 mg/kg, i.p., 15 min before EEDQ administration) was found to produce a partial protection of central D1 receptors in all brain areas investigated, including cerebrocortical regions. These data could perhaps explain the failure of clozapine to induce tardive dyskinesia upon chronic treatment.

Dopamine receptor occupancy in vivo: measurement using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).

N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) inactivates a variety of monoamine neurotransmitter receptors. In this report, protection against EEDQ-induced inactivation of D-1 and D-2 DA receptors by DA antagonists and agonists was used to obtain a measure of occupancy of these receptors in vivo by such drugs. Rats were pretreated with drugs and then given EEDQ (10 mg/kg, i.p.). Twenty-four hours after the EEDQ injections, the animals were decapitated and the number of receptors remaining was measured using conventional receptor binding assays. The D-1 antagonist SCH 23390 potently protected D-1 sites from EEDQ-induced inactivation in a dose-dependent manner. Similarly, NO-756, another D-1 antagonist, selectively protected D-1 sites from inactivation. Conversely, haloperidol, a relatively selective D-2 antagonist, protected D-2 sites from inactivation. Likewise, a number of antipsychotic DA antagonists also protected D-2 sites from inactivation. Clozapine, fluperlapine, and (+) butaclamol were effective at protecting both D-1 sites and D-2 sites. In addition, the D-1 agonist SKF 38393 protected D-1 sites from EEDQ-induced inactivation, whereas the D-2 agonist quinpirole protected D-2 sites. (-) Apomorphine, a mixed D-1/D-2 agonist, protected both sites. Thus, this type of method provides a simple means of evaluating the occupation of DA receptors by DA antagonists and agonists in vivo.

3-Methoxytyramine accumulation: effects of typical neuroleptics and various atypical compounds.

The accumulation of 3-methoxytyramine (3-MT), the O-methylated metabolite of dopamine (DA), in rat striatum was used to assess the effects of drugs on dopaminergic activity. This was accomplished by pretreating rats with pargyline to completely inhibit 3-MT catabolism. Under the conditions used, 3-MT accumulation was linear over time for at least 90 minutes. Apomorphine and gamma-butyrolactone, drugs which depress the activity of DA-containing neurons, decreased striatal 3-MT accumulation; whereas typical neuroleptics (haloperidol, fluphenazine, chlorpromazine), which increase the activity of DA-containing neurons, increased striatal 3-MT accumulation. In addition, a number of other drugs which block DA receptors and exert various atypical actions on dopaminergic functioning were examined. These "atypical" compounds (clozapine, buspirone, molindone) also increased striatal 3-MT accumulation, but were generally less potent than the typical neuroleptics examined. Moreover, the potencies of the typical neuroleptics and "atypical" compounds that were tested appear to be somewhat related to their affinities for D-2 DA receptors, as measured by their abilities to displace 3H-spiperone from rat striatal membrane preparations. Interestingly, this relationship was less evident when NaCl was omitted from the 3H-spiperone binding assay buffer. The potential antipsychotic drugs, BW 234U and SCH 23390, were also investigated for their effects on 3-MT accumulation and 3H-spiperone binding, and they were relatively inactive in both of these measures of dopaminergic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

D-1 dopamine receptor stimulation elevates plasma prolactin levels.

SKF 38393, a D-1 dopamine receptor agonist, produced dose-dependent elevations in plasma prolactin concentrations. Following the administration of SCH 23390, a D-1 antagonist, plasma prolactin concentrations tended to decrease; and low doses of SCH 23390 completely prevented the SKF 38393-induced elevations in plasma prolactin. These observations suggest that D-1 receptor stimulation can promote prolactin secretion.

D-1 and D-2 dopamine receptor blockade: interactive effects in vitro and in vivo.

Haloperidol, at low concentrations that block D-2 dopamine (DA) receptors but not D-1 DA receptors (less than 10 microM), potentiated the enhancement of adenylate cyclase activity produced by the D-1 agonist SKF 38393. Low concentrations of haloperidol (less than or equal to 5 microM) also potentiated the K+-evoked release of [3H]acetylcholine from superfused striatal tissue slices. Both of these effects of haloperidol were blocked by nanomolar concentrations of SCH 23390, a D-1 receptor antagonist. In addition, SCH 23390 reduced the ability of haloperidol to antagonize the inhibition of [3H]acetylcholine release produced by the DA agonist apomorphine. By itself, SCH 23390 did not alter either basal adenylate cyclase activity or the K+-evoked release of [3H]acetylcholine. These findings suggest that SCH 23390 can attenuate in vitro responses to D-2 receptor blockade. Likewise, in vivo, very low doses (less than 1 microgram/kg) of SCH 23390 reduced the ability of haloperidol to elevate striatal DA metabolite concentrations and plasma prolactin concentrations. Thus, D-1 receptor blockade may attenuate the effects of D-2 DA receptor blockade both in vitro and in vivo.

Apomorphine enantiomers' effects on dopamine metabolism: receptor and non-receptor related actions.

The enantiomers of apomorphine (APO) inhibited dopamine synthesis in rat striatal synaptosomes, with R(-)-APO being about twice as potent as S(+)-APO. Sulpiride, a DA receptor antagonist, partially antagonized the inhibitory effects of only (-)-APO, suggesting that (-)-APO's, but not (+)-APO's, effects on dopamine synthesis may be at least partially receptor-mediated. The addition of 6-methyl-5,6,7,8-tetrahydrobiopterine (6-MPH4), an artificial cofactor for tyrosine hydroxylase, partially antagonized the inhibitory effects of both enantiomers, being considerably more effective against the (+)enantiomer. These data suggest that the APO enantiomers may directly inhibit enzymes within the synaptosome which regulate dopamine synthesis. Furthermore, investigations measuring DA synthesis rates in synaptosomes that had been pre-incubated with (-)-APO and then washed to remove the (-)-APO in the medium, indicate that (-)-APO may be retained by synaptosomes. Preliminary studies measuring the accumulation of [3H](-)-APO by synaptosomes also suggest that synaptosomes can accumulate APO. Although both APO enantiomers suppressed DA synthesis in vitro, only (-)-APO reduced striatal DA metabolite concentrations in vivo, and this reduction was prevented by haloperidol, a DA receptor antagonist. In addition, 6-MPH4 prevented the decrease in the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) produced by (-)-APO but not the decrease in the DA metabolite homovanillic acid (HVA).

Homocysteine prevents apomorphine-induced decreases in dopamine metabolites.

Low doses of apomorphine reduced dopamine (DA) metabolite concentrations in mouse and rat striata. These reductions were prevented by pretreatment with homocysteine at a dosage which had been shown to inhibit protein carboxyl and other methylation reactions in brain. By itself, homocysteine did not alter DA metabolite concentrations. These findings are discussed with regard to the possible role of cerebral methylation reactions in mediating DA autoreceptor functions.

Modulation of dopamine receptor supersensitivity by chronic insulin: implication in schizophrenia.

Haloperidol-induced increases in the number of dopamine receptors, as measured by [3H]spiperone binding to striatal membranes, do not occur in rats repeatedly treated with insulin in doses eliciting pronounced hypoglycemia. Given alone, however, insulin has no effect on [3H]spiperone binding in normal rats. These findings demonstrate a modulating effect of insulin on brain dopamine receptor sensitization. This effect might be relevant to the mechanism of insulin coma therapy in schizophrenia and is consistent with and supports the dopaminergic hypothesis of this disorder.

Rapid automated high-performance liquid chromatographic analysis of cyclic adenosine 3',5'-monophosphate. Synthesis in brain tissues.

A rapid and sensitive automated system for measuring cyclic adenosine 3',5'-monophosphate (cAMP) synthesized from either radiolabelled adenine or adenosine 5'-triphosphate (ATP) in intact and broken cell tissue preparations, respectively, is described. After incubation with radiolabelled precursor, tissue samples are deproteinized and then injected directly onto a reversed-phase high-performance liquid chromatographic column. The column effluent fraction which contains cAMP is collected into scintillation vials and assayed for tritium by liquid scintillation spectrometry. Since the high-performance liquid chromatographic and fraction collection procedures are automated, over fifty samples can be analyzed in duplicate in a single day. The utility of this assay is illustrated by investigations of the effects of beta-adrenergic receptor stimulation on cAMP synthesis in tissue slices prepared from rat cerebral cortex and dopamine on cAMP synthesis in striatal membrane preparations.

Inhibition of protein carboxylmethylation and dopamine autoreceptor functioning.

S-Adenosyl-L-homocysteine (SAH, 2-100 microM) greatly inhibited protein carboxymethylation (PCM) in rat striatal synaptosomes, but did not alter the ability of apomorphine and other DA agonists to inhibit DA synthesis. SAH (10 microM) also did not significantly alter the ability of either 0.5 or 1.0 microM apomorphine to inhibit DA release from superfused rat striatal tissue slices, but it did antagonize the response to 5.0 microM apomorphine. The former two concentrations of apomorphine predominantly affected only DA release, whereas the latter concentration suppressed both DA and acetylcholine release. These findings are discussed with regard to the possible relationship between DA autoreceptor functioning and PCM activity.

Dopamine receptor subtypes: in vivo biochemical evidence for functional interaction.

SKF 38393, a selective D-1 dopamine (DA) agonist, enhanced the ability of spiperone, a D-2 antagonist, to increase rat striatal DA metabolite concentrations. Conversely, SKF 38393 reduced the abilities of pergolide and LY 171555, D-2 agonists, to decrease striatal DA metabolite concentrations. These findings are discussed in terms of a possible functional interaction between D-1 and D-2 DA receptors.

Alterations in dopamine metabolism after chronic administration of haloperidol. Possible role of increased autoreceptor sensitivity.

Basal levels of the metabolites of dopamine (DA) were reduced following the chronic administration of haloperidol. The ability of small doses (50 or 100 micrograms/kg) of apomorphine to reduce the concentrations of the metabolite of DA, homovanillic acid (HVA), was also enhanced following the chronic administration of haloperidol. In addition, the accumulation of DA and of the metabolite of DA, 3-methoxytyramine (3-MT), in rats treated with pargyline was reduced following the chronic administration of haloperidol, suggesting that basal turnover and release of DA also may be reduced. These findings are discussed in relation to possible changes in the sensitivity of DA autoreceptors and the activity of DA-containing neurons.

Dopamine synthesis in synaptosomes: relation of autoreceptor functioning to pH, membrane depolarization, and intrasynaptosomal dopamine content.

Factors affecting dopamine (DA) synthesis in rat striatal synaptosomes were examined by measuring the conversion of [3H]tyrosine (Tyr) to [3H]DA. Any [3H]DA that was synthesized was extracted into a toluene-based scintillation cocktail and quantitated by liquid scintillation spectrometry. The extraction was facilitated using di-(2-ethylhexyl) phosphoric acid (DEHP), a liquid cation exchanger. DA, apomorphine, and other DA agonists were much less potent inhibitors of DA synthesis in striatal synaptosomes at pH 6.2 than at pH 7.2. 3-(3-Hydroxyphenyl)-N-n-propylpiperidine (3-PPP), a putative DA autoreceptor agonist, was inactive at pH 6.2. However, at pH 7.2, 3-PPP did inhibit DA synthesis. This inhibition was reversed by sulpiride, a DA receptor antagonist, but not by benztropine, a DA uptake blocker, suggesting that 3-PPP inhibits DA synthesis by stimulating the DA autoreceptor. DA release from synaptosomes was much greater at pH 6.2 than at pH 7.2, most probably because the synaptosomal membrane appears to be depolarized at pH 6.2, as measured by the accumulation of [3H]tetraphenylphosphonium ions. Since tyrosine hydroxylase is inhibited by DA, this finding suggested that low assay buffer pH (i.e., pH 6.2) might interfere with the ability of 3-PPP and other DA agonists to inhibit DA synthesis, by promoting DA release. Likewise, reserpine and tetrabenazine, compounds which disrupt vesicular DA storage, were much less effective inhibitors of DA synthesis at pH 6.2 (high basal DA release). Moreover, D-amphetamine and high buffer potassium concentrations, treatments which promote DA release, also interfered with the ability of 3-PPP to inhibit DA synthesis. Thus, modulation of the release of DA in equilibrium with tyrosine hydroxylase may be a mechanism by which the DA autoreceptor regulates DA synthesis.