Lumateperone-mediated effects on prefrontal glutamatergic receptor-mediated neurotransmission: A dopamine D1 receptor dependent mechanism.

Lumateperone is a novel drug approved for the treatment of schizophrenia in adults and depressive episodes associated with bipolar depression in adults, as monotherapy and as adjunctive therapy with lithium or valproate treatment in the United States. Lumateperone simultaneously modulates key neurotransmitters, such as serotonin, dopamine, and glutamate, implicated in serious mental illness. In patients with schizophrenia, lumateperone was shown to improve positive symptoms along with negative and depressive symptoms, while also enhancing prosocial behavior. Moreover, in patients with bipolar I or II disorder, lumateperone improved depressive symptoms as well. To further understand the mechanisms related to lumateperone's clinical response, the aim of this study was to investigate the effect of lumateperone on dopaminergic- and glutamatergic signaling in the rat medial prefrontal cortex (mPFC). We used the conditioned avoidance response (CAR) test to determine the antipsychotic-like effect of lumateperone, electrophysiology in vitro to study lumateperone's effects on NMDA- and AMPA-induced currents in the mPFC, and the neurochemical techniques microdialysis and amperometry to measure dopamine- and glutamate release in the rat mPFC. Our results demonstrate that lumateperone; i) significantly suppressed CAR in rats, indicating an antipsychotic-like effect, ii) facilitated NMDA and AMPA receptor-mediated currents in the mPFC, in a dopamine D1-dependent manner, and iii) significantly increased dopamine and glutamate release in the rat mPFC. To the extent that these findings can be translated to humans, the ability of lumateperone to activate these pathways may contribute to its demonstrated effectiveness in safely improving symptoms related to neuropsychiatric disorder including mood alterations.

Biochemical effects in brain of low doses of haloperidol are qualitatively similar to those of high doses.

Typical and atypical antipsychotic drugs (APD) show differential effects in brain on both dopamine output and activation of Fos-like immunoreactivity (FLI) in dopamine nerve terminal regions. Typical APD increase dopamine output preferentially in the core and atypical APD increase dopamine output preferentially in the shell of the nucleus accumbens (NAC). Whereas both typical and atypical APD increase FLI in NAC, typicals cause FLI activation in the striatum and atypicals cause FLI activation in the prefrontal cortex. Clinically, low doses of haloperidol cause less side-effects than higher doses, and low-dose haloperidol has been suggested as a cost-effective alternative to atypical APD. Here, in vivo voltammetry in anaesthetised, pargyline-pretreated rats was used to measure dopamine output in the two subdivisions of the NAC and, in addition, immunohistochemistry was used to assess FLI activation in dopamine target areas, following acute haloperidol administration. Haloperidol, 0.001 and 0.01 mg/kg i.v., caused a significantly higher dopamine output in the core than in the shell of the NAC. Moreover, haloperidol 0.05 and 0.5, but not 0.005, mg/kg s.c. increased FLI in the NAC and the striatum, but not in the medial prefrontal cortex. Thus, even extremely low doses of haloperidol generate, in principle, the same biochemical effects in brain as higher doses, and these effects remain different from those of atypical APD. These biological data indicate that clinical differences between haloperidol and atypicals are qualitative rather than dose-dependent. Consequently, our results do not support the use of low-dose haloperidol as replacement for atypical APD in the treatment of schizophrenia.

Effects of competitive and non-competitive NMDA receptor antagonists on dopamine output in the shell and core subdivisions of the nucleus accumbens.

The effects of acute intravenous administration of the non-competitive NMDA receptor antagonists, phencyclidine (PCP), dizocilpine (MK-801; (+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,b)cyclohepten-5,10-imine), and the competitive NMDA receptor antagonist CGP 39551 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid) on extracellular dopamine concentrations were analyzed in the shell and core subdivisions of the nucleus accumbens (NAC), associated with limbic and motor functions, respectively. Extracellular dopamine concentrations were assessed utilizing differential normal pulse voltammetry in chloral hydrate anesthetized, pargyline pretreated rats. Intravenous administration of PCP (0.5 mg/kg) or MK-801 (0.1 mg/kg) both significantly elevated extracellular dopamine levels in the NAC shell but not in the core. However, administration of relatively low doses of the competitive NMDA receptor antagonist CGP 39551 (2.5 mg/kg) failed to affect dopamine output in either region. However, when a higher dose (10 mg/kg) was administered a significant elevation in dopamine output was obtained in the shell compared to the core. Our data demonstrate that non-competitive NMDA receptor antagonists evoke an accumbal dopamine output that is selective to limbic cortical related NAC regions. This profile is shared also by competitive NMDA receptor antagonists when given in high, but not low doses. Our results are compatible with the reported elicitation of PCP-like behavioral effects by competitive NMDA receptor antagonists when administered in relatively high doses. Moreover, these findings suggest that differences in the regional accumbal dopamine output between competitive and non-competitive NMDA receptor antagonists may be essentially attributable to the relative degree of NMDA receptor antagonism achieved by the drugs. This experimental model may afford a biochemical means to assess the psychotomimetic liability of NMDA receptor antagonists, a side effect that may reduce their usefulness as neuroprotective agents.

Effects of antipsychotic drugs on cholecystokinin and preprotachykinin (substance P) mRNA expression in the rat hippocampal formation.

To assess the involvement of substance P (SP) and cholecystokinin (CCK) in the effects of antipsychotic drugs, preprotachykinin-A (PPT-A) and CCK mRNA expression was studied in the hippocampal formation using in situ hybridisation following 21 daily i.p. injections with the typical antipsychotic drug haloperidol (1 mg/kg) and the atypical drug clozapine (15 mg/kg). PPT-A mRNA levels were increased in the hippocampal CA3 subregion and in the entorhinal cortex after haloperidol, whereas a decrease was observed in the CA1 after clozapine. CCK mRNA levels increased in the CA1, the entorhinal cortex and in hilus, following both haloperidol and clozapine. It is suggested that earlier findings of increased SP levels in the hippocampal formation of schizophrenics may be a consequence of haloperidol treatment and that reduced hippocampal CCK and CCK mRNA levels found earlier in schizophrenics do not result from antipsychotic drug treatment. These results are consonant to the hypothesis that increased cortical CCK transmission may be beneficial in the treatment of psychosis.

Effects of atypical antipsychotic drugs on dopamine output in the shell and core of the nucleus accumbens: role of 5-HT(2A) and alpha(1)-adrenoceptor antagonism.

The effects of acute intravenous administration of several new, atypical antipsychotic drugs (APDs): olanzapine (0.05 and 1.0 mg/kg), sertindole (0.1 and 1.0 mg/kg) and quetiapine (0.25 and 2.5 mg/kg), a selective 5-HT(2A) receptor antagonist, M100907 (0.03 and 0.3 mg/kg), and an alpha(1)-adrenoceptor antagonist, prazosin (0.3 mg/kg), on regional dopamine output were examined in the two subdivisions of the nucleus accumbens (NAC), the core and shell, which seem associated with motor control and limbic functions, respectively, by using in vivo differential normal pulse voltammetry in anaesthetised, pargyline-pretreated rats. Both quetiapine and sertindole, in the two doses used, caused a more pronounced dopamine release in the shell than in the core region of the NAC. In contrast, the low dose of olanzapine increased dopamine output almost to the same extent in both regions, whereas the high dose increased dopamine output to a greater extent in the core. M100907 selectively increased dopamine output in the shell. Also, prazosin significantly increased dopamine output in the shell, but not in the core. The results indicate that both 5-HT(2A) and alpha(1)-adrenoceptor antagonism may play an important role in the preferential effect of atypical APDs on dopamine output in the shell versus the core of the NAC.

Effect of chronic antipsychotic drug treatment on preprosomatostatin and preprotachykinin A mRNA levels in the medial prefrontal cortex, the nucleus accumbens and the caudate putamen of the rat.

In situ hybridization histochemistry was used to study the expression of preprosomatostatin (PPSOM) and preprotachykinin A (PPT-A) mRNA in the medial prefrontal cortex (mPFC), the nucleus accumbens (NAC) and the caudate putamen (CP) of the rat after chronic (21 days) treatment with the classical antipsychotic drug haloperidol (1 mg/kg i.p.), the atypical antipsychotic drugs clozapine (15 mg/kg i.p.) and amperozide (5 mg/kg i.p.), and the selective dopamine (DA)-D2/D3 receptor antagonist raclopride (2 mg/kg i.p.). Whereas amperozide markedly elevated the numerical density of PPSOM mRNA expressing neurons in the mPFC (52%), the other drugs did not significantly affect PPSOM mRNA levels in any of the brain regions studied. Amperozide also altered PPT-A mRNA expression in the mPFC, i.e. a decrease (22%) was found. Of the other drugs tested only haloperidol significantly decreased PPT-A mRNA levels in the NAC shell (14%), in the dorso-lateral CP (19%) and in the medial CP (15%). In view of the differences between amperozide and the other drugs studied, as regards both pre-clinical and clinical characteristics, we suggest that the specific effects of amperozide on PPSOM and PPT-A mRNA in the mPFC may be related to its 5-HT releasing action in the frontal cortex, an effect possibly caused by its alpha2-adrenoceptor blocking activity. This effect, in turn, may be related to an antidepressant-like action that this compound exhibits in animal studies. The decrease in PPT-A mRNA levels seen after the haloperidol treatment is probably due to its potent DA-D2 receptor antagonism and may be related to side-effects, rather than therapeutic effects of this drug.

Differential actions of typical and atypical antipsychotic drugs on dopamine release in the core and shell of the nucleus accumbens.

The effects of acute administration of typical and atypical antipsychotic drugs on extracellular dopamine (DA) concentrations in brain were examined in two subdivisions of the nucleus accumbens (NAC), the core and the shell, which are largely associated with motor control and limbic functions, respectively, by using in vivo differential normal pulse voltammetry in anesthetized, pargyline pretreated rats. The following drugs were studied: haloperidol (0.1 and 1.0 mg/kg), clozapine (1.0 and 5.0 mg/kg), amperozide (1.0 and 2.0 mg/kg), risperidone (0.1 and 1.0 mg/kg), the selective 5-HT2A/5-HT2C receptor antagonist ritanserin (1 mg/kg) and the selective DA-D2/D3 receptor antagonist raclopride (10 and 320 micrograms/kg). Drugs with predominantly high 5-HT2 receptor antagonistic action, such as amperozide and ritanserin, as well as low doses of either risperidone or clozapine increased DA concentrations to a greater extent in the shell than in the core subdivision of the NAC. In contrast, drugs with a more potent D2 receptor antagonistic action, such as haloperidol and raclopride, as well as high doses of either risperidone or clozapine, elicited a larger DA increase in the core than in the shell. Consequently, atypical antipsychotics characterized by potent 5-HT2 receptor antagonism can be differentiated from typical antipsychotic drugs on the basis of their preferential effect on DA transmission in the shell region of the NAC.

Regulation of neuronal adenylate cyclase.

It appears that several components function in a spirit of integrated cooperation toward the intracellular regulation of neurotransmitter responsiveness. We have demonstrated that cytoskeletal proteins might interact with GNs and that GNs and GNi might interact with one another. At this juncture, it appears that both of these phenomena might occur only in cells of neural origin. Calmodulin and antidepressants may also affect adenylate cyclase in nervous tissue alone. The effects of AAGTP are different in nervous tissue from other tissues, and experiments with that nucleotide have led to the discovery of a new, 32 kDa GTP-binding protein which appears only in neural crest cells. Appreciation of the intricacies of signal transduction through the adenylate cyclase system are developing along with our understanding of that system. When combined with the complexity of neurotransmitter responsiveness, comprehension of the combined systems remains in its infancy, destined to grow as well as to surprise and delight all who are interested.

(Na+ + K+)-ATPase in artificial lipid vesicles: influence of the concentration of mono- and divalent cations on the pumping rate.

(Na+ + K+)-ATPase from kidney outer medulla was incorporated into artificial dioleoylphosphatidylcholine vesicles. Transport activity was induced by adding ATP to the external medium. A voltage-sensitive dye was used to detect the ATP-driven potassium extrusion in the presence of valinomycin. The observed substrate-protein interactions of the reconstituted (Na+ + K+)-ATPase largely agree with that from native tissues. An agreement between ATP hydrolysis and transport activity is given for concentration dependences of sodium, potassium, magnesium and calcium ions. The only significant deviations were observed in the influence of pH. Protons were found to have different influence on transport, enzymatic activity and phosphorylation of the enzyme. The transport studies showed a twofold interaction of protons with the protein: competition with sodium at the cytoplasmic ion binding sites, a non competitive inhibition of transport which is not correlated with protein phosphorylation.

Exchange of guanine nucleotide between GTP-binding proteins that regulate neuronal adenylate cyclase.

GTP-binding proteins have been demonstrated to stimulate and inhibit rat brain adenylate cyclase without the prior addition of hormone. Exposure of rat cerebral cortex membranes to hydrolysis-resistant GTP analogs results in inhibition (or stimulation) of adenylate cyclase, which persists subsequent to buffer washing. The hydrolysis-resistant GTP photoaffinity probe P3-(4-azidoanilido)-P1-5' GTP (AAGTP) can promote a similar persistent inhibition of adenylate cyclase, and, after removal of unbound AAGTP and subsequent UV photolysis, AAGTP is covalently linked to the 40-kDa inhibitory GTP binding protein, GNi (inhibitory guanine nucleotide binding regulatory subunit of adenylate cyclase). Under conditions where the persistent inhibition of adenylate cyclase is overcome by subsequent incubation with 5'-guanylyl imidodiphosphate or NaF, AAGTP bound to the 40-kDa GNi protein is diminished while that bound to the 42-kDa stimulatory GTP-binding protein (GNs) is increased. Additionally, we have identified a 32-kDa protein that binds AAGTP with an affinity similar to that of GNs. This protein does not appear to be a byproduct of proteolysis as demonstrated by Staphylococcus aureus V8 protease digestion experiments, and it is not a substrate for ADP-ribosylation by bacterial toxins. The sum of the AAGTP bound by the GNi and GNs proteins is constant, and the transfer of nonphotoactivated AAGTP to GNs from GNi is stable to buffer washing. Furthermore, this alteration in the AAGTP-labeling pattern corresponds to the shift in adenylate cyclase from inhibition to stimulation. These data raise the possibility that hydrolysis-resistant GTP analogs might be exchanged directly between the GNi and GNs and that there exists some interaction between those proteins in the regulation of adenylate cyclase activity.

Effects of the ATP, ADP and inorganic phosphate on the transport rate of the Na+,K+-pump.

(Na+ + K+)-ATPase from kidney outer medulla was incorporated into artificial dioleoylphosphatidylcholine vesicles. In the reconstituted system the pump can be activated by adding ATP to the external medium. ATP-driven potassium extrusion by the Na+,K+-pump was studied using a voltage-sensitive dye in the presence of valinomycin. ADP strongly reduced the turnover rate of the pump with a concentration for half-maximal inhibition of cD,1/2 = 0.1 mM. cD,1/2 was found to be virtually independent of ATP concentration, indicating that the inhibition is non-competitive with respect to ATP. The non-competitive inhibition by ADP can be explained on the basis of the Post-Albers reaction cycle of the Na+,K+-pump, assuming that the main action of ADP is the reversal of the phosphorylation step. A similar 'product inhibition' was observed with inorganic phosphate, but at much higher concentrations (cP,1/2 = 14 mM).

(Na+ + K+)-ATPase in artificial lipid vesicles: influence of lipid structure on pumping rate.

(Na+ + K+)-ATPase from kidney outer medulla was incorporated into tightly-sealed, single-shelled lipid vesicles by a detergent-dialysis procedure. The rate of ATP-driven potassium extrusion from vesicles formed from different phosphatidylcholines (PC) was measured optically, using a voltage-sensitive dye in the presence of valinomycin. High transport rates were observed for di(18:1)PC, di(20:1)PC and di(22:1)PC, whereas vesicles formed from di(14:1)PC and di(16:1)PC were virtually inactive. The variation of pumping activity with lipid structure mainly results from differences in the amount of enzyme incorporated with the correct orientation into the vesicle membrane, and to a lesser extent from lipid-dependent variations of the intrinsic turnover rate of the enzyme. The activation energy of ion transport decreases in the order di(16:1)PC, di(18:1)PC, di(20:1)PC approximately equal to di(22:1)PC.

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase.

A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K+ so that the membrane voltage approaches the Nernst potential for K+. With constant external K+ concentration, the time course of internal K+ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K+ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.

Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal membranes.

The ability of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer into the intravesicular space was investigated by using trypsin as a proteolytic probe. We found that under defined conditions trypsin is able to dissect the translocation activity of microsomal membranes into components that can be separated into two fractions, one soluble and the other membrane bound. The trypsinized membrane fraction has lost its translocation activity. Addition of the trypsin-generated soluble fraction, however, results in reconstitution of translocation activity. These results are compatible with the notion proposed in the signal hypothesis that the translocation activity of the microsomal membrane resides in transmembrane protein(s). We propose that trypsin effects solubilization from the membrane of cytosol-exposed domain(s) involved in recognition of the signal sequence or ribosome or both, leaving behind membrane-integrated domain(s) that provide the environment for the passage of the nascent chain across the membrane. Signal peptidase activity was unaffected by trypsinization of microsomal vesicles consistent with a localization of the active site of this enzyme on the cisternal side of the vesicles.

Part II. Behavioral and electrophysiological characteristics of the addicted neonate.

The neonatal withdrawal period was characterized by heightened auditory responsiveness and orientation, lowered overall alertness and poor attentiveness to and following of visual stimuli. Electroencephalographic recordings revealed high-frequency dysynchronous activity suggestive of cns irritability. Analysis of evoked response data further corroborated the behavioral findings with evidence for low arousal value of visual stimulation in the vertex frequency characteristics and poorly defined occipital responses. Auditory evoked responses appeared better integrated, and a significant correlation was found between auditory orienting ability and latency of the P2 component. The long-range developmental significance of these neonatal characteristics awaits further follow-up investigation.

Self-stimulation alters human sensory brain responses.

Human electrocortical potentials evoked by self-administered auditory and visual stimuli manifest much smaller amplitude and faster poststimulus timing than do average brain responses evoked by identical machine-delivered stimuli. Auditory evoked potentials show this "self-stimulation effect" to a greater degree than do visual responses. For visual evoked potentials, the effect appears greater at the vertex association area than over the occipital cortex. Individual differences in the magnitude of the "self-stimutlation effect" relate to level of intelligence.