Andrea Balla

High dose D-serine in the treatment of schizophrenia

BACKGROUND D-serine is an allosteric modulator of the brain N-methyl-d-aspartate (NMDA) receptor and a potential novel treatment of schizophrenia. Double-blind studies have been performed at 30 mg/kg/day (approximately 2 g/day) with encouraging results, but no formal dose escalation studies have been performed. We describe the first evaluation of the efficacy and safety of d-serine at doses >30 mg/kg/day; a 4-week, open-label trial of adjunctive D-serine (30, 60 or 120 mg/kg/day). METHODS 42 antipsychotic-stabilized patients with schizophrenia or schizoaffective disorder participated. PANSS was obtained bi-weekly and neuropsychological (MATRICS) was obtained pre- and post medication phase. The pharmacokinetics/pharmacodynamics (PK/PD), and safety of doses> or =30 mg/kg was also evaluated. RESULTS Significant improvement in symptoms and neuropsychological measures was noted across doses. On the PANSS, improvement was observed for positive (p=0.006;d=0.46), negative (p<0.001;d=0.68), general (p=0.001;d=0.53), and total (p<0.0001;d=0.74) symptoms. On MATRICS, while only non-significant improvement was noted at 30 mg/kg, highly significant, large effect size improvement was noted on the composite score (p<0.01;d=1.0) for doses> or =60 mg/kg, leading to a significant dose-by-time interaction (p<0.01). In PK analyses, significant dose-dependent increases in plasma D-serine levels were seen during the study, predictive of significantly increased brain levels. Furthermore, increases in plasma levels correlated with improved symptomatic and neuropsychological function. DISCUSSION These findings support double-blind investigation of D-serine at doses> or =60 mg/kg/d, and suggest effectiveness in treatment of both persistent symptoms and neurocognitive dysfunction.

Inhibition of System A-mediated glycine transport in cortical synaptosomes by therapeutic concentrations of clozapine: implications for mechanisms of action

Clozapine is an atypical antipsychotic with particular efficacy in schizophrenia, possibly related to potentiation of brain N-methyl-D-aspartate receptor (NMDAR) -mediated neurotransmission. NMDARs are regulated in vivo by glycine, which is regulated in turn by glycine transporters. The present study investigates transport processes regulating glycine uptake into rat brain synaptosomes, along with effects of clozapine on synaptosomal glycine transport. Amino-acid uptake of amino acids was assessed in rat brain P2 synaptosomal preparations using a radiotransport assay. Synaptosomal glycine transport was inhibited by a series of amino acids and by the selective System A antagonist MeAIB (2-methyl-aminoisobutyric acid). Clozapine inhibited transport of both glycine and MeAIB, but not other amino acids, at concentrations associated with preferential clinical response (0.5–1 μg/ml). By contrast, other antipsychotics studied were ineffective. The novel glycine transport inhibitor N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine (NFPS) produced biphasic inhibition of [3H]glycine transport, with IC50 values of approximately 25 nM and 25 μM, respectively. NFPS inhibition of [3H]MeAIB was monophasic with a single IC50 value of 31 μM. Clozapine significantly inhibited [3H]glycine binding even in the presence of 100 nM NFPS. In conclusion, this study suggests first that System A transporters, or a subset thereof, may play a critical role in regulation of synaptic glycine levels and by extension of NMDA receptor regulation, and second that System A antagonism may contribute to the differential clinical efficacy of clozapine compared with other typical or atypical antipsychotics.

Reversal of phencyclidine-induced dopaminergic dysregulation by N-methyl-D-aspartate receptor/glycine-site agonists.

N-methyl-D-aspartate (NMDA) receptors may play a critical role in the pathophysiology of schizophrenia. In rodents, NMDA receptor antagonists, such as phencyclidine (PCP), induce dopaminergic dysregulation that resembles the pattern observed in schizophrenia. The present study investigates the degree to which concurrent treatment with NMDA modulators, such as glycine and the recently developed glycine transport antagonist N[3-(4″-fluorophenyl)-3-(4″-phenylphenoxy)propyl]sarcosine (NFPS) prevents dopaminergic dysregulation observed following chronic (3 months) or subchronic (2 weeks) PCP administration. Both chronic and subchronic treatment with PCP in the absence of glycine or NFPS led to significant potentiation of amphetamine-induced dopamine release in the prefrontal cortex and striatum, similar to that observed in schizophrenia. Treatment with either high-dose glycine or NFPS along with PCP prevented PCP effects. These findings demonstrate effective doses of glycine for use in animal models of schizophrenia, and support recent clinical studies showing the effectiveness of NMDA agonists in the treatment of persistent symptoms of schizophrenia.

Continuous Phencyclidine Treatment Induces Schizophrenia-Like Hyperreactivity of Striatal Dopamine Release

Functional dopaminergic hyperactivity is a key feature of schizophrenia. Recent in vivo imaging studies have demonstrated greater striatal dopamine release in response to amphetamine challenge in schizophrenia subjects than in normal controls. N-methyl-D-aspartate (NMDA) receptors are known to play a prominent role in regulation of striatal dopamine release. In humans, NMDA antagonists induce a psychotic state that closely resembles schizophrenia. The present study investigates the degree to which chronic continuous administration of the NMDA antagonist phencyclidine (PCP) induces schizophrenia-like hyperreactivity of striatal dopamine release to amphetamine in rodents. Rats were treated with 10 or 15 mg/kg/d PCP for two weeks by osmotic minipump, and striatal dopamine release to amphetamine challenge (1 mg/kg) was monitored by microdialysis. PCP-treated rats showed significant enhancement in amphetamine-induced dopamine release, along with significantly enhanced locomotor activity. These findings support the concept that NMDA receptor dysfunction may contribute to dopaminergic dysfunction in schizophrenia.

Subchronic continuous phencyclidine administration potentiates amphetamine-induced frontal cortex dopamine release.

Functional dopaminergic hyperactivity is a key feature of schizophrenia. Etiology of this dopaminergic hyperactivity, however, is unknown. We have recently demonstrated that subchronic phencyclidine (PCP) treatment in rodents induces striatal dopaminergic hyperactivity similar to that observed in schizophrenia. The present study investigates the ability of PCP to potentiate amphetamine-induced dopamine release in prefrontal cortex (PFC) and nucleus accumbens (NAc) shell. Prefrontal dopaminergic hyperactivity is postulated to underlie cognitive dysfunction in schizophrenia. In contrast, the degree of NAc involvement is unknown and recent studies have suggested that PCP-induced hyperactivity in rodents may correlate with PFC, rather than NAc, dopamine levels. Rats were treated with 5–20 mg/kg/day PCP for 3–14 days by osmotic minipump. PFC and NAc dopamine release to amphetamine challenge (1 mg/kg) was monitored by in vivo microdialysis and HPLC-EC. Doses of 10 mg/kg/day and above produced serum PCP concentrations (50–150 ng/ml) most associated with PCP psychosis in humans. PCP-treated rats showed significant, dose-dependent enhancement in amphetamine-induced dopamine release in PFC but not NAc, along with significantly enhanced locomotor activity. Enhanced response was observed following 3-day, as well as 14-day, treatment and resolved within 4 days of PCP treatment withdrawal. These findings support the concept that endogenous NMDA receptor dysfunction could account for the pattern of dopaminergic dysfunction observed in schizophrenia, and suggest that even short duration abuse of PCP-like agents may greatly potentiate behavioral effects of psychostimulants in drug abuse situations. Finally, these studies provide a model system in which to evaluate effects of potential psychotherapeutic agents.

GABAB/NMDA receptor interaction in the regulation of extracellular dopamine levels in rodent prefrontal cortex and striatum

Deficits in N-methyl-D-aspartate receptor (NMDAR)-mediated neurotransmission may underlie dopaminergic hyperactivity in schizophrenia. Dysregulation of the GABAergic system has also been implicated. In this study we investigated a role for GABA(B) receptors as an intermediate step in the pathway leading from NMDAR stimulation to DA regulation. Since glycine (GLY) has been found to ameliorate treatment resistant negative symptoms in schizophrenia, we treated a group of rats with 16% GLY food for 2 weeks. DA levels in prefrontal cortex (PFC) and striatum (STR) were assessed by dual-probe microdialysis and HPLC-EC in freely moving rats. Infusion of the GABA(B) receptor agonists SKF97541 and baclofen into PFC and STR significantly reduced basal DA, an effect that was reversed by the antagonist, CGP52432. In PFC, GABA(B) agonists also reduced AMPH-induced DA release following treatment with either 1 or 5 mg/kg AMPH. Similar effects were seen following subchronic glycine treatment in the absence, but not presence of CGP52432 during 5 mg/kg AMPH treatment. In STR SKF97541 decreased only the 1 mg/kg AMPH-induced DA release. Subchronic GLY treatment in STR leads to a significant reduction in basal DA levels, but did not affect AMPH (5 mg/kg)-induced release. Our findings support a model in which NMDA/glycine-site agonists modulate DA release in part through presynaptic GABA(B) receptors on DA terminals, with both GABA(B) ligands and GLY significantly modulating AMPH-induced DA release. Both sites, therefore, may represent appropriate targets for drug development in schizophrenia and substance abuse disorders.

A novel alanine-insensitive D-serine transporter in rat brain synaptosomal membranes.

D-Serine is an endogenous modulator of brain N-methyl-D-aspartate receptors. This study investigates serine transport in brain synaptosomal fractions. Saturable, temperature-dependent uptake of both D- and L-[3H]serine was observed. Alanine was only partially effective in blocking transport, arguing against involvement of system ASC. Inhibitors of the systems A, L and GLY were also ineffective. Saturation studies suggested a submillimolar K(m) for both D- and L-serine. These data suggest the presence of a novel serine transport system in rodent synaptosomes.

Functional connectivity fMRI in mouse brain at 7T using isoflurane.

Although many resting state fMRI human studies have been published, the number of such rodent studies is considerably less. The reason for this is the severe technical challenge of high magnetic field small rodent imaging. Local magnetic field susceptibility changes at air tissue boundaries cause image distortion and signal losses. The current study reports measures of functional connectivity in mice using only isoflurane for the anesthetic. Because all anesthetic agents will alter cerebral blood flow and cerebral metabolism, the impact these changes have on neuronal connectivity has yet to be fully understood, however this work reports for the first time that reliable functional connectivity measures in mouse brain can be obtained with isoflurane.

Characterization of interactions between phencyclidine and amphetamine in rodent prefrontal cortex and striatum: implications in NMDA/glycine-site-mediated dopaminergic dysregulation and dopamine transporter function.

N-Methyl-D-aspartate (NMDA) antagonists induced behavioral and neurochemical changes in rodents that serve as animal models of schizophrenia. Chronic phencyclidine (PCP, 15 mg/(kg day) for 3 weeks via Alzet osmotic pump) administration enhances the amphetamine (AMPH)-induced dopamine (DA) efflux in prefrontal cortex (PFC), similar to that observed in schizophrenia. NMDA/glycine-site agonists, such as glycine (GLY), administered via dietary supplementation, reverse the enhanced effect. The present study investigated mechanisms of glycine-induced reversal of PCP-induced stimulation of AMPH-induced DA release, using simultaneous measurement of DA and AMPH in brain microdialysate, as well as peripheral and tissue AMPH levels. PCP treatment, by itself, increased peripheral and central AMPH levels, presumably via interaction with hepatic enzymes (e.g. cytochrome P450 CYP2C11). GLY (16% diet) had no effect on peripheral AMPH levels in the presence of PCP. Nevertheless, GLY significantly reduced extracellular/tissue AMPH ratios in both PFC and striatum (STR), especially following PCP administration, suggesting a feedback mediated effect on the dopamine transporter. GLY also inhibited acute AMPH (5 mg/kg)-induced DA release in PFC, but not STR. These findings suggest that GLY may modulate DA release in brain by producing feedback regulation of dopamine transporter function, possibly via potentiation of NMDA-stimulated GABA release and presynaptic GABAB receptor activation. The present studies also demonstrate pharmacokinetic interaction between AMPH and PCP, which may be of both clinical and research relevance.

Physiological release of striatal acetylcholine (in vivo): effect of somatostatin on dopaminergic-cholinergic interaction

The effects of somatostatin (SOM) on the release of acetylcholine (ACh) and dopamine (DA) from striatum of freely moving rats were studied by transversal microdialysis. Acetylcholine (ACh) and dopamine (DA) were detected by high performance liquid chromatography (HPLC) with electrochemical detection. Somatostatin (0.1, 0.5 and 1 microM), administered locally through the microdialysis probe to the striatum, was able to release dose-dependently ACh from the cholinergic neurons of the striatum. The increase in the extracellular levels of ACh produced by 1 microM SOM in the striatum reached a maximum of 200%. ACh-releasing effect of SOM was completely inhibited by tetrodotoxin indicating that neuronal firing is involved in its effect. Local infusion of sulpiride, 10 microM, D(2) receptor antagonist, potentiated (about 100%) the SOM (1 microM)-induced release of ACh. SOM, 1 microM, was more effective in enhancing the release of ACh in the striatum (two-fold increase) after degeneration of the nigrostriatal DA pathway with 6-hydroxydopamine (6-OHDA) (250 microg/animal, i.c.v.). The D(2) receptor agonists bromcriptine, 10 microM, or apomorphine, 10 microM, completely antagonize SOM-induced release. SOM, 1 microM, enhanced the release of DA (about 400%). These findings indicate that SOM is capable of releasing both ACh and DA in the striatum, however, its effect on ACh release is partially masked unless the D(2) receptor-mediated tonic inhibitory effect of released DA from the nigro-striatal pathway is attenuated.