Christopher P. Ford

Properties and Opioid Inhibition of Mesolimbic Dopamine Neurons Vary according to Target Location

The mesolimbic dopamine system, which mediates the rewarding properties of nearly all drugs of abuse, originates in the ventral tegmental area (VTA) and sends major projections to both the nucleus accumbens (NAc) and the basolateral amygdala (BLA). To address whether differences occur between neurons that project to these separate areas, retrograde microspheres were injected to either the BLA or the NAc of DBA/2J mice. Whole-cell recordings were made from labeled VTA dopamine neurons. We found that identified neurons that projected to the BLA and NAc originated within different quadrants of the VTA with neither group exhibiting large-amplitude h-currents. Neurons that projected to the NAc exhibited a greater outward current in response to the κ-opioid agonist (5α,7α,8α)-(+)-N-methyl-N-[7-(pyrrolidinyl)-1-oxaspiro [4,5]dec-8-yl]-benzeneacetamide (U69593; 200 nm), whereas neurons that projected to the BLA exhibited greater inhibition to the μ/δ opioid agonist [Met5] enkephalin (ME; 3 μm). In addition, we found that the presynaptic inhibition of GABAergic transmission at both GABAA and GABAB receptors was differentially regulated by U69593 between the two groups. When dopamine IPSCs were examined, U69593 caused a greater inhibition in NAc- than BLA-projecting neurons. ME had no effect on either. Finally, the regulation of extracellular dopamine by dopamine uptake transporters was equal across the VTA. These results suggest that opioids differentially inhibit mesolimbic neurons depending on their target projections. Identifying the properties of projecting mesolimbic VTA dopamine neurons is crucial to understanding the action of drugs of abuse.

Control of Extracellular Dopamine at Dendrite and Axon Terminals

Midbrain dopamine neurons release dopamine from both axons and dendrites. The mechanism underlying release at these different sites has been proposed to differ. This study used electrochemical and electrophysiological methods to compare the time course and calcium dependence of somatodendritic dopamine release in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) to that of axonal dopamine release in the dorsal striatum. The amount of dopamine released in the striatum was ∼20-fold greater than in cell body regions of the VTA or SNc. However, the calcium dependence and time to peak of the dopamine transients were similar. These results illustrate an unexpected overall similarity in the mechanisms of dopamine release in the striatum and cell body regions. To examine how diffusion regulates the time course of dopamine following release, dextran was added to the extracellular solution to slow diffusion. In the VTA, dextran slowed the rate of rise and fall of the extracellular dopamine transient as measured by fast-scan cyclic voltammetry yet did not alter the kinetics of the dopamine-dependent IPSC. Dextran failed to significantly alter the time course of the rise and fall of the dopamine transient in the striatum, suggesting a more influential role for reuptake in the striatum. The conclusion is that the time course of dopamine within the extracellular space of the VTA is dependent on both diffusion and reuptake, whereas the activation of D2 receptors on dopamine neurons is primarily limited by reuptake.

Dopaminergic Modulation of Axon Initial Segment Calcium Channels Regulates Action Potential Initiation

Action potentials initiate in the axon initial segment (AIS), a specialized compartment enriched with Na(+) and K(+) channels. Recently, we found that T- and R-type Ca(2+) channels are concentrated in the AIS, where they contribute to local subthreshold membrane depolarization and thereby influence action potential initiation. While periods of high-frequency activity can alter availability of AIS voltage-gated channels, mechanisms for long-term modulation of AIS channel function remain unknown. Here, we examined the regulatory pathways that control AIS Ca(2+) channel activity in brainstem interneurons. T-type Ca(2+) channels were downregulated by dopamine receptor activation acting via protein kinase C, which in turn reduced neuronal output. These effects occurred without altering AIS Na(+) or somatodendritic T-type channel activity and could be mediated by endogenous dopamine sources present in the auditory brainstem. This pathway represents a new mechanism to inhibit neurons by specifically regulating Ca(2+) channels directly involved in action potential initiation.

The Time Course of Dopamine Transmission in the Ventral Tegmental Area

Synaptic transmission mediated by G-protein coupled receptors (GPCR) is not generally thought to be point-to-point. To determine the extent over which dopamine signals in the midbrain, the present study examined the concentration and time course of dopamine that underlies a D2-receptor IPSC (D2-IPSC) in the ventral tegmental area. Extracellular dopamine was measured electrochemically while simultaneously recording D2-IPSCs. The presence of dopamine was brief relative to the IPSC, suggesting that G-protein dependent potassium channel activation determined the IPSC time course. The activation kinetics of D2 receptor-dependent potassium current was studied using outside-out patch recordings with rapid application of dopamine. Dopamine applied at a minimum concentration of 10 μm for a maximum of 100 ms mimicked the IPSC. Higher concentrations applied for as little as 5 ms did not change the kinetics of the current. The results indicate that both the intrinsic kinetics of G-protein coupled receptor signaling and a rapidly rising high concentration of dopamine determine the time course of the IPSC. Thus, dopamine transmission in the midbrain is more localized then previously proposed.

CRF enhancement of GIRK channel-mediated transmission in dopamine neurons.

Dopamine neurons in the ventral midbrain contribute to learning and memory of natural and drug-related rewards. Corticotropin-releasing factor (CRF), a stress-related peptide, is thought to be involved in aspects of relapse following drug withdrawal, but the cellular actions are poorly understood. This study investigates the action of CRF on G-protein-linked inhibitory postsynaptic currents (IPSCs) mediated by GIRK (Kir3) channels in dopamine neurons. CRF enhanced the amplitude and slowed the kinetics of IPSCs following activation of D2-dopamine and GABAB receptors. This action was postsynaptic and dependent on the CRF1 receptor. The enhancement induced by CRF was attenuated by repeated in vivo exposures to psychostimulants or restraint stress. The results indicate that CRF influences dopamine- and GABA-mediated inhibition in the midbrain, suggesting implications for the chronic actions of psychostimulants and stress on dopamine-mediated behaviors.

Possible role of phosphatidylinositol 4,5 bisphosphate in luteinizing hormone releasing hormone-mediated M-current inhibition in bullfrog sympathetic neurons.

Luteinizing hormone releasing hormone (LHRH) is a physiological modulator of neuronal excitability in bullfrog sympathetic ganglia (BFSG). Actions of LHRH involve suppression of the noninactivating, voltage‐dependent M‐type K+ channel conductance (gM). We found, using whole‐cell recordings from these neurons, that LHRH‐induced suppression of gM was attenuated by the phospholipase C (PLC) inhibitor U73122 (10 µm) but not by the inactive isomer U73343 (10 µm). Buffering internal Ca2+ to 117 nm with intracellular 20 mm BAPTA + 8 mm Ca2+ or to < 10 nm with intracellular 20 mm BAPTA + 0.4 mm Ca2+ did not attenuate LHRH‐induced gM suppression. Suppression of gM by LHRH was not antagonized by the inositol 1,4,5 trisphosphate (InsP3) receptor antagonist heparin (∼ 300 µm). Preventing phosphatidylinositol‐4,5‐bisphosphate (PIP2) synthesis by blocking phosphatidylinositol‐4‐kinase with wortmannin (10 µm) or with the nonhydrolysable ATP analogue AMP‐PNP (3 mm) prolonged recovery of LHRH‐induced gM suppression. This effect was not produced by blocking phosphatidyl inositol‐3‐kinase with LY294002 (10 µm). Rundown of gM was attenuated when cells were dialysed with 240 µm di‐octanoyl PIP2 or 240 µm di‐octanoyl phosphatidylinositol‐3,4,5‐trisphosphate (PIP3) but not with 240 µm di‐octanoyl phosphatidylcholine. LHRH‐induced gM suppression was competitively antagonized by dialysis with 240 µm di‐octanoyl PIP2, but not with di‐octanoyl phosphatidylcholine. These results would be expected if LHRH‐induced gM suppression reflects a PLC‐mediated decrease in plasma membrane PIP2 levels.

Presynaptic regulation of dendrodendritic dopamine transmission

The amount of dopamine release from terminals in the forebrain following an electrical stimulus is variable. This dynamic regulation, both between and within trains of electrical stimuli, has fostered the notion that burst firing of dopamine neurons in vivo may be a determinant of dopamine release in projection areas. In the present study dendritic dopamine release was examined in the substantia nigra and ventral tegmental area in mouse brain slices using whole‐cell recording of a dopamine‐mediated inhibitory postsynaptic current (IPSC). Paired stimuli produced a depression of the IPSC that was not observed with paired pulses of exogenously applied dopamine. Increasing the number of electrical stimuli from one to five produced an increase in the amplitude the dopamine IPSC but the increase was less than additive, indicating a depression of transmission with each successive stimulus. Analysis with fast‐scan cyclic voltammetry demonstrated that presynaptic D2‐autoreceptors did not contribute to the depression. Facilitation of the IPSC was observed only after the probability of release was reduced. Thus the regulation of dopamine release in the cell body region was dependent on dopamine neuron impulse activity. Under circumstance where there was initially little activity the probability of dopamine release was high and repetitive activation resulted in depression of further release. With increased activity, the release probability decreased and a burst of activity caused a relative facilitation of dopamine release. This form of regulation would be expected to limit activity within the cell body region.

Normalizing dopamine D2 receptor-mediated responses in D2 null mutant mice by virus-mediated receptor restoration: Comparing D2L and D2S

D2 receptor null mutant (Drd2(-/-)) mice have altered responses to the rewarding and locomotor effects of psychostimulant drugs, which is evidence of a necessary role for D2 receptors in these behaviors. Furthermore, work with mice that constitutively express only the D2 receptor short form (D2S), as a result of genetic deletion of the long form (D2L), provides the basis for a current model in which D2L is thought to be the postsynaptic D2 receptor on medium spiny neurons in the basal forebrain, and D2S the autoreceptor that regulates the activity of dopamine neurons and dopamine synthesis and release. Because constitutive genetic deletion of the D2 or D2L receptor may cause compensatory changes that influence functional outcomes, our approach is to identify aspects of the abnormal phenotype of a Drd2(-/-) mouse that can be normalized by virus-mediated D2 receptor expression. Drd2(-/-) mice are deficient in basal and methamphetamine-induced locomotor activation and lack D2 receptor agonist-induced activation of G protein-regulated inward rectifying potassium channels (GIRKs) in dopaminergic neurons. Here we show that virus-mediated expression of D2L in the nucleus accumbens significantly restored methamphetamine-induced locomotor activation, but not basal locomotor activity, compared to mice receiving the control virus. It also restored the effect of methamphetamine to decrease time spent in the center of the activity chamber in female but not male Drd2(-/-) mice. Furthermore, the effect of expression of D2S was indistinguishable from D2L. Similarly, virus-mediated expression of either D2S or D2L in substantia nigra neurons restored D2 agonist-induced activation of GIRKs. In this acute expression system, the alternatively spliced forms of the D2 receptor appear to be equally capable of acting as postsynaptic receptors and autoreceptors.

Mesoprefrontal Dopamine Neurons Distinguish Themselves

By distinguishing groups of dopamine neurons that differ in their projection patterns and intrinsic properties, Lammel and colleagues report in this issue of Neuron that mesocorticolimbic dopamine neurons of the ventral tegmental area (VTA) form a distinct subclass of dopamine cells.