A mouse model of the schizophrenia-associated 1q21.1 microdeletion syndrome exhibits altered mesolimbic dopamine transmission.

1q21.1 hemizygous microdeletion is a copy number variant leading to eightfold increased risk of schizophrenia. In order to investigate biological alterations induced by this microdeletion, we generated a novel mouse model (Df(h1q21)/+) and characterized it in a broad test battery focusing on schizophrenia-related assays. Df(h1q21)/+ mice displayed increased hyperactivity in response to amphetamine challenge and increased sensitivity to the disruptive effects of amphetamine and phencyclidine hydrochloride (PCP) on prepulse inhibition. Probing of the direct dopamine (DA) pathway using the DA D1 receptor agonist SKF-81297 revealed no differences in induced locomotor activity compared to wild-type mice, but Df(h1q21)/+ mice showed increased sensitivity to the DA D2 receptor agonist quinpirole and the D1/D2 agonist apomorphine. Electrophysiological characterization of DA neuron firing in the ventral tegmental area revealed more spontaneously active DA neurons and increased firing variability in Df(h1q21)/+ mice, and decreased feedback reduction of DA neuron firing in response to amphetamine. In a range of other assays, Df(h1q21)/+ mice showed no difference from wild-type mice: gross brain morphology and basic functions such as reflexes, ASR, thermal pain sensitivity, and motor performance were unaltered. Similarly, anxiety related measures, baseline prepulse inhibition, and seizure threshold were unaltered. In addition to the central nervous system-related phenotypes, Df(h1q21)/+ mice exhibited reduced head-to tail length, which is reminiscent of the short stature reported in humans with 1q21.1 deletion. With aspects of both construct and face validity, the Df(h1q21)/+ model may be used to gain insight into schizophrenia-relevant alterations in dopaminergic transmission.

Effects of exogenous alpha-synuclein on stimulated dopamine overflow in dorsal striatum.

Alpha-synuclein (α-syn) is mainly a presynaptic protein that has been implicated in Parkinson's disease and various other neurodegenerative disorders. Evidence obtained in knockout mice suggests that α-syn controls plasticity of dopamine (DA) overflow in presynaptic terminals. It is also believed that α-syn spreads and may seed its aggregates from cell to cell. The effects of exogenously applied α-syn on dopaminergic neurotransmission have not been studied. We addressed this issue by microinjecting human α-syn protein into the dorsal striatum of wild-type and α-syn knockout mice and monitoring stimulated DA overflow with constant potential amperometry. The evoked DA overflow was decreased in knockout mice six days after α-syn microinjection. The maximal velocity of DA re-uptake was reduced in both genotypes. Similar results were not seen when the effects of microinjected α-syn were studied immediately after the treatment, but instead there was a trend toward an increase in both stimulated DA overflow and maximal velocity of DA re-uptake. We conclude that locally applied human α-syn affects DA overflow and the effects depend on the presence of endogenous α-syn.

Characterization of a 32 µm diameter carbon fiber electrode for in vivo fast-scan cyclic voltammetry.

Carbon fiber electrodes (CFE) are commonly used for in vivo detection of catecholamines due to their excellent electrochemical properties and biocompatibility. Fast-scan cyclic voltammetry (FSCV) combined with CFEs permits the detection of catecholamines such as dopamine (DA) with high specificity and reliability. However, advances in neuroscience constantly demand sensors with greater sensitivities and selectivities. This study investigated an untreated CFE of 32 µm diameter and 300 µm exposed length for detection of DA using FSCV. Despite the larger area of the working electrode, we observed only a small increase in the background current in comparison with the commonly used CFE of 7 µm diameter and 100 µm exposed length. The sensitivity of the 32 µm CFE was 9 times greater than that of the 7 µm CFE. These larger electrodes exhibited good linearity and a 6 fold higher signal-to-noise ratio than 7 µm CFEs in vitro. The 32 µm CFE showed significantly better selectivity for DA in preference to 3,4-dihydroxyphenylacetic acid than the 7 µm CFE and similar selectivity to the 7 µm CFE for ascorbic and homovanillic acid. The electrodes displayed good temporal resolution and electrochemical stability in both in vitro and in vivo tests.

Methylphenidate modifies overflow and presynaptic compartmentalization of dopamine via an α-synuclein-dependent mechanism.

Methylphenidate (MPD) modulates dopamine (DA) overflow in part by redistributing vesicle pools, a function shared by the presynaptic protein α-synuclein (α-syn). We suggest that α-syn modifies the effect of MPD on DA neurotransmission. The effect was studied in the dorsal striatum in wild-type mice and two mouse lines lacking α-syn by using in vivo voltammetry and microdialysis. MPD (1 mg/kg) attenuated evoked DA overflow only in mice lacking α-syn but produced a similar increase in the extracellular DA levels in all three lines. A kinetic analysis showed that MPD decreased DA release per stimulus pulse in α-syn-deficient mice but increased in wild-type mice. MPD blocked DA reuptake and produced a similar increase in the apparent affinity (K(m)) for DA reuptake in all three lines. Repeated-burst stimulation redistributes vesicular storage pools and facilitates DA overflow, and this form of facilitation is significantly enhanced in α-syn knockout mice. The DA reuptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) (10 mg/kg) completely blocked the facilitation of DA overflow in all three lines, whereas MPD (1 mg/kg) selectively decreased it only in mice lacking α-syn. MPD (5 mg/kg) and GBR12909 (10 mg/kg) produced equipotent inhibition of DA reuptake (in terms of K(m)), indicating that reuptake inhibition does not explain the MPD selectivity. Our data indicate that MPD decreases DA release probability in the absence of α-syn and increases it in control animals, whereas the effect of MPD on DA reuptake is independent of α-syn. We suggest that this selectivity is based on α-syn-dependent compartmentalization of presynaptic DA.

Sub-regional differences and mechanisms of the short-term plasticity of dopamine overflow in striatum in mice lacking alpha-synuclein.

Mice lacking the pre-synaptic protein alpha-synuclein (α-syn) demonstrate enhanced facilitation of dopamine (DA) overflow in dorsal striatum following repeated, high-frequency burst stimulation of the dopaminergic pathways. Dorsal striatum is most vulnerable to neurodegeneration in Parkinson's disease. The role of α-syn in facilitation of DA overflow in the ventral striatum, which is less vulnerable to neurodegeneration, is unknown. We investigated the link between the absence of α-syn and the plasticity of DA overflow in the dorsal and ventral striatum by in vivo voltammetry and the possible mechanisms of modulation of the plasticity of DA overflow. We show that the facilitation of DA overflow following paired-burst stimulation is significantly enhanced in the dorsolateral but not in the ventral striatum of mice lacking α-syn. Re-uptake inhibitor, GBR12909, completely eliminated the facilitation of DA overflow regardless of the presence of α-syn in both dorsal and ventral striatum, indicating that re-uptake is critical for maintenance of paired-burst facilitation (PBF). Inhibition of D2 autoreceptors by haloperidol decreased PBF only in mice lacking α-syn. However, the basal function of D2 autoreceptors tested by paired-pulse depression of DA overflow was not different between the lines. Therefore, alterations in the D2 autoreceptor system do not explain the different effect of haloperidol on PBF in mice with and without α-syn. This indicates that neither re-uptake nor D2 autoreceptors differentiate the PBF between the genotypes. We propose that modification of DA vesicles in α-syn knockout mice, as reported in several studies, may be a factor underlying the enhanced PBF in these mice.

Decreased reuptake of dopamine in the dorsal striatum in the absence of α-synuclein.

The presynaptic protein alpha-synuclein (α-syn) plays a role in dopaminergic neurotransmission in the nigrostriatal dopaminergic system. Mutations in this protein have been linked to pathogenesis of Parkinson's disease. However, the details of regulation of dopamine homeostasis by α-syn and its molecular targets are generally unknown. We investigated the effect of α-syn deletion on striatal dopaminergic homeostasis. Two α-syn deficient mouse lines, one carrying a spontaneous deletion of α-syn locus and the other a transgenic α-syn knockout, were used in the study. Stimulated and basal extracellular dopamine levels were determined in the dorsal striatum by in vivo voltammetry and in vivo microdialysis, respectively. Dopamine transporter expression was studied by immunohistochemistry. Stimulated dopamine overflow and basal extracellular dopamine levels were higher in mice lacking α-syn with a concomitant decrease in dopamine transporter expression and reuptake in the dorsal striatum. We show that α-syn deletion produces significant adaptive changes in the striatal dopaminergic system via modulation of reuptake.

Dynamic regulation of dopamine and serotonin responses to salient stimuli during chronic haloperidol treatment.

Antipsychotic drugs are the clinical standard for the treatment of schizophrenia. Although these drugs work initially, many compliant patients relapse due to treatment failure. The known biomarkers can not sufficiently explain antipsychotic treatment failure. We, therefore, enquired how the dynamic responses of the neurotransmitters, dopamine and serotonin, change in relation to treatment action and failure. Rats received either short-term (2-6 d) or long-term (12-14 d) treatment with haloperidol, which resembled human D2 receptor occupancy, using osmotic mini-pumps. Dopamine and serotonin basal levels and responses to novelty, appetitive food, and to an aversive tail pinch were measured in the prefrontal cortex, nucleus accumbens and caudate putamen using in-vivo microdialysis, and the behaviour was recorded. Subsequently, we used in-vivo voltammetry to measure dopamine overflow in the nucleus accumbens. Haloperidol decreased dopamine, but not serotonin baseline levels in a time-dependent way. Salient stimuli induced dopamine and serotonin responses. Short-term haloperidol treatment attenuated the mesolimbic dopamine responses to aversive stimulation, while the responses to appetitive stimulation were largely preserved. After long-term treatment, the initial response adaptations were reversed. Similar changes were also observed at the behavioural level. In-vivo voltammetry showed that nucleus accumbens dopamine adaptations and their reversal were mediated by changes in extracellular dopamine release. Chronic haloperidol treatment, which resembles human D2 receptor occupancy, modulates dopamine and behavioural responses to aversive and appetitive stimulation depending on the duration of treatment. Specific changes in dopamine response dynamics and their reversal may be a functional substrate of antipsychotic action and failure respectively.

Neuromuscular pathology in mice lacking alpha-synuclein.

This work was undertaken in order to study the possible role of alpha-synuclein in the function of the neuro-muscular junction in skeletal muscles. Repeated stimulation of skeletal muscle motor neurons revealed signs of neuromuscular pathology in alpha-synuclein null mutated (C57Bl/6JOlaHsd) and knockout (B6;129X1-Snca(tm1Rosl)/J) mice. This stimulation produced repetitive compound muscle action potentials in both lines of alpha-synuclein deficient mice. Muscle strength and muscle coordination during ambulation were unaffected, though motor learning was slower in alpha-synuclein deficient mice in the Rotarod test. We conclude that alpha-synuclein may play a role in acetylcholine compartmentalization at the neuromuscular junction, and in the fine control of activity of skeletal muscles.

Stimulated dopamine overflow and alpha-synuclein expression in the nucleus accumbens core distinguish rats bred for differential ethanol preference.

The key neurochemical systems and structures involved in the predisposition to substance abuse and preference to ethanol (EtOH) are not known in detail but clearly dopamine (DA) is an important modulator of addiction. Recent data indicate that alpha-synuclein (alpha-syn), a pre-synaptic protein, plays a role in regulation of DA release from the pre-synaptic terminals in striatum and the expression of this protein is different after drug abuse or following abstinence. In the present work, we analysed stimulated DA overflow in the dorsal and ventral striatum in EtOH naïve alko alchohol (AA) and alko non-alchohol (ANA) rats selected for more than 100 generations for their differential EtOH preference. In the same structures, we studied the expression of alpha-syn using western blotting. AA rats, in comparison with ANA rats, showed a marked reduction of stimulated peak DA overflow and higher levels of alpha-syn in the nucleus accumbens core. In the same structure, DA re-uptake was increased in AA rats in comparison with ANA rats. The effects of EtOH at low (0.1 g/kg) and higher (3 mg/kg) doses on DA overflow measured in the nucleus accumbens shell were similar in both lines. These results indicate that high expression of alpha-syn may contribute to the reduced DA overflow and the possible activation of re-uptake in the nucleus accumbens core of AA rats in comparison with ANA rats.

Behavioural and neurochemical response of alpha-synuclein A30P transgenic mice to the effects of L-DOPA.

Transgenic mice carrying human A30P mutated alpha-synuclein demonstrate hypolocomotion and dysfunction of the presynaptic machinery of dopamine overflow, induced by reducing capacity of the dopamine storage pool. We suggested that overexpression of alpha-synuclein may change sensitivity of these mice to L-DOPA. Current study assessed behavioural and neurochemical responses in A30P mice to L-DOPA using automated activity monitoring and voltammetry. We confirmed decreased locomotion and rearing of A30P transgenic mice compared to wild-type controls. L-DOPA (10-200mg/kg, i.p.) dose-dependently lowered locomotor activity, including stereotypy, in both genotypes, but the effects were larger in A30P mice. The effects of drug on stimulated dopamine overflow were investigated in the nucleus accumbens shell. L-DOPA at the dose of 30mg/kg did not change peak dopamine overflow induced by 10Hz stimulation of the medial forebrain bundle in either genotype, but increased it at the higher (20-50Hz) frequencies of stimulation. At the higher frequencies of stimulation, L-DOPA elevated dopamine overflow significantly more in A30P mice than in the control animals. These data show that A30P transgenic mice are more sensitive to the effects of L-DOPA at both behavioural and neurochemical level.

Characterization of the striatal dopaminergic neurotransmission in MEN2B mice with elevated cerebral tissue dopamine.

The Ret receptor tyrosine kinase is the common signaling receptor for the glial cell line-derived neurotrophic factor (GDNF) family ligands. The Met918Thr mutation leads to constitutive activation of Ret and is responsible for dominantly inherited cancer syndrome MEN2B. Previously, we found that the mice carrying the mutation (MEN2B mice) have profoundly increased tissue dopamine (DA) concentrations in the striatum as well as increased striatal levels of tyrosine hydroxylase (TH) and dopamine transporter. The aim of this study was to characterize the striatal dopaminergic neurotransmission in MEN2B mice and to clarify the mechanisms by which they compensate their over-production of DA. We found that tyrosine hydroxylase activity and DA synthesis are increased in MEN2B mice. Augmented effects of alpha-methyl-para-tyrosine (alphaMT, an inhibitor of TH) and tetrabenazine (VMAT2 blocker) on DA levels suggest that also storage of DA is increased in MEN2B mice. There was no difference in the basal extracellular DA concentrations or potassium-evoked DA release between the genotypes. The effects of cocaine and haloperidol were also similar between the genotypes as assessed by in vivo microdialysis. However, with in vivo voltammetry we found increase in stimulated DA release in MEN2B mice and detailed analysis of DA overflow showed that uptake of DA was also enhanced in MEN2B mice. Thus, our data show that enhanced synthesis of DA leading to increased storage and releasable pools in pre-synaptic terminals in MEN2B mice apparently also leads to increased DA release, which in turn is compensated by higher dopamine transporter activity.

Site-specific role of catechol-O-methyltransferase in dopamine overflow within prefrontal cortex and dorsal striatum.

Accumulating evidence from clinical and preclinical studies shows that catechol-O-methyltransferase (COMT) plays a significant role in dopamine metabolism in the prefrontal cortex, but not in the striatum. However, to what extent dopamine overflow in the prefrontal cortex and striatum is controlled by enzymatic degradation versus reuptake is unknown. We used COMT deficient mice to investigate the role of COMT in these two brain regions with in vivo voltammetry. A real-time analysis of evoked dopamine overflow showed that removal of dopamine was twofold slower in the prefrontal cortex of mice lacking COMT than in wild-type mice, indicating that half of the dopamine decline in this brain region results from COMT-mediated enzymatic degradation. Lack of COMT did not influence dopamine overflow/decline in the dorsal striatum. COMT-deficient mice demonstrated a small (20-25%) but consistent increase in evoked dopamine release in the prefrontal cortex, but not in the dorsal striatum. Cocaine affected equally dopaminergic neurotransmission in the prefrontal cortex in both genotypes by prolonging 3-4 times dopamine elimination from extracellular space. Paradoxically, this happened without increase of the peak levels of evoked dopamine release. The present findings represent the first demonstration of the significant contribution of COMT in modulating the dynamics of dopamine overflow in the prefrontal cortex and underscore the therapeutic potential of manipulating COMT activity to alter dopaminergic neurotransmission in the prefrontal cortex.

Mechanics of self-stimulation and dopamine release in the nucleus accumbens.

Robust self-stimulation can be obtained from electrodes implanted in the medial forebrain bundle. We used in-vivo voltammetry to monitor stimulated dopamine release in the mouse nucleus accumbens during implantation of the stimulating electrodes. The higher the level of stimulated dopamine release during electrode implantation, the lower was the threshold for self-stimulation and the shorter the duration of the stimulation train when it was controlled by animal. We suggest that dopamine release is a reliable indicator of the proximity of the stimulating electrode to the brain reward sites. Inclusion of this indicator solves the problem of large interindividual variation in self-stimulation currents and permits a new approach to studies on mechanisms and pathways involved in brain reward.

Abnormal compartmentalization of norepinephrine in mouse dentate gyrus in alpha-synuclein knockout and A30P transgenic mice.

In the dentate gyrus of the mouse hippocampus, presynaptic recruitment of norepinephrine in response to repeated-burst stimulation can be described in terms of an interaction between storage and readily releasable pools. The dynamics of this interaction depends on neuronal activity (bursting), so that the higher the demand for norepinephrine, the faster it is delivered from the storage pool. We also found that alpha-synuclein, a presynaptic protein that plays a crucial role in dopamine compartmentalization in the striatum, is also involved in the compartmentalization of norepinephrine in the dentate gyrus. Experiments in transgenic mice with modified or absent alpha-synuclein revealed that the familial Parkinson's disease-linked alpha-synuclein mutation A30P can cause selective changes in the function of noradrenergic terminals. Addition of mutated human alpha-synuclein abolished the normal norepinephrine mobilization. There were no compensatory mechanisms available in the norepinephrine presynaptic terminals. In contrast, deletion of mouse alpha-synuclein is compensated for by increased vesicle transport from the storage pool. The effects are essentially the same as previously reported for dopaminergic terminals in the striatum, indicating that the important role of alpha-synuclein in neurotransmitter mobilization is not limited to dopaminergic terminals.

Brain reward in the absence of alpha-synuclein.

Alpha-synuclein has been implicated in the pathophysiology of Parkinson's disease. Recent studies revealed its role as a negative regulator of dopamine release in the nigrostriatal dopaminergic system. Alpha-synuclein may, however, play a more universal role in dopaminergic neurotransmission. It may represent an endogenous modulator in the mesolimbic dopaminergic system, and be involved in brain reward. We show here that the absence of alpha-synuclein resulting from spontaneous mutation in a subline of C57BL/6J mice greatly increased the rate of operant behavior during intracranial self-stimulation. The present work demonstrates that a lack of alpha-synuclein sensitized the brain reward system, implying that the levels of alpha-synuclein expression may predispose an individual to drug abuse or to a number of psychiatric diseases.

Locomotor activity and evoked dopamine release are reduced in mice overexpressing A30P-mutated human alpha-synuclein.

We have generated a transgenic mouse line overexpressing mutated human A30P alpha-synuclein under the control of the prion-related protein promoter. Immunohistology revealed mutated human A30P alpha-synuclein protein in numerous brain areas, but no gross morphological changes, Lewy bodies, or loss of dopaminergic cell bodies. The transgenic mice displayed decreased locomotion, impaired motor coordination, and balance. In vivo voltammetry showed that A30P mice responded to longer stimulation of the ascending dopaminergic pathways with less dopamine release in striatum and had a slower rate of dopamine decline after repeated stimulations or after alpha-methyl-p-tyrosine-HCl treatment. However, dopamine re-uptake or transporter levels were similar in transgenic and control mice. Our data provide evidence that overexpression of mutated human A30P alpha-synuclein in mice leads to a reduced size of the dopamine storage pool. This is in agreement with the previously postulated involvement of alpha-synuclein in the turnover of transmitter vesicles and may explain the observed motor deficits in A30P mice.

Noradrenaline overflow in mouse dentate gyrus following locus coeruleus and natural stimulation: real-time monitoring by in vivo voltammetry.

The pattern of catecholaminergic innervation of the dentate gyrus (DG) of the hippocampus, particularly the relatively dense and selective noradrenergic input, creates favourable conditions for real-time monitoring of noradrenaline (NA) release following stimulation of the locus coeruleus (LC) by in vivo voltammetry. Two electrochemically active species with different temporal characteristics were registered in the DG following electrical stimulation of the LC. Several approaches, including testing of anatomical and pharmacological specificity, coating of microelectrodes with Nafion and use of fast cyclic voltammetry, were used to verify the characteristics of electrochemical responses. The first sharp peak that appeared immediately during stimulation was definitely associated with NA overflow. The second late peak was possibly attributable to ascorbic acid. We examined the characteristics of alpha-2 adrenoceptor regulation of NA release in the DG, and showed for the first time that noradrenergic terminals resemble dopaminergic terminals in their mechanisms of increasing the refilling rate of the readily releasable pool following stimulation repeated at short intervals. Amperometric registration of NA in the DG was complicated by interference with electrical activity of hippocampus. This interference could be used, after appropriate filtration, for simultaneous recording from the same microelectrode of NA release and electrical activity of the hippocampus.

Role of alpha-synuclein in presynaptic dopamine recruitment.

Real-time monitoring of stimulated dopamine release in mice with different alpha-synuclein expression was used to study the role of alpha-synuclein in presynaptic dopamine recruitment. Repeated electrical stimulations of ascending dopaminergic pathways decreased the capacity of the readily releasable pool (RRP) and temporarily increased its refilling rate, significantly slowing the rate of dopamine decline in mice with normally expressed alpha-synuclein. Mice with alpha-synuclein null mutation demonstrated a permanent increase of the refilling rate. This increase maintained stable dopamine release during stimulation (which induced dopamine decline in other animals) and served as an adaptation to altered dopamine compartmentalization. Mice without alpha-synuclein and with overexpression of human A30P mutated alpha-synuclein had a lower capacity of the dopamine storage pool than other animals. Reducing capacity of the storage pool in transgenic A30P mice led to paradoxical effects of l-dopa, which elevated dopamine release in response to single stimulation but decreased the refilling rate of the RRP.

Atipamezole, an alpha2-adrenoceptor antagonist, augments the effects of L-DOPA on evoked dopamine release in rat striatum.

The effects of atipamezole, an alpha(2)-adrenoceptor antagonist, L-3,4-dihydroxyphenylalanine (L-DOPA) and the combination of these drugs on dopamine overflow were studied in dopaminergic presynaptic terminals of rat caudate and nucleus accumbens. Dopamine overflow evoked by 100 pulses of electrical stimulation of the medial forebrain bundle at a low (20 Hz) and high (50 Hz) frequency was measured by in vivo voltammetry. L-DOPA (15 mg/kg) increased dopamine overflow in the caudate nucleus, but this dose had no effects in the nucleus accumbens. Atipamezole (300 microg/kg) had no effects on its own on dopamine overflow, but it did increase the size of the readily releasable storage pool and the effects of L-DOPA treatment in both structures. The combination of the drugs increased dopamine overflow to a larger extent at high compared to low stimulation frequencies. We conclude that the rat caudate nucleus is more sensitive than the nucleus accumbens to the effects of L-DOPA, and the effects of L-DOPA treatment might be effectively enhanced by antagonism of alpha(2)-adrenoceptors.