There are three known high-affinity targets for cocaine: the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Decades of studies support the dopamine (DA) hypothesis that the blockade of DAT and the subsequent increase in extracellular DA primarily mediate cocaine reward and reinforcement. Contrary to expectations, DAT knockout (DAT-KO) mice and SERT or NET knockout mice still selfadminister cocaine and͞or display conditioned place preference (CPP) to cocaine, which led to the reevaluation of the DA hypothesis and the proposal of redundant reward pathways. To study the role of DAT in cocaine reward, we have generated a knockin mouse line carrying a functional DAT that is insensitive to cocaine. In these mice, cocaine suppressed locomotor activity, did not elevate extracellular DA in the nucleus accumbens, and did not produce reward as measured by CPP. This result suggests that blockade of DAT is necessary for cocaine reward in mice with a functional DAT. This mouse model is unique in that it is specifically designed to differentiate the role of DAT from the roles of NET and SERT in cocaine-induced biochemical and behavioral effects.addiction ͉ amphetamine ͉ conditioned place preference ͉ knockin C ocaine inhibits the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) with similar potencies and elevates extracellular concentrations of these monoamine neurotransmitters, thereby producing complex neurochemical and behavioral effects (1, 2). However, there is a wealth of evidence indicating that the dopaminergic system, especially DAT, is most important in mediating cocaine's addictive properties (3-6). For instance, the potencies of cocaine analogs for producing self-administration, a measure of drug reward, correlate to their affinities for binding DAT, but not SERT or NET (1, 6). Among the drugs that block all three transporters, those with a high affinity for DAT are selfadministered or produce conditioned place preference (CPP), another measure of drug reward, whereas SERT or NET selective inhibitors do not produce reward in WT animals (7-9).The generation of DAT knockout (DAT-KO) mice (10) allowed a direct test of whether DAT inhibition is required for cocaine reward. Contrary to the expectations, these mice still self-administer cocaine (11) and exhibit cocaine-induced CPP (12, 13). These results suggest that DAT inhibition is not solely required for cocaine reward, at least in DAT-KO mice, leading to the reevaluation of the dopamine (DA) hypothesis and the proposal that redundant systems might mediate cocaine reward (14-16). However, complete deletion of DAT causes tremendous adaptive changes in DA homeostasis, including alterations in DA synthesis, storage, extracellular levels, and receptor expression and functions (10, 17). These adaptive changes may significantly alter normal reward pathways. For instance, fluoxetine and nisoxetine, selective inhibitors for SERT and NET, respectively, produce CPP in DAT-KO ...

A hallmark of the GABA projection neurons of the substantia nigra pars reticulata (SNr), a key basal ganglia output nucleus, is its depolarized membrane potential and rapid spontaneous spikes that encode the basal ganglia output. Parkinsonian movement disorders are often associated with abnormalities in SNr GABA neuron firing intensity and/or pattern. A fundamental question remains regarding the molecular identity of the ion channels that drive these neurons to a depolarized membrane potential. We show here that SNr GABA projection neurons selectively express type 3 canonical transient receptor potential (TRPC3) channels. These channels are tonically active and mediate an inward, Na ϩ -dependent current, leading to a substantial depolarization in these neurons. Inhibition of TRPC3 channels induces hyperpolarization, decreases firing frequency, and increases firing irregularity. These data demonstrate that TRPC3 channels play important roles in ensuring the appropriate firing intensity and pattern in SNr GABA projection neurons that are crucial to movement control.

Substantia nigra pars reticulata (SNr) is a key basal ganglia output nucleus critical for movement control. Its γ-aminobutyric acid (GABA)-containing projection neurons intermingle with nigral dopamine (DA) neuron dendrites. Here we show that SNr GABA neurons co-express dopamine D1 and D5 receptor mRNAs and also mRNA for TRPC3 channels. Dopamine induced an inward current in these neurons and increased their firing frequency. These effects were mimicked by D1-like agonists, blocked by a D1-like antagonist. D1-like receptor blockade reduced SNr GABA neuron firing frequency and increased their firing irregularity. These D1-like effects were absent in D1 or D5 receptor knockout mice and inhibited by intracellularly applied D1 or D5 receptor antibody. These D1-like effects were also inhibited when the tonically active TRPC3 channels were inhibited by intracellularly applied TRPC3 channel antibody. Furthermore, stimulation of DA neurons induced a direct inward current in SNr GABA neurons that was sensitive to D1-like blockade. Manipulation of DA neuron activity and DA release and inhibition of dopamine reuptake affected SNr GABA neuron activity in a D1-like receptor-dependent manner. Taken together, our findings indicate that dendritically released dopamine tonically excites SNr GABA neurons via D1-D5 receptor co-activation that enhances constitutively active TRPC3 channels, forming an ultra-short SNc→SNr dopamine pathway that regulates the firing intensity and pattern of these basal ganglia output neurons.

The substantia nigra pars reticulata (SNr) is a key basal ganglia output nucleus. Inhibitory outputs from SNr are encoded in spike frequency and pattern of the inhibitory SNr projection neurons. SNr output intensity and pattern are often abnormal in movement disorders of basal ganglia origin. In Parkinson's disease, histamine innervation and histamine H3 receptor expression in SNr may be increased. However, the functional consequences of these alterations are not known. In this study, whole cell patch-clamp recordings were used to elucidate the function of different histamine receptors in SNr. Histamine increased SNr inhibitory projection neuron firing frequency and thus inhibitory output. This effect was mediated by activation of histamine H1 and H2 receptors that induced inward currents and depolarization. In contrast, histamine H3 receptor activation hyperpolarized and inhibited SNr inhibitory projection neurons, thus decreasing the intensity of basal ganglia output. By the hyperpolarization, H3 receptor activation also increased the irregularity of the interspike intervals or changed the pattern of SNr inhibitory neuron firing. H3 receptor-mediated effects were normally dominated by those mediated by H1 and H2 receptors. Furthermore, endogenously released histamine provided a tonic, H1 and H2 receptor-mediated excitation that helped keep SNr inhibitory projection neurons sufficiently depolarized and spiking regularly. These results suggest that H1 and H2 receptors and H3 receptor exert opposite effects on SNr inhibitory projection neurons. Functional balance of these different histamine receptors may contribute to the proper intensity and pattern of basal ganglia output and, as a consequence, exert important effects on motor control.

Cortical dysplasia (CD) is a common cause of intractable epilepsy in children and adults. We have studied rats irradiated in utero as a model of CD to better understand mechanisms that underlie dysplasia-associated epilepsy. Prior studies have shown a reduction in the number of cortical interneurons and in the frequency of inhibitory postsynaptic currents (IPSCs) in pyramidal cells in this model. They have also shown a reduced frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) in the surviving cortical interneurons. However, the inhibitory synaptic contacts were not examined in that study. The current experiments were performed to assess inhibitory synaptic activity in fast-spiking (FS) interneurons in irradiated rats and controls and the balance of excitatory and inhibitory synaptic activity in these cells. Whole cell recordings were obtained from layer IV FS cells in controls and comparable FS cells in irradiated rats. The frequency of spontaneous and miniature IPSCs was reduced in dysplastic cortex, but the amplitude of these currents was unchanged. Stimulus-evoked IPSCs showed short-term depression in control and short-term facilitation in dysplastic cortex. Simultaneous recording of spontaneous EPSCs and IPSCs showed a shift in the ratio of excitation-to-inhibition in favor of inhibition in FS cells from dysplastic cortex. The same shift toward inhibition was seen when miniature EPSCs and IPSCs were examined. These results show that FS cells in dysplastic cortex have a relative lack of excitatory drive. This may result in an important class of inhibitory cells that are less able to perform their normal function especially in periods of increased excitatory activity.

Nicotinic acetylcholine receptors (nAChRs) potently regulate dopamine (DA) release in the striatum and alter cocaine's ability to reinforce behaviors. Since cocaine is a weak nAChR inhibitor, we hypothesized that cocaine may alter DA release by inhibiting the nAChRs in DA terminals in the striatum and thus contribute to cocaine's reinforcing properties primarily associated with the inhibition of DA transporters. We found that biologically relevant concentrations of cocaine can mildly inhibit nAChR-mediated currents in midbrain DA neurons and consequently alter DA release in the dorsal and ventral striatum. At very high concentrations, cocaine also inhibits voltage-gated Na channels in DA neurons. Furthermore, our results show that partial inhibition of nAChRs by cocaine reduces evoked DA release. This diminution of DA release via nAChR inhibition more strongly influences release evoked at low or tonic stimulation frequencies than at higher (phasic) stimulation frequencies, particularly in the dorsolateral striatum. This cocaine-induced shift favoring phasic DA release may contribute to the enhanced saliency and motivational value of cocaine-associated memories and behaviors.

Cortical dysplasia (CD) is associated with severe epilepsy in humans, and the in utero irradiation of fetal rats provides a model of this disorder. These animals show a selective loss of inhibitory interneurons, and the surviving interneurons have a reduced excitatory synaptic drive. The current study was undertaken to see how alterations in synaptic input would affect spontaneous firing of interneurons in dysplastic cortex. We recorded spontaneous action potentials and excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs, respectively) from somatostatin (SST)-, parvalbumin (PV)-, and calretinin (CR)-immunoreactive (ir) interneurons. We found that SST- and PV-ir interneurons fired less frequently and with less regularity than controls. This corresponded to a relative imbalance in the ratio of EPSCs to IPSCs that favored inhibition. In contrast, CR-ir interneurons from CD showed no differences from controls in spontaneous firing or ratio of EPSCs to IPSCs. Additional studies demonstrated that synaptic input had a powerful effect on spontaneous firing in all interneurons. These findings demonstrate that a relative reduction in excitatory drive results in less active SST- and PV-ir interneurons in irradiated rats. This would further impair cortical inhibition in these animals and may be an important mechanism of epileptogenesis.

SUMMARYPurpose: Cortical dysplasia (CD) is a major cause of epilepsy in children and adults, but underlying mechanisms of epileptogenesis in this disorder are poorly understood. We have utilized the irradiated rat model to study an injury-based form of diffuse CD in rats. Prior studies in this model have shown reduced numbers of c-aminobutyric acid (GABA)ergic interneurons and reduced inhibitory synaptic currents in pyramidal cells in CD. We analyzed the number of excitatory and inhibitory presynaptic terminals in the neocortex of irradiated rats to better characterize altered connectivity in experimental CD. Methods: Antibodies to vesicular glutamate transporter 1 (VGLUT1), vesicular glutamate transporter 2 (VGLUT2), vesicular GABA transporter (VGAT), and parvalbumin (PV) were used to quantify glutamatergic and GABAergic presynaptic terminals in control and dysplastic cortex. Results: We found that the density of VGLUT1 terminals was increased in CD in comparison to layers IV, V, and VI in control cortex. VGLUT2 terminals were increased in CD compared to layers IV and VI. VGAT terminals were reduced in CD compared to layers II/III, IV, and V in controls as were PV-immunoreactive somata and terminals. Discussion: These findings suggest an overall increase in excitatory synaptic connectivity and decrease in inhibitory synaptic connectivity in CD in irradiated rat. We propose that these changes contribute to hyperexcitability in these animals and may contribute to epileptogenicity in some forms of human CD.