D. James Surmeier

Modulation of calcium currents by a D1 dopaminergic protein kinase/phosphatase cascade in rat neostriatal neurons

In rat neostriatal neurons, D1 dopamine receptors regulate the activity of cyclic AMP-dependent protein kinase (PKA) and protein phosphatase 1 (PP1). The influence of these signaling elements on high voltage-activated (HVA) calcium currents was studied using whole-cell voltage-clamp techniques. The application of D1 agonists or cyclic AMP analogs reversibly reduced N- and P-type Ca2+ currents. Inhibition of PKA antagonized this modulation, as did inhibition of PP1, suggesting that the D1 effect was mediated by a PKA enhancement of PP1 activity directed toward Ca2+ channels. In a subset of neurons, D1 receptor-mediated activation of PKA enhanced L-type currents. The differential regulation of HVA currents by the D1 pathway helps to explain the diversity of effects this pathway has on synaptic integration and plasticity in medium spiny neurons.

Are neostriatal dopamine receptors co-localized?

The postsynaptic effects of dopamine in the neostriatum are mediated by five G-protein-coupled receptors. The extent to which these receptors are co-localized in neostriatal neurons has become controversial. This debate has far-reaching implications for treatment strategies in disorders of dopaminergic signaling, such as Parkinsons disease and schizophrenia. This review examines the molecular and cellular evidence for and against co-localization, including new information derived from single-cell mRNA amplification and patch-clamping of isolated neurons. It is concluded that this evidence is largely consistent with co-localization of functionally significant receptors of the D1 and D2 families in the majority of neostriatal efferent neurons. This conclusion has important implications for parallel processing models of the neostriatum.

D5 Dopamine Receptors Enhance Zn2+-Sensitive GABAA Currents in Striatal Cholinergic Interneurons through a PKA/PP1 Cascade

Cholinergic interneurons have been implicated in striatally mediated associative learning. In classical conditioning paradigms, conditioned stimuli trigger a transient suppression of neuronal activity that is dependent upon an intact dopaminergic innervation. Our hypothesis was that this suppression reflected dopaminergic enhancement of sensory-linked GABAergic input. As a test, the impact of dopamine on interneuronal GABA(A) receptor function was studied by combined patch-clamp recording and single-cell reverse transcription PCR. Activation of D5 dopamine receptors reversibly enhanced a Zn2+-sensitive component of GABA(A) currents. Although dependent upon protein kinase A (PKA) activation, the modulation was blocked by protein phosphatase 1 (PP1) inhibition, suggesting it was dependent upon dephosphorylation. These results establish a novel mechanism by which intrastriatally released dopamine mediates changes in GABAergic signaling that could underlie the initial stages of associative learning.

Developmental regulation of a slowly-inactivating potassium conductance in rat neostriatal neurons.

In late embryonic and early post-natal rat neostriatal neurons, the voltage-dependent potassium currents activated by depolarization are largely attributable to a rapidly inactivating A-current and a delayed rectifier current. Over the first 4 weeks of post-natal life, a third potassium current emerges in most cells. This slowly inactivating conductance is distinct from the A-current and delayed rectifier in voltage-dependence, kinetics and pharmacology. The properties of this conductance suggest that it may be of central importance to the integrative behavior of neostriatal neurons by controlling such features as first spike latency and interspike interval.

Inwardly Rectifying Potassium (IRK) Currents Are Correlated with IRK Subunit Expression in Rat Nucleus Accumbens Medium Spiny Neurons

Inwardly rectifying K+ (IRK) channels are critical for shaping cell excitability. Whole-cell patch-clamp and single-cell RT-PCR techniques were used to characterize the inwardly rectifying K+ currents found in projection neurons of the rat nucleus accumbens. Inwardly rectifying currents were highly selective for K+ and blocked by low millimolar concentrations of Cs+ or Ba2+. In a subset of neurons, the inwardly rectifying current appeared to inactivate at hyperpolarized membrane potentials. In an attempt to identify this subset, neurons were profiled using single-cell RT-PCR. Neurons expressing substance P mRNA exhibited noninactivating inward rectifier currents, whereas neurons expressing enkephalin mRNA exhibited inactivating inward rectifier currents. The inactivation of the inward rectifier was correlated with the expression of IRK1 mRNA. These results demonstrate a clear physiological difference in the properties of medium spiny neurons and suggest that this difference could influence active state transitions driven by cortical and hippocampal excitatory input.

Two types of A-current differing in voltage-dependence are expressed by neurons of the rat neostriatum

Transient potassium currents of the A type are thought to be important in a number of physiological processes of excitable cells, including spike repolarization and synaptic integration. This functional diversity may reflect the contribution of distinct subtypes of A channel to cellular behavior. Using the whole-cell variant of the patch clamp technique, we have found that two types of A-current are expressed in rat neostriatal neurons, one that is similar to previous descriptions in mammals and a second that is activated at considerably more depolarized potentials.

Glutamate-Mediated Excitotoxic Death of Cultured Striatal Neurons Is Mediated by Non-NMDA Receptors

Considerable interest has focused on the role of glutamate-mediated excitotoxicity in neurodegenerative disorders of the basal ganglia. The in vitro data on the receptor mechanisms involved in this process, however, have been inconclusive. Some studies have indicated that excitotoxins acting at NMDA receptors kill striatal neurons and others have indicated that NMDA receptor-mediated excitotoxic death of striatal neurons is minimal in the absence of cortex. In the present study, we used a pharmacological approach to carefully reexamine this issue in 2-week-old cultures of striatal neurons dissociated from E17 rat embryos. The sensitivity of these neurons to glutamate agonists and antagonists was determined by monitoring cell loss in identified regions of the growth dishes. We found that glutamate killed striatal neurons with an EC50 of 100 microM. This loss was not mediated by NMDA receptors, since it was not reduced by the NMDA receptor antagonist APV (0.1-1.0 mM). Consistent with this result, up to 50 mM NMDA receptor-specific excitotoxin quinolinic acid (QA) did not affect neuronal survival. Depolarizing the QA-exposed neurons with 35 mM potassium chloride to enhance NMDA receptor activation by QA also did not produce neuron loss. The metabotropic glutamate receptor antagonist AP3 (500 microM) also had no effect on the striatal neuron loss produced by 100 microM glutamate. In contrast, the non-NMDA antagonist GYKI 52466 (100 microM) did block the excitotoxic effect of glutamate (100 microM). Specific AMPA and KA receptor agonists and the non-NMDA antagonist GYKI 52466 revealed that the non-NMDA receptor-mediated excitotoxic effect of glutamate was mediated by KA receptors. These results suggest that cultured striatal neurons are directly vulnerable to non-NMDA glutamate agonists, but not to NMDA and metabotropic glutamate agonists. Thus, non-NMDA receptors may play a greater role in the excitotoxic death of striatal neurons in disease and experimental animal models than previously realized.

Voltage-clamp analysis of a transient potassium current in rat neostriatal neurons.

Whole cell voltage-clamp recordings were made from cultured rat neostriatal neurons. Depolarizing voltage commands evoked transient and sustained outward K-currents. The transient K-current was activated by depolarizing commands beyond -50 mV; peak current was dependent upon holding potential. Bath application of 4-aminopyridine, but not inorganic calcium channel blockers (Cd, Co, Mn), attenuated the transient current. Reversal was near the K-equilibrium potential. These properties suggest that this transient K-current is similar to the A-current described in a number of other neurons.

Selective blockade of a slowly inactivating potassium current in striatal neurons by (±) 6-chloro-APB hydrobromide (SKF82958)

The ion channels of rat striatal neurons are known to be modulated by stimulation of D1 dopamine receptors. The susceptibility of depolarization‐activated K+ currents to be modulated by the D1 agonist, 6‐chloro‐7,8‐dihydroxy‐3‐allyl‐1‐phenyl‐2,3,4,5‐tetra‐hydro‐1H‐3‐benzazepine (APB) was investigated using whole‐cell voltage‐clamp recording techniques from acutely isolated neurons. APB (0.01–100 μM) produced a concentration‐dependent reduction in the total K+ current. At intermediate concentrations (ca. 10 μM), APB selectively depressed the slowly inactivating A‐current (IAs). A similar effect was produced by application of the D1 agonist, 7,8‐dihydroxy‐1‐phenyl‐2,3,4,5‐tetrahydro‐1‐H‐2‐benzazepine (SKF38393, 10 μM). APB reduced IAs rapidly, having onset and recovery time constants of 1.2 sec and 1.6 sec, respectively. Unexpectedly, the effect of APB could not be mimicked by application of Sp‐adenosine 3′,5′‐cyclic monophosphothioate triethylamine (Sp‐cAMPS, 100–200 μM), a membrane‐permeable analog of cyclic AMP (cAMP), or by pretreatment with forskolin (25 μM), an activator of adenylyl cyclase. The reduction in IAs also was not blocked by pretreatment with the D1 receptor antagonist, R(+)‐SCH23390 hydrochloride (SCH23390, 10–20 μM). In addition, intracellular dialysis with guanosine‐5′‐O‐(2‐thiodiphosphate (GDP‐β‐S, 200 μM) did not preclude the APB‐induced inhibition of IAs, nor did dialysis with guanosine‐5′‐O‐(3‐thiotriphosphate (GTP‐γ‐S, 400 μM) prevent reversal of the effect. The effect of APB was produced by a reduction in the maximal conductance of IAs without changing the voltage‐dependence of the current. Collectively, these results argue that APB does not inhibit IAs through D1 receptors coupled to stimulation of adenylyl cyclase, but rather by allosterically regulating or blocking the channels giving rise to this current. Synapse 29:213–224, 1998.

The expression of γ-aminobutyric acid and Leu-enkephalin immunoreactivity in primary monolayer cultures of rat striatum

Primary monolayer cultures of rat striatum were examined for gamma-aminobutyric acid (GABA) and leucine-enkephalin (L-ENK) immunoreactivity. Cultures were established on polycation-treated glass coverslips from the striata of gestational day 17 rat embryos using a serum and insulin-supplemented medium. The proportion of GABA-immunoreactive (GABA-IR) neurons increased during the first week in vitro from approximately one third to nearly one half and remained relatively constant thereafter. On the other hand, the proportion of L-ENK-IR neurons increased gradually over the culturing period, increasing from about one-fifth of the neurons initially to one-half after 3-4 weeks in vitro. The changes in the proportions of GABA- and L-ENK-IR neurons appeared to be largely a consequence of the death of non-immunoreactive neurons, not delayed expression or induction of GABA or L-ENK traits. Light microscopic analysis of somatic-proximal neuritic morphology led to a partitioning of the neuronal population into 4 groups. GABA- and L-ENK-IR groups were heterogeneous in this regard and differed only modestly.