Salvador Hernandez-Lopez

G-Protein-Coupled Receptor Modulation of Striatal CaV1.3 L-Type Ca2+ Channels Is Dependent on a Shank-Binding Domain

Voltage-gated L-type Ca2+ channels are key determinants of synaptic integration and plasticity, dendritic electrogenesis, and activity-dependent gene expression in neurons. Fulfilling these functions requires appropriate channel gating, perisynaptic targeting, and linkage to intracellular signaling cascades controlled by G-protein-coupled receptors (GPCRs). Surprisingly, little is known about how these requirements are met in neurons. The studies described here shed new light on how this is accomplished. We show that D2 dopaminergic and M1 muscarinic receptors selectively modulate a biophysically distinctive subtype of L-type Ca2+ channels (CaV1.3) in striatal medium spiny neurons. The splice variant of these channels expressed in medium spiny neurons contains cytoplasmic Src homology 3 and PDZ (postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1) domains that bind the synaptic scaffolding protein Shank. Medium spiny neurons coexpressed CaV1.3-interacting Shank isoforms that colocalized with PSD-95 and CaV1.3a channels in puncta resembling spines on which glutamatergic corticostriatal synapses are formed. The modulation of CaV1.3 channels by D2 and M1 receptors was disrupted by intracellular dialysis of a peptide designed to compete for the CaV1.3 PDZ domain but not with one targeting a related PDZ domain. The modulation also was disrupted by application of peptides targeting the Shank interaction with Homer. Upstate transitions in medium spiny neurons driven by activation of glutamatergic receptors were suppressed by genetic deletion of CaV1.3 channels or by activation of D2 dopaminergic receptors. Together, these results suggest that Shank promotes the assembly of a signaling complex at corticostriatal synapses that enables key GPCRs to regulate L-type Ca2+ channels and the integration of glutamatergic synaptic events.

D2 dopamine receptor-mediated modulation of voltage-dependent Na+ channels reduces autonomous activity in striatal cholinergic interneurons

Striatal cholinergic interneurons are critical elements of the striatal circuitry controlling motor planning, movement, and associative learning. Intrastriatal release of dopamine and inhibition of interneuron activity is thought to be a critical link between behaviorally relevant events, such as reward, and alterations in striatal function. However, the mechanisms mediating this modulation are unclear. Using a combination of electrophysiological, molecular, and computational approaches, the studies reported here show that D2 dopamine receptor modulation of Na+ currents underlying autonomous spiking contributes to a slowing of discharge rate, such as that seen in vivo. Four lines of evidence support this conclusion. First, D2 receptor stimulation in tissue slices reduced the autonomous spiking in the presence of synaptic blockers. Second, in acutely isolated neurons, D2 receptor activation led to a reduction in Na+ currents underlying pacemaking. The modulation was mediated by a protein kinase C-dependent enhancement of channel entry into a slow-inactivated state at depolarized potentials. Third, the sodium channel blocker TTX mimicked the effects of D2 receptor agonists on pacemaking. Fourth, simulation of cholinergic interneuron pacemaking revealed that a modest increase in the entry of Na+ channels into the slow-inactivated state was sufficient to account for the slowing of pacemaker discharge. These studies establish a cellular mechanism linking dopamine and the reduction in striatal cholinergic interneuron activity seen in the initial stages of associative learning.

Spontaneous Voltage Oscillations in Striatal Projection Neurons in a Rat Corticostriatal Slice

In a rat corticostriatal slice, brief, suprathreshold, repetitive cortical stimulation evoked long‐lasting plateau potentials in neostriatal neurons. Plateau potentials were often followed by spontaneous voltage transitions between two preferred membrane potentials. While the induction of plateau potentials was disrupted by non‐NMDA and NMDA glutamate receptor antagonists, the maintenance of spontaneous voltage transitions was only blocked by NMDA receptor and L‐type Ca2+ channel antagonists. The frequency and duration of depolarized events, resembling up‐states described in vivo, were increased by NMDA and L‐type Ca2+ channel agonists as well as by GABAA receptor and K+ channel antagonists. NMDA created a region of negative slope conductance and a positive slope crossing indicative of membrane bistability in the current‐voltage relationship. NMDA‐induced bistability was partially blocked by L‐type Ca2+ channel antagonists. Although evoked by synaptic stimulation, plateau potentials and voltage oscillations could not be evoked by somatic current injection – suggesting a dendritic origin. These data show that NMDA and L‐type Ca2+ conductances of spiny neurons are capable of rendering them bistable. This may help to support prolonged depolarizations and voltage oscillations under certain conditions.

Inhibitory action of dopamine involves a subthreshold Cs+-sensitive conductance in neostriatal neurons

Intracellular recordings in in vitro slice preparations of rat brain were used to compare the actions of dopamine and dopamine receptor agonists on the subthreshold membrane properties of neostriatal neurons. A reproducible response for dopaminergic agonists was evoked after firing produced by current ramp injections that induced a subthreshold voltage displacement. Dopamine (10–100 μM) decreased both firing rate and membrane slope input resistance in virtually all cells tested. Input resistance change appeared as an increase in inward rectification. Approximate reversal potential was around -87 mV. The D1 receptor agonists SKF 38393 and C1-APB (1–10 μM) mimicked both dopamine effects with a reversal potential around -89 mV. The effects were blocked by the presence of 5–10 μM caesium (Cs+) but not by 1 μM tetrodotoxin, suggesting that main D1 effects on input resistance are due to subthreshold Cs+ sensitive conductances. cAMP analogues mimicked the actions of D1 receptor agonists. The D2 agonist, quinpirole (1–10 μM), did not produce any input resistance change, nonetheless, it still produced a decrease in firing rate. This suggests that the main D2 effect on firing is due to actions on suprathreshold ion conductances. All effects were blocked by D1 and D2 antagonists, respectively. D1 or D2 effects were found in the majority of cells tested.

Presynaptic α4β2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus.

Several behavioral effects of nicotine are mediated by changes in serotonin (5-HT) release in brain areas that receive serotonergic afferents from the dorsal raphe nucleus (DRN). In vitro experiments have demonstrated that nicotine increases the firing activity in the majority of DRN 5-HT neurons and that DRN contains nicotinic acetylcholine receptors (nAChRs) located at both somata and presynaptic elements. One of the most common presynaptic effects of nicotine is to increase glutamate release. Although DRN receives profuse glutamatergic afferents, the effect of nicotine on glutamate release in the DRN has not been studied in detail. Using whole-cell recording techniques, we investigated the effects of nicotine on the glutamatergic input to 5-HT DRN neurons in rat midbrain slices. Low nicotine concentrations, in the presence of bicuculline and tetrodotoxin (TTX), increased the frequency but did not change the amplitude of glutamate-induced EPSCs, recorded from identified 5-HT neurons. Nicotine-induced increase of glutamatergic EPSC frequency persisted 10–20 min after drug withdrawal. This nicotinic effect was mimicked by exogenous administration of acetylcholine (ACh) or inhibition of ACh metabolism. In addition, the nicotine-induced increase in EPSC frequency was abolished by blockade of α4β2 nAChRs, voltage-gated calcium channels, or intracellular calcium signaling but not by α7 nAChR antagonists. These data suggest that both nicotine and endogenous ACh can increase glutamate release through activation of presynaptic α4β2 but not α7 nAChRs in the DRN. The effect involves long-term changes in synaptic function, and it is dependent on voltage-gated calcium channels and presynaptic calcium stores.

Serotoninergic dorsal raphe neurons possess functional postsynaptic nicotinic acetylcholine receptors

Very few neurons in the telencephalon have been shown to express functional postsynaptic nicotinic acetylcholine receptors (nAChRs), among them, the noradrenergic and dopaminergic neurons. However, there is no evidence for postsynaptic nAChRs on serotonergic neurons. In this study, we asked if functional nAChRs are present in serotonergic (5‐HT) and nonserotonergic (non‐5‐HT) neurons of the dorsal raphe nucleus (DRN). In rat midbrain slices, field stimulation at the tegmental pedunculopontine (PPT) nucleus evoked postsynaptic currents (eEPSCs) with different components in DRN neurons. After blocking the glutamatergic and GABAergic components, the remaining eEPSCs were blocked by mecamylamine and reduced by either the selective α7 nAChR antagonist methyllycaconitine (MLA) or the selective α4β2 nAChR antagonist dihydro‐β‐eritroidine (DHβE). Simultaneous addition of MLA and DHβE blocked all eEPSCs. Integrity of the PPT‐DRN pathway was assessed by both anterograde biocytin tracing and antidromic stimulation from the DRN. Inward currents evoked by the direct application of acetylcholine (ACh), in the presence of atropine and tetrodotoxin, consisted of two kinetically different currents: one was blocked by MLA and the other by DHβE; in both 5‐HT and non‐5‐HT DR neurons. Analysis of spontaneous (sEPSCs) and evoked (eEPSCs) synaptic events led to the conclusion that nAChRs were located at the postsynaptic membrane. The possible implications of these newly described nAChRs in various physiological processes and behavioral events, such as the wake‐sleep cycle, are discussed. Synapse 62:601–615, 2008.

Dopaminergic Modulation of the Striatal Microcircuit: Receptor-Specific Configuration of Cell Assemblies

Selection and inhibition of motor behaviors are related to the coordinated activity and compositional capabilities of striatal cell assemblies. Striatal network activity represents a main step in basal ganglia processing. The dopaminergic system differentially regulates distinct populations of striatal medium spiny neurons (MSNs) through the activation of D1- or D2-type receptors. Although postsynaptic and presynaptic actions of these receptors are clearly different in MSNs during cell-focused studies, their activation during network activity has shown inconsistent responses. Therefore, using electrophysiological techniques, functional multicell calcium imaging, and neuronal population analysis in rat corticostriatal slices, we describe the effect of selective dopaminergic receptor activation in the striatal network by observing cell assembly configurations. At the microcircuit level, during striatal network activity, the selective activation of either D1- or D2-type receptors is reflected as overall increases in neuronal synchronization. However, graph theory techniques applied to the transitions between network states revealed receptor-specific configurations of striatal cell assemblies: D1 receptor activation generated closed trajectories with high recurrence and few alternate routes favoring the selection of specific sequences, whereas D2 receptor activation created trajectories with low recurrence and more alternate pathways while promoting diverse transitions among neuronal pools. At the single-cell level, the activation of dopaminergic receptors enhanced the negative-slope conductance region (NSCR) in D1-type-responsive cells, whereas in neurons expressing D2-type receptors, the NSCR was decreased. Consequently, receptor-specific network dynamics most probably result from the interplay of postsynaptic and presynaptic dopaminergic actions.

Action of Substance P (Neurokinin-1) Receptor Activation on Rat Neostriatal Projection Neurons

Substance P (SP) acts as a neurotransmitter in the neostriatum through the axon collaterals of spiny projection neurons. However, possible direct or indirect actions of SP on the neostriatal output neurons have not been described. Targets of SP terminals within the neostriatum include interneurons, spiny neurons, afferent fibers and boutons. SP induces the release of both dopamine (DA) and acetylcholine (ACh). Since some postsynaptic actions of both DA and ACh on spiny neurons are known, we asked if activation of neostriatal NK1‐class receptors is able to reproduce them. The SP NK1‐receptor agonist, GR73632 (1 μM), had both excitatory and inhibitory actions on virtually all spiny neurons tested at resting potential. The excitatory action was blocked by atropine and coursed with an increase in firing rate and input resistance (RN). The inhibitory action was blocked by haloperidol and coursed with a reduction in firing rate and RN. Therefore, the release of both DA and ACh induced by NK1‐receptor activation modulates indirectly the excitability of the projection neurons. SP facilitates the actions of these transmitters on the spiny neuron. A residual excitatory response to the NK1‐receptor agonist was observed in 30% of a sample of neurons tested in the presence of both haloperidol and atropine. The increase in RN that accompanied this response could be observed in the presence of 1 μM TTX or 100 μM Cd2+, suggesting a direct effect. Double labeling showed that only SP‐immunoreactive neurons were facilitated by NK1‐receptor activation in these conditions. Synapse 33:26–35, 1999.

Dopamine modulates the afterhyperpolarization in neostriatal neurones.

Intracellular techniques were used to study the actions of dopaminergic D1 agonists on the afterhyperpolarization (AHP) that follows action potentials in rat neostriatal neurones. Dopamine or Cl-APB (10 μM), or 1–10 μM 6-C1-PB all increased AHP amplitude. This effect was blocked by 1 μM SCH-23390, a D1 antagonist, but not by 1 μM sulpiride, a D2 antagonist. Both 500 μM dibutyryl cAMP and 5 μM BayK 8644 induced a similar AHP increase. BayK 8644 occluded the effect of agonists. The results suggest that the action of dopamine is mediated via the recently described protein kinase A enhancement of L-type Ca2+ channels. The results partially explain the decrease in firing frequency induced by dopamine and a possible site of antagonism with cholinergic modulation.

Nicotinic modulation of serotonergic activity in the dorsal raphe nucleus.

Abstract Cholinergic signaling mediated by nicotinic receptors has been associated to a large number of physiological and behavioral processes such as learning, memory, attention, food-intake and mood disorders. Although it is well established that many nicotinic actions are mediated through an increase in serotonin (5-HT) release, the physiological mechanisms by which nicotine produces these effects are still unclear. The dorsal raphe nucleus (DRN) contains the major amount of 5-HT neurons projecting to different parts of the brain. DRN also contains nicotinic acetylcholine receptors (nAChRs) located at somatic and presynaptic elements. Nicotine produces both inhibitory and excitatory effects on different subpopulations of 5-HT DRN neurons. In this review, we describe the presynaptic and postsynaptic mechanisms by which nicotine increases the excitability of DRN neurons as well as the subtypes of nAChRs involved. We also describe the inhibitory effects of nicotine and the role of 5-HT1A receptors in this effect. These nicotinic actions modulate the activity of different neuronal subpopulations in the DRN, changing the 5-HT tone in the brain areas where these groups of neurons project. Some of the physiological implications of nicotine-induced 5-HT release are discussed.