Sara R. Jones

Cocaine self-administration in dopamine-transporter knockout mice.

The plasma membrane dopamine transporter (DAT) is responsible for clearing dopamine from the synapse. Cocaine blockade of DAT leads to increased extracellular dopamine, an effect widely considered to be the primary cause of the reinforcing and addictive properties of cocaine. In this study we tested whether these properties are limited to the dopaminergic system in mice lacking DAT. In the absence of DAT, these mice exhibit high levels of extracellular dopamine, but paradoxically still self-administer cocaine. Mapping of the sites of cocaine binding and neuronal activation suggests an involvement of serotonergic brain regions in this response. These results demonstrate that the interaction of cocaine with targets other than DAT, possibly the serotonin transporter, can initiate and sustain cocaine self-administration in these mice.

Mice lacking the norepinephrine transporter are supersensitive to psychostimulants

The action of norepinephrine (NE) is terminated, in part, by its uptake into presynaptic noradrenergic neurons by the plasma-membrane NE transporter (NET), which is a target for antidepressants and psychostimulants. Disruption of the NET gene in mice prolonged the clearance of NE and elevated extracellular levels of this catecholamine. In a classical test for antidepressant drugs, the NET-deficient (NET−/−) animals behaved like antidepressant-treated wild-type mice. Mutants were hyper-responsive to locomotor stimulation by cocaine or amphetamine. These responses were accompanied by dopamine D2/D3 receptor supersensitivity. Thus altering NET expression significantly modulates midbrain dopaminergic function, an effect that may be an important component of the actions of antidepressants and psychostimulants.

Dopamine Transporter Is Required for In Vivo MPTP Neurotoxicity: Evidence from Mice Lacking the Transporter

Abstract: The neurotoxic effect of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) was tested on mice lacking the dopamine (DA) transporter (DAT−/− mice). Striatal tissue DA content and glial fibrillary acidic protein (GFAP) mRNA expression were assessed as markers of MPTP neurotoxicity. MPTP (30 mg/kg, s.c., b.i.d.) produced an 87% decrease in tissue DA levels and a 29‐fold increase in the level of GFAP mRNA in the striatum of wild‐type animals 48 h after administration. Conversely, there were no significant changes in either parameter in DAT−/− mice. Heterozygotes demonstrated partial sensitivity to MPTP administration as shown by an intermediate value (48%) of tissue DA loss. Direct intrastriatal infusion of the active metabolite of MPTP, 1‐methyl‐4‐phenylpyridinium (MPP+; 10 mM), via a microdialysis probe produced a massive efflux of DA in wild‐type mice (>320‐fold). In the DAT−/− mice the same treatment produced a much smaller increase in extracellular DA (sixfold), which is likely secondary to tissue damage due to the implantation of the dialysis probe. These observations show that the DAT is a mandatory component for expression of MPTP toxicity in vivo.

Knockout of the Vesicular Monoamine Transporter 2 Gene Results in Neonatal Death and Supersensitivity to Cocaine and Amphetamine

Vesicular monoamine transporters are known to transport monoamines from the cytoplasm into secretory vesicles. We have used homologous recombination to generate mutant mice lacking the vesicular monoamine transporter 2 (VMAT2), the predominant form expressed in the brain. Newborn homozygotes die within a few days after birth, manifesting severely impaired monoamine storage and vesicular release. In heterozygous adult mice, extracellular striatal dopamine levels, as well as K+- and amphetamine-evoked dopamine release, are diminished. The observed changes in presynaptic homeostasis are accompanied by a pronounced supersensitivity of the mice to the locomotor effects of the dopamine agonist apomorphine, the psychostimulants cocaine and amphetamine, and ethanol. Importantly, VMAT2 heterozygous mice do not develop further sensitization to repeated cocaine administration. These observations stress the importance of VMAT2 in the maintenance of presynaptic function and suggest that these mice may provide an animal model for delineating the mechanisms of vesicular release, monoamine function, and postsynaptic sensitization associated with drug abuse.

Loss of autoreceptor functions in mice lacking the dopamine transporter

Autoreceptors provide an important inhibitory feedback mechanism for dopamine neurons by altering neuronal functions in response to changes in extracellular levels of dopamine. Elevated dopamine may be a component of several neuropsychiatric disorders. However, evidence concerning the state of autoreceptors in such conditions has remained elusive. The function of dopamine autoreceptors was assessed in mice lacking the dopamine transporter (DAT). Genetic deletion of the DAT gene in mice results in a persistent elevation in levels of extracellular dopamine. Direct assessment of impulse-, synthesis- and release-regulating autoreceptors in these mice reveals a nearly complete loss of function. These findings may provide insight into the neurochemical consequences of hyperdopaminergia.

Functional hyperdopaminergia in dopamine transporter knock-out mice

Dopamine is an important regulator of many central nervous system functions. Hyperfunction of the dopaminergic system is believed to be related to several pathological conditions. Genetic deletion of the dopamine transporter gene in mice results in a persistent extracellular hyperdopaminergic tone, that is functionally revealed as hyperactivity. The lack of a reuptake mechanism produces a marked increase in functional extracellular dopamine which results in profound plasticity of pre- and postsynaptic parameters of dopamine homeostasis. The mice lacking the dopamine transporter gene may represent an appropriate model to elucidate the molecular adaptive changes accompanying pathological states associated with hyperdopaminergic function.

Increased MPTP Neurotoxicity in Vesicular Monoamine Transporter 2 Heterozygote Knockout Mice

Abstract: The neurotoxic action of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) has been proposed to be attenuated by sequestration into intracellular vesicles by the vesicular monoamine transporter (VMAT2). The purpose of this study was to determine if mice with genetically reduced levels of VMAT2 (heterozygote knockout; VMAT2 +/−) were more vulnerable to MPTP. Striatal dopamine (DA) content, the levels of DA transporter (DAT) protein, and the expression of glial fibrillary acidic protein (GFAP) mRNA, a marker of gliosis, were assessed as markers of MPTP neurotoxicity. In all parameters measured VMAT2 +/− mice were more sensitive than their wild‐type littermates (VMAT2 +/+). Administration of MPTP (7.5, 15, or 30 mg/kg, b.i.d.) resulted in dose‐dependent reductions in striatal DA levels in both VMAT2 +/− and VMAT2 +/+ animals, but the neurotoxic potency of MPTP was approximately doubled in the VMAT2 +/− mice: 59 versus 23% DA loss 7 days after 7.5 mg/kg dose for VMAT2 +/− and VMAT2 +/+ mice, respectively. Dopaminergic nerve terminal integrity, as assessed by DAT protein expression, also revealed more drastic reductions in the VMAT2 +/− mice: 59 versus 35% loss at 7.5 mg/kg and 95 versus 58% loss at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. Expression of GFAP mRNA 2 days after MPTP was higher in the VMAT2 +/− mice than in the wild‐type: 15.8‐ versus 7.8‐fold increase at 7.5 mg/kg and 20.1‐ versus 9.6‐fold at 15 mg/kg for VMAT2 +/− and VMAT2 +/+ mice, respectively. These observations clearly demonstrate that VMAT2 +/− mice are more susceptible to the neurotoxic effects of MPTP, suggesting that VMAT2‐mediated sequestration of the neurotoxin into vesicles may play an important role in attenuating MPTP toxicity in vivo.

Comparison of Dopamine Uptake in the Basolateral Amygdaloid Nucleus, Caudate‐Putamen, and Nucleus Accumbens of the Rat

Abstract: Regional differences in the kinetics and pharmacological inhibition of dopamine uptake were investigated with fast‐scan cyclic voltammetry in both the intact rat brain and a brain slice preparation. The regions compared were the basolateral amygdaloid nucleus, caudate‐putamen, and nucleus accumbens. The frequency dependence of dopamine efflux evoked in vivo by electrical stimulation of the medial forebrain bundle was evaluated by nonlinear curve fitting with a Michaelis‐Menten‐based kinetic model. The Km for dopamine uptake was found to be significantly higher in the basolateral amygdala (0.6 µM) than in the other two regions (0.2 µM), whereas the Vmax value for dopamine uptake in the basolateral amygdala was significantly lower (0.49 µM/s vs. 3.8 and 2.4 µM/s in the caudate and accumbens, respectively). Similar kinetics were also obtained in brain slices. Addition of a dopamine uptake inhibitor, cocaine or nomifensine (10 µM), to the perfusion buffer increased the apparent Km value >25‐fold in slices of both the caudate‐putamen and nucleus accumbens. In contrast, neither uptake inhibitor had an observable effect in the basolateral amygdaloid nucleus. Thus, dopamine uptake in the rat brain is regionally distinct with regard to rate, affinity, and sensitivity to competitive inhibition.

Evoked Extracellular Dopamine In Vivo in the Medial Prefrontal Cortex

Abstract: The measurement of evoked extracellular dopamine in the medial prefrontal cortex by using fast‐scan cyclic voltammetry with carbon‐fiber microelectrodes was established and release characteristics of mesoprefrontal dopamine neurons were examined in vivo in anesthetized rats. Despite the sparse dopaminergic innervation and the presence of more dense noradrenergic and serotonergic innervations overall in the medial prefrontal cortex, the measurement of extracellular dopamine was achieved by selective recording in dopamine‐rich terminal fields and selective activation of ascending dopamine neurons. This was confirmed by electrochemical, pharmacological, and anatomical evidence. An increased release capacity for mesoprefrontal dopamine neurons was also demonstrated by the slower decay of the evoked dopamine response after inhibition of catecholamine synthesis and the maintenance of the evoked dopamine response at higher levels in the medial prefrontal cortex compared with the striatum during supraphysiological stimulation.

Dynamic Observation of Dopamine Autoreceptor Effects in Rat Striatal Slices

Abstract: Fast‐scan cyclic voltammetry has been used to measure dopamine (DA) synaptic overflow in slices of rat caudate nucleus induced by electrical stimulation with one‐, two‐, and 50‐pulse, 10‐Hz trains. Synaptic overflow in this preparation is shown to be the result of the competing effects of release and cellular uptake. Release caused by all pulses was attenuated by the D2 agonist quinpirole (1 μM). The rapid time response of the measurements (100 ms) allows the autoinhibition induced by endogenous, released DA to be resolved in real time. The concentration of DA released during the second pulse of a train was 58% of that released by the first pulse, an effect that is partially blocked by the addition of 2 μM sulpiride, a D2 antagonist, to the perfusion buffer. DA release during the first stimulus pulse is unaffected by 2 μM sulpiride, suggesting that autoreceptors are not normally occupied in this preparation. Release caused by the third pulse was 14% of the first pulse and also could be partially enhanced by 2 μM sulpiride. The duration of the inhibition of release induced by endogenous DA was estimated by varying the interval between one‐pulse stimulations until the overflow of DA induced by the second pulse was equal to that on the first; a half‐time of ∼ 17 s was found. The addition of picrotoxin (100 μM) and glutamate (10 μM) to the perfusion buffer did not affect stimulated release of DA, although the addition of atropine (100 μM) attenuated overflow for all the trains tested.