Indirect Striatal Projection Neurons Research Articles

Selective activation of striatal indirect pathway suppresses levodopa induced-dyskinesias

Levodopa (L-DOPA) administration remains the gold standard therapy for Parkinson's disease (PD). Despite several pharmacological advances in the use of L-DOPA, a high proportion of chronically treated patients continues to suffer disabling involuntary movements, namely, L-DOPA-induced dyskinesias (LIDs). As part of the effort to stop these unwanted side effects, the present study used a rodent model to identify and manipulate the striatal outflow circuitry responsible for LIDs. To do so, optogenetic technology was used to activate separately the striatal direct (D1R- expressing) and indirect (D2R- expressing) pathways in a mouse model of PD. Firstly, D1-cre or A2a-cre animals received unilateral injections of neurotoxin 6-hydroxydopamine (6-OHDA) to simulate the loss of dopamine observed in PD patients. The effects of independently stimulating each pathway were tested to see if experimental dyskinesias could be induced. Secondly, dopamine depleted A2a-cre animals received systemic L-DOPA to evoke dyskinetic movements. The ability of indirect pathway optogenetic stimulation to suppress pre-established LIDs was then tested. Selective manipulation of direct pathway evoked optodyskinesias both in dopamine depleted and intact animals, but optical inhibition of these neurons failed to suppress LIDs. On the other hand, selective activation of indirect striatal projection neurons produced an immediate and reliable suppression of LIDs. Thus, a functional dissociation has been found here whereby activation of D1R- and D2R-expressing projection neurons evokes and inhibits LIDs respectively, supporting the notion of tight interaction between the two striatal efferent systems in both normal and pathological conditions. This points to the importance of maintaining an equilibrium in the activity of both striatal pathways to produce normal movement. Finally, the ability of selective indirect pathway optogenetic activation to block the expression of LIDs in an animal model of PD sheds light on intrinsic mechanisms responsible for striatal-based dyskinesias and identifies a potential therapeutic target for suppressing LIDs in PD patients.

Dopamine D2 and Adenosine A2A Receptors Interaction on Ca2+ Current Modulation in a Rodent Model of Parkinsonism.

Adenosine A1 and A2A receptors are expressed in striatal projection neurons (SPNs). A1 receptors are located in direct (dSPN) and indirect SPNs (iSNP). A2A receptors are only present in iSPNs. Dopamine D2 receptors are also expressed in iSPNs and interactions between D2 and A2A receptors have received attention. iSPNs activity increases during parkinsonism (PD) and A2A receptors may be responsible by enhancing Ca2+ currents (iCa2+). Therefore, A2A receptors blockade is a therapeutic approach. We asked whether A2A receptors need the interaction with D2 receptors (D2R) to exert their actions. By using isolated and identified iSPNs to avoid indirect influences, we show that D2R action habilitates A2A receptors (A2AR) modulation. iCa2+ through voltage gated Ca2+ channels (CaV) was used as a signal to observe this interaction. Voltage-clamp recordings in acutely dissociated iSPNs, current-clamp recordings in slices and calcium imaging in transgenic A2A-Cre mice, showed that D2R reduction in iCa2+ endows A2AR to restore iCa2+ on iSPNs showing an antagonistic interaction between D2 and A2A receptors. A2A receptors were blocked by the antagonist istradefylline, however, this blockade differed in control and dopamine-depleted iSPNs: istradefylline reduced D2R modulation in parkinsonian animals as compared to controls. Calcium imaging recordings show that istradefylline occludes D2R actions in the parkinsonian circuitry and this effect depends on the order of drugs application. Thus, while D2 activation enables A2A receptors action, blockade of A2AR induces a reduction in the action of D2 agonists, confirming a complex interaction.Summary StatementA2A receptor required previous D2 receptor activation to modulate Ca2+ currents. Istradefylline decreases pramipexole modulation on Ca2+ currents. Istradefylline reduces A2A + neurons activity in striatial microcircuit, but pramipexole failed to further reduce neuronal activity.

Striatal indirect pathway mediates exploration via collicular competition.

The ability to suppress actions that lead to a negative outcome and explore alternative actions is necessary for optimal decision making. Although the basal ganglia have been implicated in these processes1-5, the circuit mechanisms underlying action selection and exploration remain unclear. Here, using a simple lateralized licking task, we show that indirect striatal projection neurons (iSPN) in the basal ganglia contribute to these processes through modulation of the superior colliculus (SC). Optogenetic activation of iSPNs suppresses contraversive licking and promotes ipsiversive licking. Activity in lateral superior colliculus (lSC), a region downstream of the basal ganglia, is necessary for task performance and predicts lick direction. Furthermore, iSPN activation suppresses ipsilateral lSC, but surprisingly excites contralateral lSC, explaining the emergence of ipsiversive licking. Optogenetic inactivation reveals inter-collicular competition whereby each hemisphere of the superior colliculus inhibits the other, thus allowing the indirect pathway to disinhibit the contralateral lSC and trigger licking. Finally, inactivating iSPNs impairs suppression of devalued but previously rewarded licking and reduces exploratory licking. Our results reveal that iSPNs engage the competitive interaction between lSC hemispheres to trigger a motor action and suggest a general circuit mechanism for exploration during action selection.

Differences in synaptic integration between direct and indirect striatal projection neurons: Role of CaV 3 channels.

Different corticostriatal suprathreshold responses in direct and indirect striatal projection neurons (SPNs) of rodents have been reported. Responses consist in prolonged synaptic potentials of polysynaptic and intrinsic origin, in which voltage-gated Ca2 ⁺ currents play a role. Recording simultaneous Ca2 ⁺ imaging and voltage responses at the soma, while activating the corticostriatal pathway, we show that encoding of synaptic responses into trains of action potentials (APs) is different in SPNs: firing of APs in D1-SPNs increase gradually, in parallel with Ca2 ⁺ entry, as a function of stimulus intensity. In contrast, D2-SPNs attain a maximum number of evoked spikes at low stimulus intensities, Ca2 ⁺ entry is limited, and both remain the same in spite of increasing stimulus strength. Stimulus needs to reach certain intensity, to have propagated Ca2 ⁺ potentials to the soma plus a sudden step in Ca2 ⁺ entry, without changing the number of fired APs, phenomena never seen in D1-SPNs. Constant firing in spite of changing stimulus, suggested the involvement of underlying inactivating potentials. We found that Caᵥ3 currents contribute to Ca2+ entry in both classes of SPNs, but have a more notable effect in D2-SPNs, where a low-threshold spike was disclosed. Blockade of CaV 3 channels retarded the steep rise in firing in D2-SPNs. Inhibition block increased the number of spikes fired by D2-SPNs, without changing firing in D1-SPNs. These differences in synaptic integration enable a biophysical dissimilarity: dendritic inhibition appears to be more relevant for D2-SPNs. This may imply distinctions in the set of interneurons affecting each SPN class.

Correction: Suarez et al., “Differential Synaptic Remodeling by Dopamine in Direct and Indirect Striatal Projection Neurons in Pitx3−/−Mice, a Genetic Model of Parkinson's Disease”

Correction: Suarez et al., “Differential Synaptic Remodeling by Dopamine in Direct and Indirect Striatal Projection Neurons in Pitx3^−/−Mice, a Genetic Model of Parkinson's Disease”

Differential Synaptic Remodeling by Dopamine in Direct and Indirect Striatal Projection Neurons in Pitx3-/- Mice, a Genetic Model of Parkinson's Disease.

In toxin-based models of Parkinson's disease (PD), striatal projection neurons (SPNs) exhibit dendritic atrophy and spine loss concurrent with an increase in excitability. Chronic l-DOPA treatment that induces dyskinesia selectively restores spine density and excitability in indirect pathway SPNs (iSPNs), whereas spine loss and hyperexcitability persist in direct pathway SPNs (dSPNs). These alterations have only been characterized in toxin-based models of PD, raising the possibility that they are an artifact of exposure to the toxin, which may engage compensatory mechanisms independent of the PD-like pathology or due to the loss of dopaminergic afferents. To test all these, we studied the synaptic remodeling in Pitx3-/- or aphakia mice, a genetic model of PD, in which most of the dopamine neurons in the substantia nigra fail to fully differentiate and to innervate the striatum. We made 3D reconstructions of the dendritic arbor and measured excitability in identified SPNs located in dorsal striatum of BAC-Pitx3-/- mice treated with saline or l-DOPA. Both dSPNs and iSPNs from BAC-Pitx3-/- mice had shorter dendritic trees, lower spine density, and more action potentials than their counterparts from WT mice. Chronic l-DOPA treatment restored spine density and firing rate in iSPNs. By contrast, in dSPNs, spine loss and hyperexcitability persisted following l-DOPA treatment, which is similar to what happens in 6-OHDA WT mice. This indicates that dopamine-mediated synaptic remodeling and plasticity is independent of dopamine innervation during SPN development and that Pitx3-/- mice are a good model because they develop the same pathology described in the toxins-based models and in human postmortem studies of advanced PD.SIGNIFICANCE STATEMENT As the only genetic model of Parkinson's disease (PD) that develops dyskinesia, Pitx3-/- mice reproduce the behavioral effects seen in humans and are a good system for studying dopamine-induced synaptic remodeling. The studies we present here establish that the structural and functional synaptic plasticity that occur in striatal projection neurons in PD and in l-DOPA-induced dyskinesia are specifically due to modulation of the neurotransmitter dopamine and are not artifacts of the use of chemical toxins in PD models. In addition, our findings provide evidence that synaptic plasticity in the Pitx3-/- mouse is similar to that seen in toxin models despite its lack of dopaminergic innervation of the striatum during development. Pitx3-/- mice reproduced the alterations described in patients with advanced PD and in well accepted toxin-based models of PD and dyskinesia. These results further consolidate the fidelity of the Pitx3-/- mouse as a PD model in which to study the morphological and physiological remodeling of striatal projection neurons by administration of l-DOPA and other drugs.

Contribution of different classes of glutamate receptors in the corticostriatal polysynaptic responses from striatal direct and indirect projection neurons

BackgroundPrevious work showed differences in the polysynaptic activation of GABAergic synapses during corticostriatal suprathreshold responses in direct and indirect striatal projection neurons (dSPNs and iSPNs). Here, we now show differences and similarities in the polysynaptic activation of cortical glutamatergic synapses on the same responses. Corticostriatal contacts have been extensively studied. However, several questions remain unanswered, e.g.: what are the differences and similarities in the responses to glutamate in dSPNs and iSPNs? Does glutamatergic synaptic activation exhibits a distribution of latencies over time in vitro? That would be a strong suggestion of polysynaptic cortical convergence. What is the role of kainate receptors in corticostriatal transmission? Current-clamp recordings were used to answer these questions. One hypothesis was: if prolonged synaptic activation distributed along time was present, then it would be mainly generated from the cortex, and not from the striatum.ResultsBy isolating responses from AMPA-receptors out of the complex suprathreshold response of SPNs, it is shown that a single cortical stimulus induces early and late synaptic activation lasting hundreds of milliseconds. Prolonged responses depended on cortical stimulation because they could not be elicited using intrastriatal stimulation, even if GABAergic transmission was blocked. Thus, the results are not explained by differences in evoked inhibition. Moreover, inhibitory participation was larger after cortical than after intrastriatal stimulation. A strong activation of interneurons was obtained from the cortex, demonstrating that polysynaptic activation includes the striatum. Prolonged kainate (KA) receptor responses were also elicited from the cortex. Responses of dSPNs and iSPNs did not depend on the cortical area stimulated. In contrast to AMPA-receptors, responses from NMDA- and KA-receptors do not exhibit early and late responses, but generate slow responses that contribute to plateau depolarizations.ConclusionsAs it has been established in previous physiological studies in vivo, synaptic invasion over different latencies, spanning hundreds of milliseconds after a single stimulus strongly indicates convergent polysynaptic activation. Interconnected cortical neurons converging on the same SPNs may explain prolonged corticostriatal responses. Glutamate receptors participation in these responses is described as well as differences and similarities between dSPNs and iSPNs.

GABAergic inputs from direct and indirect striatal projection neurons onto cholinergic interneurons in the primate putamen.

Striatal cholinergic interneurons (ChIs) are involved in reward-dependent learning and the regulation of attention. The activity of these neurons is modulated by intrinsic and extrinsic γ-aminobutyric acid (GABA)ergic and glutamatergic afferents, but the source and relative prevalence of these diverse regulatory inputs remain to be characterized. To address this issue, we performed a quantitative ultrastructural analysis of the GABAergic and glutamatergic innervation of ChIs in the postcommissural putamen of rhesus monkeys. Postembedding immunogold localization of GABA combined with peroxidase immunostaining for choline acetyltransferase showed that 60% of all synaptic inputs to ChIs originate from GABAergic terminals, whereas 21% are from putatively glutamatergic terminals that establish asymmetric synapses, and 19% from other (non-GABAergic) sources of symmetric synapses. Double pre-embedding immunoelectron microscopy using substance P and Met-/Leu-enkephalin antibodies to label GABAergic terminals from collaterals of "direct" and "indirect" striatal projection neurons, respectively, revealed that 47% of the indirect pathway terminals and 36% of the direct pathway terminals target ChIs. Together, substance P- and enkephalin-positive terminals represent 24% of all synapses onto ChIs in the monkey putamen. These findings show that ChIs receive prominent GABAergic inputs from multiple origins, including a significant contingent from axon collaterals of direct and indirect pathway projection neurons.

Dopamine Differentially Modulates the Excitability of Striatal Neurons of the Direct and Indirect Pathways in Lamprey

The functions of the basal ganglia are critically dependent on dopamine. In mammals, dopamine differentially modulates the excitability of the direct and indirect striatal projection neurons, and these populations selectively express dopamine D1 and D2 receptors, respectively. Although the detailed organization of the basal ganglia is conserved throughout the vertebrate phylum, it was unknown whether the differential dopamine modulation of the direct and indirect pathways is present in non-mammalian species. We aim here to determine whether the receptor expression and opposing dopaminergic modulation of the direct and indirect pathways is present in one of the phylogenetically oldest vertebrates, the river lamprey. Using in situ hybridization and patch-clamp recordings, we show that D1 receptors are almost exclusively expressed in the striatal neurons projecting directly to the homolog of the substantia nigra pars reticulata. In addition, the majority of striatal neurons projecting to the homolog of the globus pallidus interna/globus pallidus externa express D1 or D2 receptors. As in mammals, application of dopamine receptor agonists differentially modulates the excitability of these neurons, increasing the excitability of the D1-expressing neurons and decreasing the excitability of D2-expressing neurons. Our results suggest that the segregated expression of the D1 and D2 receptors in the direct and indirect striatal projection neurons has been conserved across the vertebrate phylum. Because dopamine receptor agonists differentially modulate these pathways, increasing the excitability of the direct pathway and decreasing the excitability of the indirect pathway, this organization may be conserved as a mechanism that biases the networks toward action selection.

The Balance of Striatal Feedback Transmission Is Disrupted in a Model of Parkinsonism

Inhibitory connections among striatal projection neurons (SPNs) called "feedback inhibition," have been proposed to endow the striatal microcircuit with computational capabilities, such as motor sequence selection, filtering, and the emergence of alternating network states. These properties are disrupted in models of Parkinsonism. However, the impact of feedback inhibition in the striatal network has remained under debate. Here, we test this inhibition at the microcircuit level. We used optical and electrophysiological recordings in mice and rats to demonstrate the action of striatal feedback transmission in normal and pathological conditions. Dynamic calcium imaging with single-cell resolution revealed the synchronous activation of a pool of identified SPNs by antidromic stimulation. Using bacterial artificial chromosome-transgenic mice, we demonstrate that the activated neuron pool equally possessed cells from the direct and indirect basal ganglia pathways. This pool inhibits itself because of its own GABA release when stimuli are frequent enough, demonstrating functional and significant inhibition. Blockade of GABAA receptors doubled the number of responsive neurons to the same stimulus, revealing a second postsynaptic neuron pool whose firing was being arrested by the first pool. Stronger connections arise from indirect SPNs. Dopamine deprivation impaired striatal feedback transmission disrupting the ability of a neuronal pool to arrest the firing of another neuronal pool. We demonstrate that feedback inhibition among SPNs is strong enough to control the firing of cell ensembles in the striatal microcircuit. However, to be effective, feedback inhibition should arise from synchronized pools of SPNs whose targets are other SPNs pools.

D1 Dopamine Receptor Supersensitivity in the Dopamine-Depleted Striatum Results from a Switch in the Regulation of ERK1/2/MAP Kinase

Dopamine effects in the striatum are mediated principally through the D1 and D2 dopamine receptor subtypes, which are segregated to the direct and indirect striatal projection neurons. After degeneration of the nigrostriatal dopamine system, direct pathway neurons display a supersensitive response to D1 dopamine receptor agonists, which is demonstrated by the induction of immediate early genes (IEGs), such as c-fos. Here we show, using analysis of receptor-mediated signal transduction, including protein phosphorylation and induction of IEGs, that D1 dopamine receptor supersensitivity is attributable to a switch to ERK1/2/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) in direct pathway neurons. Normally, in the dopamine-intact striatum, activation of ERK1/2/MAP kinase is shown to be restricted to indirect and not direct pathway neurons in response to stimulation of corticostriatal afferents. Moreover, in the dopamine-intact striatum, treatment with full D1 dopamine receptor agonists or stimulation of nigrostriatal dopaminergic afferents, both of which result in the induction of IEGs in direct striatal projection neurons, does not activate ERK1/2/MAP kinase. However, after degeneration of the nigrostriatal dopaminergic pathway, ERK1/2/MAP kinase is activated in direct pathway neurons in response to D1 dopamine receptor agonists either alone or when combined with stimulation of corticostriatal afferents. Inhibitors of MEK (MAP kinase kinase), which is responsible for phosphorylation of ERK1/2/MAP kinase, blocks D1 dopamine receptor agonist activation of ERK1/2/MAP kinase in the dopamine-depleted striatum, as well as the supersensitive induction of IEGs. These results demonstrate that dopamine input to the striatum maintains distinct forms of protein kinase-mediated gene regulation in the direct and indirect striatal projection neurons.

Biotinylated m4-toxin demonstrates more M4 muscarinic receptor protein on direct than indirect striatal projection neurons

The striatum has nearly equal numbers of striatonigral and striatopallidal projection neurons. All are GABAergic and inhibitory, but they lie in separate neuronal circuits (‘direct’ and ‘indirect’, respectively) that appear to exert opposite effects on movement. Methods are needed to evaluate the function of each circuit. A potential way to control striatonigral neurons selectively is via M4 muscarinic receptors. The striatum has many more M4 receptors than other tissues, they are located on approximately half of all projection neurons, and mRNA for M4 receptors is prevalent only in striatonigral neurons. In order to more rigorously compare the distribution of M4 receptors on rat neurons in these pathways a toxin that binds with very high specificity to M4 receptors (m4-toxin) was biotinylated for use as a selective probe for M4 receptor protein. Pooled biotin–toxin complexes were found to retain high M4-specificity and affinity. Neurons were first labeled by retrograde transport of fluorescent microbeads (FluoSpheres) injected into the substantia nigra and globus pallidus. Coincident labeling of only 4% of the cells confirmed the validity of the retrograde labeling technique. Labeled neurons were probed for M4 receptor protein using biotinylated m4-toxin and fluorescent avidin. M4 receptors were found on 14% of indirect and 86% of direct neurons. It may be concluded that there is a relative abundance of M4 receptors controlling the direct pathway. This work supports the hypothesis that M4-selective drugs will prove useful to control the function of striatonigral neurons in the direct projection pathway.

Localization of m4 muscarinic receptors on direct ⪢ indirect striatal projection neurons

Localization of m4 muscarinic receptors on direct ⪢ indirect striatal projection neurons