Dopamine Autoreceptor Signal Transduction and Regulation

Louis A. Chiodo, Arthur S. Freeman, and Benjamin S. Bunney

The electrophysiological investigation of dopamine (DA) neurons within the mammalian brain has spanned some 24 years. This area of investigation, when coupled with the dramatic advances in our knowledge of the biochemistry, molecular biology, anatomy, and behavior of these neuronal systems, has yielded many insights into the regulation of these cells and their role in normal and perhaps aberrant behavior. Several guiding principles have emerged in our conceptualization of the membrane physiology of these clinically important cells. One such principle is that these cells possess presynaptic dopamine receptors that serve as autoreceptors [a term coined by Arvid Carlsson (9)]. That is, DA neurons have receptors that are sensitive to the neurotransmitter released by these cells—DA itself. The analysis of the precise role that autoreceptors play in regulating the physiological activity of these neurons has received extensive study (13); see also Electrophysiological Properties of Midbrain Dopamine Neurons), in part, because changes in these regulatory mechanisms may be involved in clinical disorders in which dopaminergic neuronal systems have been implicated. We now know that most mesencephalic DA neurons possess DA autoreceptors that modulate membrane excitability (thereby controlling the level of spontaneous action potential generation), DA synthesis, and DA release (see Biochemical Pharmacology of Midbrain Dopamine Neurons). In contrast to most midbrain DA cells, those that project to certain frontal cortical regions possess only release-modulating nerve terminal autoreceptors (12, 24). Given the current conceptual understanding that DA autoreceptors play a critical self-regulatory (self-inhibitory) role in these cells, why do subpopulations of mesencephalic DA neurons lack certain functional autoreceptor mechanisms? Moreover, how is the expression of these autoreceptormodulated functions determined, and how might they be altered? Although the answers to these questions are yet to be found, an understanding has been gained as to the coupling of DA autoreceptors, located on the somal and dendritic membranes of these cells (called somatodendritic DA autoreceptors), to transmembrane ionic currents that underlie the ability of these receptors to alter DA cell excitability. We will review the current understanding of the regulation of both potassium- and calcium-dependent currents in DA neurons and discuss our present knowledge of the signal transduction mechanisms that are involved.

In recent years, it has become apparent that we need to revise our concept of autoreceptors with respect to DA neurons. It has been demonstrated that the peptides cholecystokinin (CCK) and neurotensin are differentially colocalized with DA in subpopulations of midbrain DA neurons. These peptides can be released from the DA neuron and stimulate their specific receptors also located on the DA cell membrane. Thus, we now need to conceptualize DA neurons as possessing not only DA autoreceptors, but also CCK and neurotensin autoreceptors. Moreover, these peptides have the ability to modulate DA autoreceptor function. Recent studies of the effects of these peptides on impulse-regulating DA autoreceptors will be discussed.

In the early 1970s, it was observed that the local microiontophoretic application of either DA or the mixed D1/D2 receptor agonist apomorphine inhibited the spontaneous electrical activity of mesencephalic DA neurons when applied onto the soma and dendrites of these cells (1, 2). Since then, this effect has been demonstrated numerous times (for review, see ref. 13), and it is now taken as a hallmark attribute of this neurochemical class of cells (i.e., these cells possess somatodendritic DA autoreceptors that regulate impulse flow as part of their normal electrophysiological behavior). These receptors are of the D2 receptor subtype that is part of the superfamily of G-protein-coupled receptors (59; see also Molecular Biology of the Dopamine Receptor Subtypes and Signal Transduction Pathways for Catecholamine Receptors). In support of this classification, pretreatment with pertussis toxin, which inactivates both Gi and Go, blocks the inhibitory effects of somatodendritic autoreceptors (34).

Several lines of evidence have shown that D2 DA receptor activation increases potassium conductances in a variety of tissues, including the MMQ clonal pituitary cell line (43), dissociated striatal neurons (23), and lactotrophs (11). Similarly, it has been observed that DA autoreceptor stimulation increases potassium conductances in mesencephalic DA neurons in both (a) the in vitro slice (38) and (b) primary dissociated cell culture preparations (14, 17). The whole-cell potassium conductance of these cells is mediated by several distinct potassium currents (15, 17, 51, 61). Indeed, it is now known that mesencephalic DA neurons possess an anomalous rectifier current functioning at hyperpolarized membrane potentials (IANOM), two different calcium-dependent outward currents (one that is apamin-sensitive, termed IAHP) that are important in the afterhyperpolarization that follows the action potential, the delayed rectifier (IK), a transient A current (IA), and apparently an ATP-sensitive current (IATP).

The regulation of distinct potassium currents has recently been studied in some detail. It is now known that direct stimulation of the DA somatodendritic autoreceptor increases at least three different potassium currents: IK, IANOM, and IA (18, 42). It was shown recently that the coupling of D2 receptors to both IA and IK utilize a common signal transduction pathway that involves Go (18, 42). Thus, the increase in the magnitude of both these currents is pertussis-toxin-sensitive, blocked by intracellular application of GDPbS, mimicked by GTPgS, and abolished by the intracellular application of an antibody directed against the Goa subunit (see ). At this time, it is not clear whether the activated a subunit of Go directly influences both IA and IK channels or influences the observed whole-cell current via some additional intracellular mechanism. Although direct a-subunit modulation of potassium channels has been shown in other tissues (6, 37, 44, 65, 67), the activated subunit could influence other cellular transduction systems. It would appear, however, that changes in intracellular levels of cAMP are probably not involved (58).

It has been known for some time that DA neurons possess both high- and low-threshold calcium conductances (14, 27, 28). These conductances are generally thought to be critically involved in the regulation of DA cell excitability. It has been shown that DA cells possess three different calcium currents: IN, IT, and IL (39, 40). IT is a low-voltage-activated current (activated at -50 mV) that displays a rapid inactivation and is blocked partially by amiloride or nickel. IL may be activated by depolarizing voltage steps from a holding potential of -40 mV. This current slowly and incompletely inactivates, and is blocked completely by nifedipine. IN is also observed in DA neurons, and it activates at the same thresholds as IL, but it requires prior hyperpolarization of the membrane (to -90 mV) and is w-conotoxin-sensitive. Both IL and IN, but not IT, are reduced by stimulation of the DA autoreceptor (). The coupling of the DA autoreceptor to these currents involves a pertussis-toxin-sensitive G protein because pertussis toxin pretreatment blocks the coupling of the somatodendritic autoreceptor to both IN and IL (). Additional studies are required to determine which G proteins are utilized and whether the different currents are coupled to the DA autoreceptor by similar or different transduction pathways.

Analysis of the D2 receptor gene has shown that at least two molecular forms of this receptor are produced via alternative splicing of mRNA (8, 44, 45, 59). These two distinct isoforms are termed D2-short (D2S) and D2-long (D2L) and differ by a 29-amino-acid sequence contained within the third cytoplasmic loop. Given that this cytoplasmic loop is critically important for receptor interactions with G proteins, it has been hypothesized that these receptor isoforms may couple to effector systems within cells via different signal transduction pathways. Because the mRNA for these receptors are colocalized within the same neurons (including DA neurons; see refs. 25, 47, 48, 49, and 64) and selective pharmacological agents are not available (20, 26), individual expression of these isoforms in a single cell line has been used by several groups to begin to examine this issue. When expressed in NG108-15 neuroblastoma glioma hybrid cells, the D2 selectivity of these receptors is maintained and these receptors couple to potassium channels in the cell membrane. In this expression system, it has been shown that the D2S isoform couples to potassium currents via a pertussis-toxin-insensitive mechanism (10), whereas D2L receptors couple to the same currents via a pertussis-toxin-sensitive process (41). These observations raise the possibility that the D2 receptor isoforms, when expressed in the same cells (as is the case with the DA neurons), can influence transmembrane currents in similar ways but via independent transduction pathways. The precise nature of D2 isoform modulation of a variety of physiological events in the DA neuron will surely be studied extensively over the next few years. The information obtained will likely change our understanding of the molecular diversity of DA autoreceptor regulation in these cells. The use of expression systems such as the NG108 cell line should prove to be of great value in these investigations.

Recent work by several groups has demonstrated that DA neurons in the midbrain possess not only mRNA for both isoforms of the D2 receptors, but also a message for the closely related D3 receptor (5, 63). This has raised the intriguing possibility that DA neurons possess a variety of functionally distinct DA autoreceptors. To date, it has been hard to ascertain the role of D3 receptors in vivo because the available drugs that are relatively selective for these receptors also exert strong actions at D2 receptors. Thus, initial studies have begun to examine these D3 receptors following their stable expression in NG108-15 cells (19). This work has shown that the D3 receptor couples to outward potassium currents in these cells in a manner similar to that observed for D2S and D2L. The coupling of this receptor to potassium currents is pertussis-toxin-sensitive, and the intracellular application of an antibody directed against the Goa subunit completely blocked D3-mediated inhibition of these currents.

Many DA neurons in the rat ventral tegmental area and medial substantia nigra pars compacta contain the peptide cotransmitter CCK (30, 53, 54), which is present in the brain mainly in its sulfated octapeptide form (4, 21, 22, 50). A subpopulation of these DA/CCK neurons contains a second peptide cotransmitter, neurotensin (52). In addition to the acknowledged autoregulatory role of nonsynaptic somatodendritically released DA, the similar release of these peptides may result in additional phasic autoregulatory influences on the transmembrane currents and impulse activity of DA neurons. The midbrain also receives a sparse input from CCK-containing axon terminals (52) and receives a more dense input from neurotensin-containing terminals (see below and Colocalization in Dopamine Neurons).

Exogenously administered CCK and neurotensin influence the sensitivity of impulse-regulating DA autoreceptors to DA agonist-induced inhibition of firing rate. To date, these studies have been limited to extracellular electrophysiological experiments. After intravenous (i.v.) administration to rats, CCK potentiates the inhibitory effects of i.v. administration of the mixed DA agonist apomorphine (32, 33), and of the D2 agonist quinpirole (36), on DA cell firing rate. Microiontophoresis of CCK into the vicinity of the somatodendritic region of DA cells also potentiates the inhibitory effects of similarly applied DA (16). Bath-applied CCK potentiates the inhibitory effects of DA and quinpirole in midbrain brain slices in vitro (7, 66). Although not proof, these studies suggest that the effect of CCK on DA autoreceptor-mediated inhibition of impulse flow is due to a direct interaction with the DA cell membrane. Similarly, a substantial portion of the excitatory effects of i.v. CCK on DA cell firing rate was concluded to be due to direct effects on DA cells (31). Intracellular studies are required to elucidate the nature of the specific ionic conductances involved in the interaction of CCK with autoreceptor mechanisms.

CCK binds to both CCK-A and CCK-B receptors (46). Unsulfated CCK and CCK tetrapeptide are selective CCK-B agonists, and they have been used to explore the question of which receptor subtype is associated with the modulatory effects of CCK on DA autoreceptor function. There are reports of involvement of CCK-A receptors (36) and CCK-B receptors (33) in these effects.

In contrast to CCK, the tridecapeptide neurotensin attenuates the inhibitory effects of DA autoreceptor stimulation on DA neuronal firing rate. Intracerebroventricular administration of neurotensin antagonizes the inhibitory effects of i.v. quinpirole on DA cell activity (56). In in vitro midbrain slices, neurotensin also attenuates the inhibitory effects of DA (57) and the DA agonist BHT 920 (55) on DA cell firing rate. Neurotensin exerts this effect through a cAMP-dependent mechanism: This effect is mimicked by 8-Br-cAMP, by forskolin, by inhibition of phosphodiesterase, and by inhibition of protein kinase A (58).

In the ventral tegmental area, it appears that all neurotensin-immunoreactive perikarya also contain DA (3, 29, 52)). Some of these perikarya are in direct apposition to another DA neuron (3), which provides the anatomical basis for intercellular effects of somatodendritic neurotensin and DA on DA cells. In addition, neurotensin-containing axon terminals exist in the midbrain (3, 29, 35, 52, 68). About one-third of the targets of these terminals are DA cells (3). As with CCK, detailed analysis of the effects of neurotensin on transmembrane ionic currents should enhance our understanding of the mechanism of action of this peptide in the modulation of impulse regulating DA autoreceptors. The opposing effects of CCK and neurotensin on DA autoreceptor function may represent a fine-tuning mechanism for the control of DA autoreceptor sensitivity.

As reviewed above, our understanding of the nature of somatodendritic autoreceptor modulation of DA cell activity has changed over the last few years. We now know that a variety of specific potassium and calcium currents in these cells are modulated by DA autoreceptor stimulation. The increase in the magnitudes of IA and IK, when coupled with the decrease in magnitudes of IL and IN, serves as a direct and effective means of decreasing DA cell membrane excitability. It remains to be determined which G proteins are involved in the coupling of DA autoreceptors with inward calcium currents. An understanding of the transduction mechanisms that influence these inward currents is critical to determining which individual currents are affected by autoreceptor stimulation. For example, it is now clear that, because IA and IK share a common transduction pathway, they are modulated simultaneously by agonist stimulation of the autoreceptor. If IL and IN are shown to be associated with G proteins different from those associated with IA and IK, it would suggest that the autoreceptor-mediated increase in outward and decrease in inward currents could be affected differentially.

published 2000