TSSK3, a novel target for male contraception, is required for spermiogenesis.

We have previously shown that members of the family of testis-specific serine/threonine kinases (TSSKs) are post-meiotically expressed in testicular germ cells and in mature sperm in mammals. The restricted post-meiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggest that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the TSSK6 knock-out (KO) mice and of the double TSSK1/TSSK2 KO. The aim of this study was to develop KO mouse models of TSSK3 and to validate this kinase as a target for the development of a male contraceptive. We used CRISPR/Cas9 technology to generate the TSSK3 KO allele on B6D2F1 background mice. Male heterozygous pups were used to establish three independent TSSK3 KO lines. After natural mating of TSSK3 KO males, females that presented a plug (indicative of mating) were monitored for the following 24 days and no pregnancies or pups were found. Sperm numbers were drastically reduced in all three KO lines and, remarkably, round spermatids were detected in the cauda epididymis of KO mice. From the small population of sperm recovered, severe morphology defects were detected. Our results indicate an essential role of TSSK3 in spermiogenesis and support this kinase as a suitable candidate for the development of novel nonhormonal male contraceptives.

TSSK6 is required for γH2AX formation and the histone-to-protamine transition during spermiogenesis.

Spermiogenesis includes transcriptional silencing, chromatin condensation and extensive morphological changes as spermatids transform into sperm. Chromatin condensation involves histone hyperacetylation, transitory DNA breaks, histone H2AX (also known as H2AFX) phosphorylation at Ser139 (γH2AX), and replacement of histones by protamines. Previously, we have reported that the spermatid protein kinase TSSK6 is essential for fertility in mice, but its specific role in spermiogenesis is unknown. Here, we show that TSSK6 expression is spatiotemporally coincident with γH2AX formation in the nuclei of developing mouse spermatids. RNA-sequencing analysis demonstrates that genetic ablation of Tssk6 does not impact gene expression or silencing in spermatids. However, loss of TSSK6 blocks γH2AX formation, even though the timing and level of the transient DNA breaks is unaltered. Further, Tssk6-knockout sperm contained increased levels of histones H3 and H4, and protamine 2 precursor and intermediate(s) indicative of a defective histone-to-protamine transition. These results demonstrate that TSSK6 is required for γH2AX formation during spermiogenesis, and also link γH2AX to the histone-to-protamine transition and male fertility.

Heat shock protein 90 functions to stabilize and activate the testis-specific serine/threonine kinases, a family of kinases essential for male fertility.

Spermiogenesis is characterized by a profound morphological differentiation of the haploid spermatid into spermatozoa. The testis-specific serine/threonine kinases (TSSKs) comprise a family of post-meiotic kinases expressed in spermatids, are critical to spermiogenesis, and are required for male fertility in mammals. To explore the role of heat shock protein 90 (HSP90) in regulation of TSSKs, the stability and catalytic activity of epitope-tagged murine TSSKs were assessed in 293T and COS-7 cells. TSSK1, -2, -4, and -6 (small serine/threonine kinase) were all found to associate with HSP90, and pharmacological inhibition of HSP90 function using the highly specific drugs 17-AAG, SNX-5422, or NVP-AUY922 reduced TSSK protein levels in cells. The attenuation of HSP90 function abolished the catalytic activities of TSSK4 and -6 but did not significantly alter the specific activities of TSSK1 and -2. Inhibition of HSP90 resulted in increased TSSK ubiquitination and proteasomal degradation, indicating that HSP90 acts to control ubiquitin-mediated catabolism of the TSSKs. To study HSP90 and TSSKs in germ cells, a mouse primary spermatid culture model was developed and characterized. Using specific antibodies against murine TSSK2 and -6, it was demonstrated that HSP90 inhibition resulted in a marked decrease of the endogenous kinases in spermatids. Together, our findings demonstrate that HSP90 plays a broad and critical role in stabilization and activation of the TSSK family of protein kinases.

Identification of a novel HSP70-binding cochaperone critical to HSP90-mediated activation of small serine/threonine kinase.

We previously reported the identification of small serine/threonine kinase (SSTK) that is expressed in postmeiotic germ cells, associates with HSP90, and is indispensable for male fertility. Sperm from SSTK-null mice cannot fertilize eggs in vitro and are incapable of fusing with eggs that lack zona pellucida. Here, using the yeast two-hybrid screen, we have discovered a novel SSTK-interacting protein (SIP) that is expressed exclusively in testis. The gene encoding SIP is restricted to mammals and encodes a 125-amino acid polypeptide with a predicted tetratricopeptide repeat domain. SIP is co-localized with SSTK in the cytoplasm of spermatids as they undergo restructuring and chromatin condensation, but unlike SSTK, is not retained in the mature sperm. SIP binds to SSTK with high affinity (K(d) ∼10 nM), and the proteins associate with each other when co-expressed in cells. In vitro, SIP inhibited SSTK kinase activity, whereas the presence of SIP in cells resulted in enzymatic activation of SSTK without affecting Akt or MAPK activity. SIP was found to be associated with cellular HSP70, and analyses with purified proteins revealed that SIP directly bound HSP70. Importantly, SSTK recruited SIP onto HSP90, and treatment of cells with the specific HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin, completely abolished SSTK catalytic activity. Hence, these findings demonstrate that HSP90 is essential for functional maturation of the kinase and identify SIP as a cochaperone that is critical to the HSP90-mediated activation of SSTK.

Examining the cost of community-based tuberculosis treatment in South Africa.

SETTING: While South Africa has improved access to tuberculosis (TB) treatment and care, the 2015 treatment success rate for multidrug-resistant TB (MDR-TB) remains low, at 55%. Community-based TB treatment and care improves patient retention compared to the standard of care alone.OBJECTIVE: To assess the cost of a USAID-funded community-based TB model in Nelson Mandela Bay Health District (NMBHD), Eastern Cape Province, South Africa compared to the national standard of care alone.DESIGN: We estimated the cost of community-based DR-TB treatment and adherence support compared to the standard of care alone.RESULTS: Average overall costs were US$2827 lower per patient on the community-based model than the standard of care alone.CONCLUSION: The per-patient cost of the community-based model is lower than the standard of care alone. Assuming the costs and effects of a community-based model implemented in NMBHD were observed at a larger scale, implementing the model could reduce overall health system costs.

Distribution of RNA binding protein MOEP19 in the oocyte cortex and early embryo indicates pre-patterning related to blastomere polarity and trophectoderm specification.

We report the cloning and characterization of MOEP19, a novel 19 kDa RNA binding protein that marks a defined cortical cytoplasmic domain in oocytes and provides evidence of mammalian oocyte polarity and a form of pre-patterning that persists in zygotes and early embryos through the morula stage. MOEP19 contains a eukaryotic type KH-domain, typical of the KH-domain type I superfamily of RNA binding proteins, and both recombinant and native MOEP19 bind polynucleotides. By immunofluorescence, MOEP19 protein was first detected in primary follicles throughout the ooplasm. As oocytes expanded in size during oogenesis, MOEP19 increased in concentration. MOEP19 localized in the ovulated egg and early zygote as a symmetrical spherical cortical domain underlying the oolemma, deep to the zone of cortical granules. MOEP19 remained restricted to a cortical cytoplasmic crescent in blastomeres of 2-, 4- and 8-cell embryos. The MOEP19 domain was absent in regions underlying cell contacts. In morulae, the MOEP19 domain was found at the apex of outer, polarized blastomeres but was undetectable in blastomeres of the inner cell mass. In early blastocysts, MOEP19 localized in both mural and polar trophectoderm and a subset of embryos showed inner cell mass localization. MOEP19 concentration dramatically declined in late blastocysts. When blastomeres of 4- to 8-cell stages were dissociated, the polarized MOEP19 domain assumed a symmetrically spherical localization, while overnight culture of dissociated blastomeres resulted in formation of re-aggregated embryos in which polarity of the MOEP19 domain was re-established at the blastomere apices. MOEP19 showed no evidence of translation in ovulated eggs, indicating that MOEP19 is a maternal effect gene. The persistence during early development of the MOEP19 cortical oocyte domain as a cortical crescent in blastomers suggests an intrinsic pre-patterning in the egg that is related to the apical-basolateral polarity of the embryo. Although the RNAs bound to MOEP19 are presently unknown, we predict that the MOEP19 domain directs RNAs essential for normal embryonic development to specific locations in the oocyte and early embryo.

TSKS concentrates in spermatid centrioles during flagellogenesis.

Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 and 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of Tssk1 and 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.

Targeted deletion of Tssk1 and 2 causes male infertility due to haploinsufficiency.

Targeted deletion of Tssk1 and 2 resulted in male chimeras which produced sperm/spermatogenic cells bearing the mutant allele, however this allele was never transmitted to offspring, indicating infertility due to haploinsufficiency. Morphological defects in chimeras included failure to form elongated spermatids, apoptosis of spermatocytes and spermatids, and the appearance of numerous round cells in the epididymal lumen. Characterization of TSSK2 and its interactions with the substrate, TSKS, were further investigated in human and mouse. The presence of both kinase and substrate in the testis was confirmed, while persistence of both proteins in spermatozoa was revealed for the first time. In vivo binding interactions between TSSK2 and TSKS were established through co-immunoprecipitation of TSSK2/TSKS complexes from both human sperm and mouse testis extracts. A role for the human TSKS N-terminus in enzyme binding was defined by deletion mapping. TSKS immunoprecipitated from both mouse testis and human sperm extracts was actively phosphorylated. Ser281 was identified as a phosphorylation site in mouse TSKS. These results confirm both TSSK 2 and TSKS persist in sperm, define the critical role of TSKS' N-terminus in enzyme interaction, identify Ser 281 as a TSKS phosphorylation site and indicate an indispensable role for TSSK 1 and 2 in spermiogenesis.

A relationship between weak attentional control and cognitive distortions, explained by negative affect.

People high in negative affect (anxiety or depression) show cognitive distortions, specific thinking errors which contribute to the maintenance of their condition. It is thought that weak attentional control is a risk factor for negative affect and emotional disorders, because weak attentional control exaggerates the expression of attentional bias, another cognitive feature of emotional disorders. We wondered whether weak attentional control might similarly exaggerate the expression of cognitive distortions. In two samples of students from Turkey and the UK, we found that weak attentional control was indeed related to cognitive distortions, but this relationship was explained by both variables' relationships with negative affect. This suggests that weak attentional control, while related to negative affect, does not necessarily exaggerate all of its cognitive features. There seems to be a limit on the affective consequences of poor attentional control, which may limit its clinical usefulness as a risk factor for emotional disorders.

Biochemical and structural characterization of apolipoprotein A-I binding protein, a novel phosphoprotein with a potential role in sperm capacitation.

The physiological changes that sperm undergo in the female reproductive tract rendering them fertilization-competent constitute the phenomenon of capacitation. Cholesterol efflux from the sperm surface and protein kinase A (PKA)-dependent phosphorylation play major regulatory roles in capacitation, but the link between these two phenomena is unknown. We report that apolipoprotein A-I binding protein (AI-BP) is phosphorylated downstream to PKA activation, localizes to both sperm head and tail domains, and is released from the sperm into the media during in vitro capacitation. AI-BP interacts with apolipoprotein A-I, the component of high-density lipoprotein involved in cholesterol transport. The crystal structure demonstrates that the subunit of the AI-BP homodimer has a Rossmann-like fold. The protein surface has a large two compartment cavity lined with conserved residues. This cavity is likely to constitute an active site, suggesting that AI-BP functions as an enzyme. The presence of AI-BP in sperm, its phosphorylation by PKA, and its release during capacitation suggest that AI-BP plays an important role in capacitation possibly providing a link between protein phosphorylation and cholesterol efflux.

Distribution of fluphenazine and its metabolites in brain regions and other tissues of the rat.

Rats were given 5, 10, or 20 mg/kg oral doses of fluphenazine (FLU) dihydrochloride daily for 15 days. FLU and its sulfoxide (FL-SO), 7-hydroxy (7-OH-FLU) and N4'-oxide (FLU-NO) metabolites were assayed in plasma, liver, kidney, fat, whole brain, and brain regions by specific and sensitive radioimmunoassays (RIA). All metabolites were detected in tissues at higher levels than in plasma, and the levels increased with dose. FLU was 10- to 27-fold higher in brain regions than in plasma. Brain vs plasma levels of FLU correlated more closely than levels of its metabolites. Liver contained the highest levels of all analytes at all doses. FLU-SO was the major metabolite in brain regions (24% to 96% of FLU) and accumulated in fat 43 to 75 times more than FLU. Levels of 7-OH-FLU and FLU-NO were very low in brain (1% to 20% of FLU). FLU-SO and FLU-NO had only 1% to 3% the affinity for D1 and D2 receptors, but 7-OH-FLU had 20% the D2 and 5% the D1 affinity of FLU. The low affinity for dopamine receptors and low brain-levels of metabolites of FLU indicate that they are not likely to contribute importantly to pharmacologic responses of FLU. Also, the estimated relative "activity factor" for these compounds in the brain indicated that the contribution to neuropharmacologic activity by metabolites is less than 1% of FLU. Consequently, clinical monitoring of plasma FLU alone may be sufficient.

Long-term effects of S(+)N-n-propylnorapomorphine compared with typical and atypical antipsychotics: differential increases of cerebrocortical D2-like and striatolimbic D4-like dopamine receptors.

Changes in D2-like dopamine (DA) receptor binding in rat brain regions were compared by quantitative in vitro receptor autoradiography after 21-d treatment with a typical (fluphenazine), atypical (clozapine), or candidate atypical antipsychotic (S[+]-N-n-propylnorapomorphine, [+]-NPA). Fluphenazine treatment significantly increased binding of the D2,3,4 radioligands [3H]nemonapride and [3H]spiperone in caudate-putamen (CPu: 22%, 32%), nucleus accumbens (ACC: 67%, 52%), olfactory tubercle (OT: 53%, 43%), and medial prefrontal cerebral cortex (MPC: 46%, 47%) but not dorsolateral frontal cortex (DFC). D2-like binding in MPC was also increased by (+)-NPA (49%, 39%) and clozapine (60%, 40%), but not in DFC, CPu, ACC, or OT. Binding of D2,3-selective [3H]raclopride increased less after fluphenazine in ACC (27%) and CPu (16%) than with the nonselective radioligands, and not after clozapine or (+)-NPA. D3-selective binding of [3H]R (+)-7-OH-DPAT was not changed with any treatment or region including islands of Calleja. Binding of [3H]nemonapride or [3H]spiperone under D4-selective conditions (with 300 nM S[-]-raclopride and other masking agents, at sites occluded by D4 ligand L-745,870), was increased by fluphenazine, (+)-NPA, clozapine in ACC (120%, 76%, 70%, respectively), and CPu (54%, 37%, 35%), but not in OT, DFC or MPC. These results support the hypothesis that cerebrocortical D2-like and striatolimbic D4-like receptors contribute to antipsychotic actions of both typical and atypical drugs and encourage further consideration of S(+)aporphines as potential atypical antipsychotics.

Bromocriptine antagonizes behavioral effects of cocaine in the rat.

Rats given cocaine or bromocriptine under conditions of low basal arousal showed dose-dependent increases of locomotor activity for less than or equal to 1 hours, followed by depression of activity that diminished gradually over the next 2 hours. Arousal was related biphasically to dose (maximum at ca. 5 mg/kg) but depression increased monophasically with the dose of either agent. Both behavioral arousal and depression induced by cocaine were antagonized by bromocriptine, even at doses lacking behavioral effects alone (ID50 = 1.0 and 0.36 mg/kg [1.3 and 0.5 mumol/kg], respectively). Bromocriptine blocked depression of locomotion even when given after the initial stimulation by cocaine. Bromocriptine induced very weak stereotypy, and neither increased nor blocked stereotypy induced by cocaine or apomorphine. Cocaine, at maximally effective doses, did not deplete catecholamines or serotonin in brain regions at times of maximum behavioral arousal or depression, nor did bromocriptine after metabolic turnover of dopamine. Bromocriptine antagonized arousal induced by direct injection of dopamine into the nucleus accumbens. The ability of bromocriptine to block both the behavioral arousal and depression induced by cocaine may reflect activity of bromocriptine as a mixed agonist-antagonist with limited intrinsic agonistic activity at central dopaminergic D2 receptors, perhaps with particular reference to limbic mechanisms.

Fatty acid derivatives of clozapine: prolonged antidopaminergic activity of docosahexaenoylclozapine in the rat.

Stable amides of clozapine derived from fatty acids prominent in cerebral tissue might enhance the central activity of clozapine and reduce its exposure to peripheral tissues. Such derivatives might enhance the safety of this unique drug, which is the only agent with securely established superior antipsychotic effectiveness, but with a risk of potentially lethal systemic toxicity. Amide derivatives of clozapine were prepared from structurally varied fatty acid chlorides and evaluated for ability to inhibit behavioral arousal in rat induced by dopamine agonist apomorphine and to induce catalepsy. Their duration-of-action and potency were compared to free clozapine, and concentrations of clozapine were assayed in brain and blood. Selected agents were also evaluated for affinity at dopamine receptors and other potential drug-target sites. Clozapine-N-amides of linoleic, myristic, oleic, and palmitic acids had moderate initial central depressant activity but by 6 h, failed to inhibit arousal induced by apomorphine. However, the docosahexaenoic acid (DHA) derivative was orally bioavailable, 10-times more potent (ED(50) 5.0 micromol/kg) than clozapine itself, and very long-acting (>/= 24 h) against apomorphine, and did not induce catalepsy. DHA itself was inactive behaviorally. Clozapine showed expected dopamine receptor affinities, but DHA-clozapine was inactive at these and other potential target sites. After systemic administration of DHA-clozapine, serum levels of free clozapine were very low, and brain concentrations somewhat lower than after administering clozapine. DHA-clozapine is a long-acting central depressant with powerful and prolonged antidopaminergic activity after oral administration or injection without inducing catalepsy, and it markedly reduced peripheral exposure to free clozapine. It lacked the receptor-affinities shown by clozapine, suggesting that DHA-clozapine may be a precursor of free, pharmacologically active clozapine. Such agents may represent potential antipsychotic drugs with improved central/peripheral distribution, and possibly enhanced safety.

Lack of increase in dopamine transporter binding or function in rat brain tissue after treatment with blockers of neuronal uptake of dopamine.

Rats were pretreated daily for 10 days with a dopamine (DA) uptake blocker ([+]amphetamine, benztropine, cocaine, GBR-12909, mazindol, or nomifensine) or control vehicle and, after 1-4 days of no treatment, striatal tissue was fractionated to provide synaptosomes and membranes for assays of transport of 3H-DA or binding of 3H-GBR-12935. There were no significant increases of apparent maxima for uptake (Vmax) or binding (Bmax) or consistent changes in ligand affinity. Pharmacologic characterization of 3H-GBR-12935 binding extended the impression that this ligand has high affinity and selectivity for many agents which block neuronal uptake of DA uptake and much less for those which interact with DA receptors or other amine transporters. The results suggest that dopamine transporters are not regulated in the same way as receptors, nor influenced similarly toward upregulation and supersensitization by repeated treatment with antagonists.

Differential effects of ascorbate and EDTA on high-affinity binding of 3H-apomorphine and 3H-ADTN to calf caudate membranes.

Recent reports describe effects of ascorbate on binding of dopaminergic agonists to membrane preparations of brain tissue. We now report that with calf caudate membranes, in the absence of EDTA, ascorbate (up to 10 mM) lowered the binding of [3H] (-)apomorphine (3H-APO) by up to 34%, and the proportion "specific," from 69% to 36%, and lowered binding of [3H] (+/-)2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (3H-ADTN) by up to 38%, with little effect on the proportion of its specific binding. EDTA (5 mM), alone, also reduced binding of both ligands by 40-50% but had no effect on the proportion specific. With ascorbate plus EDTA, although total binding was lowered, the proportion of specific binding of 3H-APO rose to a maximum of 82% at 2.5 mM ascorbate, whereas that of 3H-ADTN was only slightly increased. Moreover, TLC revealed that the antioxidants were required during incubation to prevent temperature- and time-dependent degradation of APO much more than ADTN. Thus, while each additive, alone, lowered binding of 3H-APO and of 3H-ADTN, ascorbate plus EDTA increased the proportion of specific binding, especially of 3H-APO, and protected both from degradation.

Effects of cations on high-affinity binding of 3H-ADTN to dopaminergic sites in calf caudate tissue.

Effects of cations on binding of 0.1-10 nM 3H-ADTN to calf caudate membranes included decreased apparent Bmax by [Na+] greater than or equal to 100 mM, little effect on Kd or on affinity of other dopamine (DA) agonists (DA and apomorphine), decreased slopes of inhibition curves produced by agonists, but increased affinity of the antagonists (+)butaclamol; in contrast, low (10-20 mM) [Na+] did not decrease Bmax, increased ligand and agonist affinity and specific binding, and gave steep monophasic inhibition curves for DA agonists. K+, Li+, and Rb+ had little effect at a wide range of concentrations. Mg++ and Ca++, in physiologic concentrations, moderately increased binding of 3H-ADTN, as did microM Mn++ or Co++; the latter ions inhibited binding at greater than or equal to 10 mM, as did Cu++ (IC50 = 10 microM). The results extend impressions that physiologic [Na+] favors binding of DA antagonists and diminishes binding of agonists, but optimal agonist binding occurred at low [Na+] (10-20 mM), while divalent cations had complex actions.

Alkylation of rat dopamine transporters and blockade of dopamine uptake by EEDQ.

Effects of the alkylating agent EEDQ (N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline) on levels of dopamine transporter (DA(T)) and function were examined in caudate-putamen (CPu) tissue from rat brain. EEDQ produced profound, dose-dependent decreases in DA(T) binding in homogenates (IC(50)=78 microM) and frozen sections (IC(75)=200 microM) that were not reversed by washing. EEDQ also blocked uptake of [(3)H]DA in CPu synaptosomes (IC(50)=17 microM). However, single (10 mg/kg) or repeated administration of EEDQ in vivo (15 mg/kg/day x 3) did not alter DA(T) levels or DA uptake in CPu. Pretreatment of rats with alpha-methyl-p-tyrosine and reserpine to deplete endogenous dopamine also failed to lower DA(T) levels in CPu after injections of EEDQ. EEDQ is an effective alkylating agent for DA(T) in vitro, but not to evaluate metabolic turnover or function of DA(T) in vivo. The results encourage development of selective and in vivo-active DA(T)-alkylating agents.

R(-)2-fluoro-N-n-propylnorapomorphine: a very potent and D2-selective dopamine agonist.

A series of 2-substituted N-n-propylnorapomorphine (NPA) derivatives were synthesized and compared with other DA agonists for affinity to D1 and D2 dopamine (DA) receptors in rat brain corpus striatum tissue. The 2-substituents tested reduced D1 affinity similarly, but enhanced D2 affinity in the rank order: F greater than OH greater than Br greater than OCH3 greater than H greater than or equal to NH2. The extraordinarily high D2 affinity (Ki = 12 pM) and D2 vs. D1 selectivity (57,500) of 2-F-NPA far-exceeded that of all other DA agonists tested, and it was about 10-times more potent than NPA in vivo.

Receptor affinity, neurochemistry and behavioral characteristics of the enantiomers of thioridazine: evidence for different stereoselectivities at D1 and D2 receptors in rat brain.

The binding characteristics of the enantiomers of thioridazine were assessed in the brain of the rat using competitive radioreceptor assays with tritiated ligands selective for dopamine D1 (SCH-23390), D2 (spiperone), norepinephrine alpha-1 (prazosin) and muscarinic (quinuclinidinyl benzilate) receptors. (+)-Thioridazine was shown to have 2.7 and 4.5 times higher affinity than (-)-thioridazine for D2 and alpha-1 receptors, respectively. In contrast, (-)-thioridazine had 10 times higher affinity for the D1 receptor. Both enantiomers showed similar affinities for the muscarinic receptor. In a second experiment, thioridazine, dopamine, norepinephrine, serotonin and their metabolites were assayed in the brain of the rat after acute administration of the enantiomers of thioridazine and the assessment of catalepsy. (+)-Thioridazine was 4.1 times as potent as (-)-thioridazine in elevating the turnover of dopamine in the striatum, but neither enantiomer affected the other monoamines. The concentration of thioridazine and its metabolites in the brain, for a given dose, was similar for both enantiomers. (-)-Thioridazine induced slightly more catalepsy than (+)-thioridazine and appeared to be more toxic at large doses. While racemic thioridazine had an intermediate effect between that of its two enantiomers in the binding and neurochemical assays, it appeared to induce more catalepsy than either enantiomer, suggesting a synergistic effect in this behavioral assay. It was concluded that (+)- and (-)-thioridazine act as partially selective D2 and D1 antagonists, respectively. Therefore, clinical administration of only one enantiomer of thioridazine, rather than the currently prescribed racemate, may result in an improved therapeutic profile and so be worthy of further investigation.