Gary W. Miller

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 transporters and neuronal injury

The plasma membrane dopamine transporter (DAT) and the vesicular monoamine transporter (VMAT2) are essential for normal dopamine neurotransmission. DAT terminates the actions of dopamine by rapidly removing dopamine from the synapse, whereas VMAT2 loads cytoplasmic dopamine into vesicles for storage and subsequent release. Recent data suggest that perturbation of the tightly regulated balance between these two transporters predisposes the neurone to damage by a variety of insults. Most notable is the selective degeneration of DAT- and VMAT2-expressing dopamine nerve terminals in the striatum thought to underlie Parkinsons disease. DAT and VMAT2 expression can predict the selective vulnerability of neuronal populations, which suggests that therapeutic strategies aimed at altering DAT and VMAT2 function could have significant benefits in a variety of disorders.

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.

Forced Nonuse in Unilateral Parkinsonian Rats Exacerbates Injury

Diagnosis of Parkinsons disease (PD) is based on the presentation of clinical symptoms such as bradykinesia, resting tremor, and rigidity. However, one feature of PD that often begins years before diagnosis is decreased physical activity. We hypothesized that this depressed activity is not only a symptom of the early dopaminergic loss but also a catalyst in the degenerative process. Two experiments were performed to test this hypothesis. First, rats were exposed to a mild dose of 6-hydroxydopamine unilaterally into the nigrostriatal dopamine (DA) projections, which would normally result in an ∼20% DA loss and no detectable behavioral asymmetries. A subset of these lesioned animals then had a cast applied for 7 d to the contralateral forelimb. After the cast was removed, these animals displayed long-term behavioral asymmetry and exacerbation of neurochemical loss (∼60% depletion). Second, a group of animals received a high dose of 6-hydroxydopamine that normally would yield a severe loss of nigrostriatal terminals (∼90% loss) and chronic sensorimotor deficits. During the first 7 d after neurotoxin exposure, a subset of these animals were forced to rely on the contralateral forelimb, a procedure we have previously reported to protect DA terminals and behavioral function. Some of these rats then had the use of their “recovered” forelimb restricted during the second or third week after lesioning. This precipitated a severe and chronic loss of DA terminals and functional deficits. These results suggest decreased physical activity not only is a symptom of PD but also may act to potentiate the underlying degeneration.

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.

Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine

Overt behavioral symptoms of Parkinsons disease (PD) do not occur until over 80% of the striatal dopamine content has been lost. Diagnosis of the disorder relies on identifying clinical symptoms including akinesia, resting tremor, and rigidity. In retrospect, behavioral deficits are observed several years prior to diagnosis. Behavioral manifestations in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, such as changes in general locomotor activity and rotorod performance, require large doses of MPTP and are often transient. We hypothesized that, as in PD, subtle behavioral changes also occur in the MPTP model. In this paper, we demonstrate that mice treated with moderate doses of the dopaminergic toxin MPTP display deficits in behavioral parameters that are significantly correlated with the loss of striatal dopamine. In addition, these behavioral measures are correlated to dopamine transporter, vesicular monoamine transporter, and tyrosine hydroxylase expression and are improved following L-DOPA administration. Detection of dopamine-modulated behavioral changes in moderately depleted MPTP mice will allow for more efficacious use of this model in PD research.

Grid performance test to measure behavioral impairment in the MPTP-treated-mouse model of parkinsonism.

Behavioral impairments in mice following administration of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) require large depletions in striatal dopamine content and are often transient. In this paper, we describe a simple and inexpensive test that measures long-term behavioral deficits in mice treated with moderate doses of MPTP. These measures are significantly correlated with the loss of striatal dopamine and immunoreactivity of the dopamine transporter, vesicular monoamine transporter and tyrosine hydroxylase. In addition, behavioral impairments on the measures were reversed following L-DOPA administration. Employment of this test will allow for more efficacious use of mice in PD research, as well as provide more sensitive measures of behavioral improvement following potential therapeutic or neuroprotective interventions.

Inhibitory effect of reserpine on dopamine transporter function.

Previous studies indicate that reserpine may disrupt dopamine transporter activity. Results presented herein reveal that it also inhibits potently synaptosomal [3H]dopamine uptake. In addition, reserpine administration to rats decreased the V(max) of synaptosomal dopamine transport, as assessed ex vivo 12 h after treatment. This decrease appeared, at least in part, dissociated from concurrent inhibition of the vesicular monoamine transporter-2 (VMAT-2). In separate experiments, synaptosomal dopamine uptake did not differ between wild-type and heterozygous VMAT-2 knockout mice, and reserpine treatment did not inhibit [3H]dopamine uptake into cells heterogously expressing the human dopamine transporter. Taken together, these data suggest that reserpine may transiently alter dopamine transporter function in a noncompetitive, indirect manner.

l-DOPA Does Not Cause Neurotoxicity in VMAT2 Heterozygote Knockout Mice

One of the most useful treatments of Parkinsons disease (PD) is dihydroxyphenylalanine (L-DOPA) administration. However, L-DOPA has been suggested to be toxic to dopamine (DA) neurons and perhaps contribute to the progression of the disease. Sequestration of DA and dopaminergic neurotoxins into vesicles by the vesicular monoamine transporter 2 (VMAT2) is a key factor in preventing cellular damage. Mice with reduced expression of VMAT2 (VMAT2 heterozygote knockout mice; VMAT2 (+/-)) are more sensitive to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine. In this study, we subjected VMAT2 (+/-) mice to subchronic administration of L-DOPA to determine if it was toxic in this model. VMAT2 wild-type (VMAT2 (+/+)) and VMAT2 (+/-) mice were given i.p. injections of L-DOPA:carbidopa (50:5 mg/kg) three times a day for 28 days. Biochemical analysis revealed a significant increase in striatal DA levels in both groups of mice treated with L-DOPA. L-DOPA treatment significantly decreased DAT levels in VMAT2 (+/+) mice, but not in VMAT2 (+/-) mice. VMAT2 protein levels, an index of terminal integrity and the number of tyrosine hydroxylase (TH)-positive nigral cells remained unchanged after L-DOPA treatment. These data indicate that in an animal model that displays increased susceptibility to dopaminergic injury, a subchronic administration of L-DOPA does not induce toxicity.