Direct Pathway Striatal Projection Neurons Research Articles

Uncovering the ferroptosis related mechanism of laduviglusib in the cell-type-specific targets of the striatum in Huntington’s disease

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder featured by abnormal movements, arising from the extensive neuronal loss and glial dysfunction in the striatum. Although the causes and pathogenetic mechanisms of HD are well established, the development of disease-modifying pharmacological therapies for HD remains a formidable challenge. Laduviglusib has demonstrated neuroprotective effects through the enhancement of mitochondrial function in the striatum of HD animal models. Ferroptosis is a nonapoptotic form of cell death that occurs as a consequence of lethal iron-dependent lipid peroxidation and mitochondrial dysfunction. However, the ferroptosis-related mechanisms underlying the neuroprotective effects of laduviglusib in the striatum of HD patients remain largely uncharted. In this study, we leveraged single-nucleus RNA sequencing data obtained from the striatum of HD patients in stages 2–4 to identify differentially expressed genes within distinct cell-type. We subsequently integrated these differentially expressed genes of HD, laduviglusib target genes and ferroptosis-related genes to predict the ferroptosis-related mechanisms underpinning the neuroprotective effects of laduviglusib in HD patients. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses unveiled that the effects of laduviglusib on direct pathway striatal projection neurons (dSPNs) is mainly associated with Th17 cell differentiation pathways. Conversely, its impact on indirect pathway striatal projection neurons (iSPNs) extends to the Neurotrophin signaling pathway, FoxO signaling pathway, and reactive oxygen species pathway. In microglia, laduviglusib appears to contribute to HD pathology via mechanisms related to Th17 cell differentiation and the FoxO signaling pathway. Further, molecular docking results indicated favorable binding of laduviglusib with PARP1 (associated with dSPNs and iSPNs), SCD (associated with astrocytes), ALOX5 (associated with microglia), and HIF1A (associated with dSPNs, iSPNs, and microglia). In addition, the KEGG results suggest that laduviglusib may enhance mitochondrial function and protect against neuronal loss by targeting ferroptosis-related signaling pathways, particularly mediated by ALOX5 in microglia. These findings provide valuable insights into the potential mechanisms through which laduviglusib exerts its effects on distinct cell-types within the HD striatum.

Dopamine-mediated plasticity preserves excitatory connections to direct pathway striatal projection neurons and motor function in a mouse model of Parkinson's disease.

The cardinal symptoms of Parkinson's disease (PD) such as bradykinesia and akinesia are debilitating, and treatment options remain inadequate. The loss of nigrostriatal dopamine neurons in PD produces motor symptoms by shifting the balance of striatal output from the direct (go) to indirect (no-go) pathway in large part through changes in the excitatory connections and intrinsic excitabilities of the striatal projection neurons (SPNs). Here, we report using two different experimental models that a transient increase in striatal dopamine and enhanced D1 receptor activation, during 6-OHDA dopamine depletion, prevent the loss of mature spines and dendritic arbors on direct pathway projection neurons (dSPNs) and normal motor behavior for up to 5 months. The primary motor cortex and midline thalamic nuclei provide the major excitatory connections to SPNs. Using ChR2-assisted circuit mapping to measure inputs from motor cortex M1 to dorsolateral dSPNs, we observed a dramatic reduction in both experimental model mice and controls following dopamine depletion. Changes in the intrinsic excitabilities of SPNs were also similar to controls following dopamine depletion. Future work will examine thalamic connections to dSPNs. The findings reported here reveal previously unappreciated plasticity mechanisms within the basal ganglia that can be leveraged to treat the motor symptoms of PD.

Role of M4 -receptor cholinergic signaling in direct pathway striatal projection neurons during dopamine depletion.

Direct pathway striatal projection neurons (dSPNs) are characterized by the expression of dopamine (DA) class 1 receptors (D1 R), as well as cholinergic muscarinic M1 and M4 receptors (M1 R, M4 R). D1 R enhances neuronal firing through phosphorylation of voltage-gate calcium channels (CaV 1 Ca2+ channels) activating Gs proteins and protein kinase A (PKA). Concurrently, PKA suppresses phosphatase PP-1 through DARPP-32, thus extending this facilitatory modulation. M1 R also influences Ca2+ channels in SPNs through Gq proteins and protein kinase C. However, the signaling mechanisms of M4 R in dSPNs are less understood. Two pathways are attributed to M4 R: an inhibitory one through Gi/o proteins, and a facilitatory one via the cyclin Cdk5. Our study reveals that a previously observed facilitatory modulation via CaV 1 Ca2+ channels is linked to the Cdk5 pathway in dSPNs. This result could be significant in treating parkinsonism. Therefore, we questioned whether this effect persists post DA-depletion in experimental parkinsonism. Our findings indicate that in such conditions, M4 R activation leads to a decrease in Ca2+ current and an increased M4 R protein level, contrasting with the control response. Nevertheless, parkinsonian and control actions are inhibited by the Cdk5 inhibitor roscovitine, suggesting Cdk5's role in both conditions. Cdk5 may activate PP-1 via PKA inhibition in DA depletion. Indeed, we found that inhibiting PP-1 restores control M4 R actions, implying that PP-1 is overly active via M4 Rs in DA-depleted condition. These insights contribute to understanding how DA-depletion alters modulatory signaling in striatal neurons. Additional working hypotheses are discussed.

Pathway-specific alterations in striatal excitability and cholinergic modulation in a SAPAP3 mouse model of compulsive motor behavior

Deletion of the obsessive-compulsive disorder (OCD)-associated gene SAP90/PSD-95-associated protein 3 (Sapap3), which encodes a postsynaptic anchoring protein at corticostriatal synapses, causes OCD-like motor behaviors in mice. While corticostriatal synaptic dysfunction is central to this phenotype, the striatum efficiently adapts to pathological changes, often in ways that expand upon the original circuit impairment. Here, we show that SAPAP3 deletion causes non-synaptic and pathway-specific alterations in dorsolateral striatum circuit function. While somatic excitability was elevated in striatal projection neurons (SPNs), dendritic excitability was exclusively enhanced in direct pathway SPNs. Layered on top of this, cholinergic modulation was altered in opposing ways: striatal cholinergic interneuron density and evoked acetylcholine release were elevated, while basal muscarinic modulation of SPNs was reduced. These data describe how SAPAP3 deletion alters the striatal landscape upon which impaired corticostriatal inputs will act, offering a basis for how pathological synaptic integration and unbalanced striatal output underlying OCD-like behaviors may be shaped.

A non-canonical striatopallidal Go pathway that supports motor control

In the classical model of the basal ganglia, direct pathway striatal projection neurons (dSPNs) send projections to the substantia nigra (SNr) and entopeduncular nucleus to regulate motor function. Recent studies have re-established that dSPNs also possess axon collaterals within the globus pallidus (GPe) (bridging collaterals), yet the significance of these collaterals for behavior is unknown. Here we use in vivo optical and chemogenetic tools combined with deep learning approaches in mice to dissect the roles of dSPN GPe collaterals in motor function. We find that dSPNs projecting to the SNr send synchronous motor-related information to the GPe via axon collaterals. Inhibition of native activity in dSPN GPe terminals impairs motor activity and function via regulation of Npas1 neurons. We propose a model by which dSPN GPe axon collaterals (striatopallidal Go pathway) act in concert with the canonical terminals in the SNr to support motor control by inhibiting Npas1 neurons.

Striatal Direct Pathway Targets Npas1+ Pallidal Neurons.

The classic basal ganglia circuit model asserts a complete segregation of the two striatal output pathways. Empirical data argue that, in addition to indirect-pathway striatal projection neurons (iSPNs), direct-pathway striatal projection neurons (dSPNs) innervate the external globus pallidus (GPe). However, the functions of the latter were not known. In this study, we interrogated the organization principles of striatopallidal projections and their roles in full-body movement in mice (both males and females). In contrast to the canonical motor-promoting response of dSPNs in the dorsomedial striatum (DMSdSPNs), optogenetic stimulation of dSPNs in the dorsolateral striatum (DLSdSPNs) suppressed locomotion. Circuit analyses revealed that dSPNs selectively target Npas1+ neurons in the GPe. In a chronic 6-hydroxydopamine lesion model of Parkinson's disease, the dSPN-Npas1+ projection was dramatically strengthened. As DLSdSPN-Npas1+ projection suppresses movement, the enhancement of this projection represents a circuit mechanism for the hypokinetic symptoms of Parkinson's disease that has not been previously considered. In sum, our results suggest that dSPN input to the GPe is a critical circuit component that is involved in the regulation of movement in both healthy and parkinsonian states.SIGNIFICANCE STATEMENT In the classic basal ganglia model, the striatum is described as a divergent structure: it controls motor and adaptive functions through two segregated, opposing output streams. However, the experimental results that show the projection from direct-pathway neurons to the external pallidum have been largely ignored. Here, we showed that this striatopallidal subpathway targets a select subset of neurons in the external pallidum and is motor-suppressing. We found that this subpathway undergoes changes in a Parkinson's disease model. In particular, our results suggest that the increase in strength of this subpathway contributes to the slowness or reduced movements observed in Parkinson's disease.

Predicting complex spikes in striatal projection neurons of the direct pathway following neuromodulation by acetylcholine and dopamine.

The input structure of the basal ganglia, striatum, receives dense neuromodulatory input in the form of dopamine and acetylcholine. The two systems are tightly connected, for example, synchronized activity of cholinergic interneurons, leading to increased acetylcholine release, has been shown to directly trigger dopamine release from dopaminergic terminals in striatum. Both signals are further needed for induction of locomotion. High dopamine concentration leads to increased excitability of the direct pathway striatal projection neurons. High cholinergic tone inhibits various potassium channels further increasing the excitability of striatal projection neurons. Here, we investigate the combined effect of concurrent high acetylcholine and dopamine using biophysically detailed models based on rodent data. The aim of the study is to investigate how neuromodulation affects dendritic integration. The result shows that neuromodulation paired with synaptic activation of dendrites can give rise to complex spiking patterns, resembling spike shapes seen in the hippocampus. In the hippocampus, these complex spikes are associated with behavioral time scale plasticity and place cell tuning. We further investigate the mechanisms behind the complex spikes and find that there are two components, one axo-somatic and one dendritic in origin.

Mammalian Target of Rapamycin-RhoA Signaling Impairments in Direct Striatal Projection Neurons Induce Altered Behaviors and Striatal Physiology in Mice

BackgroundAs an integrator of molecular pathways, mTOR (mammalian target of rapamycin) has been associated with diseases including neurodevelopmental, psychiatric, and neurodegenerative disorders such as autism spectrum disorder, schizophrenia, and Huntington’s disease. An important brain area involved in all these diseases is the striatum. However, the mechanisms behind how mTOR is involved in striatal physiology and its relative role in distinct neuronal populations in these striatal-related diseases still remain to be clarified. MethodsUsing Drd1-Cre mTOR-conditional knockout male mice, we combined behavioral, biochemical, electrophysiological, and morphological analysis aiming to untangle the role of mTOR in direct pathway striatal projection neurons and how this would impact on striatal physiology. ResultsOur results indicate deep behavioral changes in absence of mTOR in Drd1-expressing neurons such as decreased spontaneous locomotion, impaired social interaction, and decreased marble-burying behavior. These alterations were accompanied by a Kv1.1-induced increase in the fast phase of afterhyperpolarization and coincident decreased distal spine density in striatal direct pathway striatal projection neurons. The physiological changes were mechanistically independent of protein synthesis but sensitive to pharmacological blockade of transforming protein RhoA activity. ConclusionsThese results identify mTOR signaling as an important regulator of striatal functions through an intricate mechanism involving RhoA and culminating in Kv1.1 overfunction, which could be targeted to treat striatal-related monogenic disorders associated with the mTOR signaling pathway.

Chemogenetic Targeting of Dorsomedial Direct-pathway Striatal Projection Neurons Selectively Elicits Rotational Behavior in Mice

The striatum of the basal ganglia is pivotal for voluntary movements and is implicated in debilitating movement disorders such as Parkinsonism and dystonia. Striatum projects to downstream nuclei through direct (dSPN) and indirect (iSPN) pathway projection neurons thought to exert opposite effects on movement. In rodent models of striatal function, unilateral dopamine deprivation induces ipsiversive rotational behavior. The dSPNs of the dorsal striatum are believed to engage distinct motor programs but underlying mechanisms remain unclear. Here, we show by employing chemogenetics [Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)] that unilateral inhibition of dorsomedial dSPNs is sufficient to selectively impair contraversive movement and elicit ipsiversive rotational behavior in mice. Adeno-associated virus (AAV) encoding Cre-dependent Gi-coupled DREADD was injected unilaterally into the dorsomedial striatum of Drd1-Cre mice, resulting in expression of the modified human M4 muscarinic receptor (hM4Di) in ∼20% of dorsostriatal dSPNs. Upon hM4Di activation, a striking positive linear correlation was found between turn ratio and viral expression, which corroborates a relationship between unilateral inhibition of dorsomedial dSPNs and rotational behavior. Bursts of ipsiversive rotations were interspersed with normal ambulation. However, partial unilateral inhibition of ∼20% of dorsostriatal dSPNs did not affect horizontal and vertical locomotion or forelimb use preference. Overall, our results substantiate a unique role of dSPNs in promoting response bias in rotational behavior and show this to be a highly sensitive measure of dSPN performance.

Hyperactive Response of Direct Pathway Striatal Projection Neurons to L-dopa and D1 Agonism in Freely Moving Parkinsonian Mice.

Dopamine (DA) profoundly stimulates motor function as demonstrated by the hypokinetic motor symptoms in Parkinson's disease (PD) and by the hyperkinetic motor side effects during dopaminergic treatment of PD. Dopamine (DA) receptor-bypassing, optogenetics- and chemogenetics-induced spike firing of striatal DA D1 receptor (D1R)-expressing, direct pathway medium spiny neurons (dSPNs or dMSNs) promotes movements. However, the endogenous D1R-mediated effects, let alone those of DA replacement, on dSPN spike activity in freely-moving animals is not established. Here we show that using transcription factor Pitx3 null mutant (Pitx3Null) mice as a model for severe and consistent DA denervation in the dorsal striatum in Parkinson's disease, antidromically identified striatonigral neurons (D1R-expressing dSPNs) had a lower baseline spike firing rate than that in DA-intact normal mice, and these neurons increased their spike firing more strongly in Pitx3Null mice than in WT mice in response to injection of L-dopa or the D1R agonist, SKF81297; the increase in spike firing temporally coincided with the motor-stimulating effects of L-dopa and SKF81297. Taken together, these results provide the first evidence from freely moving animals that in parkinsonian striatum, identified behavior-promoting dSPNs become hyperactive upon the administration of L-dopa or a D1 agonist, likely contributing to the profound dopaminergic motor stimulation in parkinsonian animals and PD patients.

Disrupted striatal neuron inputs and outputs in Huntington's disease.

Huntington's disease (HD) is a hereditary progressive neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for the protein huntingtin, resulting in a pathogenic expansion of the polyglutamine tract in the N-terminus of this protein. The HD pathology resulting from the mutation is most prominent in the striatal part of the basal ganglia, and progressive differential dysfunction and loss of striatal projection neurons and interneurons account for the progression of motor deficits seen in this disease. The present review summarizes current understanding regarding the progression in striatal neuron dysfunction and loss, based on studies both in human HD victims and in genetic mouse models of HD. We review evidence on early loss of inputs to striatum from cortex and thalamus, which may be the basis of the mild premanifest bradykinesia in HD, as well as on the subsequent loss of indirect pathway striatal projection neurons and their outputs to the external pallidal segment, which appears to be the basis of the chorea seen in early symptomatic HD. Later loss of direct pathway striatal projection neurons and their output to the internal pallidal segment account for the severe akinesia seen late in HD. Loss of parvalbuminergic striatal interneurons may contribute to the late dystonia and rigidity.

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.

'Synaptic autocontrol'.

Dale's principle, as stated by the English neuroscientist Henry Dale, posited that a presynaptic neuron will have the same chemical action on all its connections regardless of the class of target cell. Dale's statement was often interpreted as a single synapse releasing a single class of chemical messenger. There is now a vast body of experimental evidence that challenges Dale's principle. New observations reveal that we do not completely understand the complexity and extent of the mechanisms in place to control the activity of postsynaptic and presynaptic elements. Existing modelling strategies still do not take into account several of these new variables. To begin with, there are many synapses that cannot be reduced to either simple excitatory (glutamate) or inhibitory (GABA) inputs. For instance, it is now clear that, under some conditions, these two transmitters may be co-released by the same synapse (Gutiérrez, 2005). In addition, synapses in many brain regions co-release peptides; new work in this issue of The Journal of Physiology by Hawes et al. (2017) provides a unique perspective on peptide actions in the striatum. Most investigators implicitly assume that synaptic plasticity encoded as long-term potentiation (LTP) or long-term depression (LTD) is an ongoing feature in neuronal circuits and is a first step in the formation of functionally connected neuronal ensembles. By either reinforcing or weakening synapses, synaptic connections can be preferentially tuned to the flow of activity within these ensembles to produce specific activity patterns and reverberations. These circuits are shaped by synaptic changes that occur during learning and conform to procedural and declarative memories underlying goal-directed actions, routines and habits. Addiction, in some ways, falls into this category. Yet, we know that neuropeptides play key roles in many of the circuits implicated in addiction. What are the specific contributions of these peptides? Do they interact with activity-dependent plasticity mechanisms such as LTP/LTD? Hawes et al. (2017) provide a novel answer to this long-standing question. They show that corticostriatal LTP is modulated by dynorphin. Dynorphin is one of the peptides co-released by direct pathway striatal projection neurons (dSPNs), which synthesize and release the fast channel-gating transmitter GABA. The other co-released peptide by dSPNs is substance P (SP). In contrast, indirect pathway neurons (iSPNs) release enkephalins. Using voltammetry Hawes et al. show that dynorphin decreases dopamine release via κ-opioid receptors (KORs), and using optogenetics they also show that a small fraction of channel rhodopsin transfected D1-Cre dSPNs are capable of releasing enough endogenous dynorphin to decrease dopamine release. This reduction in dopamine release results in a decrease in corticostriatal LTP in neighbouring SPNs, suggesting that opioids modulate learning by controlling the number of synapses that develop LTP. This means that local release of peptides may tune circuits by impacting ongoing plasticity between specific synapses. One wonders if similar mechanisms can be found for LTD. Further in their Discussion, Hawes et al. reveal a potentially more complex scenario, that dSPNs also release SP which has opposite actions to dynorphin: increase in glutamate, acetylcholine and dopamine release. What is the possible outcome of releasing peptides with opposed actions? The authors’ speculation is interesting and experimentally testable: their spatio-temporal profiles may be different, SP acts briefly and before dynorphin, implying that LTP or LTD may be facilitated during specific temporal windows due to peptides released, perhaps interfering with spike-timing-dependent plasticity. This would determine the size of the neuronal ensembles that are formed, thus helping the subject to make hierarchical learning: augmenting or filtering different inputs at a given moment depending on context. In the case of dynorphin, this property may serve to control ensemble activation during addictive behaviour, limiting a runaway enhancement of striatal activity in response to drugs of abuse. Clearly, this mechanism can be explored as a therapeutic target for treating addiction. More generally, the field should not only investigate whether peptides released by synapses facilitate or restrain synaptic transmission, but also direct efforts towards whether additional processes such as synaptic plasticity are being modified. None declared. CONACyT Frontera-57, DGAPA-UNAM IN205610.

Selective neuronal expression of the SoxE factor, Sox8, in direct pathway striatal projection neurons of the developing mouse brain

AbstractThe striatum is the major component of the basal ganglia and is well known to play a key role in the control of motor function via balanced output from the indirect (iSPNs) and direct pathway striatal projection neurons (dSPNs). Little is known, however, about the molecular genetic mechanisms that control the formation of the iSPNs versus dSPNs. We show here that the SoxE family member, Sox8, is co‐expressed with the dSPN markers, Isl1 and Ebf1, in the developing striatum. Moreover, dSPNs, as marked by Isl1‐cre fate map, express Sox8 in the embryonic striatum and Sox8‐EGFP BAC transgenic mice specifically reveal the direct pathway axons during development. These EGFP+ axons are first observed to reach their midbrain target, the substantia nigra pars reticulata (SNr), at E14 in the mouse with a robust connection observed already at birth. The selective expression of EGFP in dSPNs of Sox8‐EGFP BAC mice is maintained at postnatal timepoints. Sox8 is known to be expressed in oligodendrocyte precursor cells (OPCs) together with other SoxE factors and we show here that the EGFP signal co‐localizes with the OPC markers throughout the brain. Finally, we show that Sox8‐EGFP BAC mice can be used to interrogate the altered dSPN development in Isl1 conditional mutants including aberrant axonal projections detected already at embryonic timepoints. Thus, Sox8 represents an early and specific marker of embryonic dSPNs and the Sox8‐EGFP BAC transgenic mice are an excellent tool to study the development of basal ganglia circuitry.

Long‐term plasticity of corticostriatal synapses is modulated by pathway‐specific co‐release of opioids through κ‐opioid receptors

Both endogenous opioids and opiate drugs of abuse modulate learning of habitual and goal-directed actions, and can also modify long-term plasticity of corticostriatal synapses. Striatal projection neurons of the direct pathway co-release the opioid neuropeptide dynorphin which can inhibit dopamine release via κ-opioid receptors. Theta-burst stimulation of corticostriatal fibres produces long-term potentiation (LTP) in striatal projection neurons when measured using whole-cell patch recording. Optogenetic activation of direct pathway striatal projection neurons inhibits LTP while reducing dopamine release. Because the endogenous release of opioids is activity dependent, this modulation of synaptic plasticity represents a negative feedback mechanism that may limit runaway enhancement of striatal neuron activity in response to drugs of abuse. Synaptic plasticity in the striatum adjusts behaviour adaptively during skill learning, or maladaptively in the case of addiction. Just as dopamine plays a critical role in synaptic plasticity underlying normal skill learning and addiction, endogenous and exogenous opiates also modulate learning and addiction-related striatal plasticity. Though the role of opioid receptors in long-term depression in striatum has been characterized, their effect on long-term potentiation (LTP) remains unknown. In particular, direct pathway (dopamine D1 receptor-containing; D1R-) spiny projection neurons (SPNs) co-release the opioid neuropeptide dynorphin, which acts at presynaptic κ-opioid receptors (KORs) on dopaminergic afferents and can negatively regulate dopamine release. Therefore, we evaluated the interaction of co-released dynorphin and KOR on striatal LTP. We optogenetically facilitate the release of endogenous dynorphin from D1R-SPNs in brain slice while using whole-cell patch recording to measure changes in the synaptic response of SPNs following theta-burst stimulation (TBS) of cortical afferents. Our results demonstrate that TBS evokes corticostriatal LTP, and that optogenetic activation of D1R-SPNs during induction impairs LTP. Additional experiments demonstrate that optogenetic activation of D1R-SPNs reduces stimulation-evoked dopamine release and that bath application of a KOR antagonist provides full rescue of both LTP induction and dopamine release during optogenetic activation of D1R-SPNs. These results suggest that an increase in the opioid neuropeptide dynorphin is responsible for reduced TBS LTP and illustrate a physiological phenomenon whereby heightened D1R-SPN activity can regulate corticostriatal plasticity. Our findings have important implications for learning in addictive states marked by elevated direct pathway activation.

Selective neuronal expression of the SoxE factor, Sox8, in direct pathway striatal projection neurons of the developing mouse brain.

The striatum is the major component of the basal ganglia and is well known to play a key role in the control of motor function via balanced output from the indirect (iSPNs) and direct pathway striatal projection neurons (dSPNs). Little is known, however, about the molecular genetic mechanisms that control the formation of the iSPNs versus dSPNs. We show here that the SoxE family member, Sox8, is co-expressed with the dSPN markers, Isl1 and Ebf1, in the developing striatum. Moreover, dSPNs, as marked by Isl1-cre fate map, express Sox8 in the embryonic striatum and Sox8-EGFP BAC transgenic mice specifically reveal the direct pathway axons during development. These EGFP+ axons are first observed to reach their midbrain target, the substantia nigra pars reticulata (SNr), at E14 in the mouse with a robust connection observed already at birth. The selective expression of EGFP in dSPNs of Sox8-EGFP BAC mice is maintained at postnatal timepoints. Sox8 is known to be expressed in oligodendrocyte precursor cells (OPCs) together with other SoxE factors and we show here that the EGFP signal co-localizes with the OPC markers throughout the brain. Finally, we show that Sox8-EGFP BAC mice can be used to interrogate the altered dSPN development in Isl1 conditional mutants including aberrant axonal projections detected already at embryonic timepoints. Thus, Sox8 represents an early and specific marker of embryonic dSPNs and the Sox8-EGFP BAC transgenic mice are an excellent tool to study the development of basal ganglia circuitry.

Foxo1 is a downstream effector of Isl1 in direct pathway striatal projection neuron development within the embryonic mouse telencephalon

Recent studies have shown that the LIM-homeodomain transcription factor Isl1 is required for the survival and differentiation of direct pathway striatonigral neurons during embryonic development. The downstream effectors of Isl1 in these processes are presently unknown. We show here that Foxo1, a transcription factor that has been implicated in cell survival, is expressed in striatal projection neurons (SPNs) that derive from the Isl1 lineage (i.e. direct pathway SPNs). Moreover, Isl1 conditional knockouts (cKOs) show a severe loss of Foxo1 expression at E15.5 with a modest recovery by E18.5. Although Foxo1 is enriched in the direct pathway SPNs at embryonic stages, it is expressed in both direct and indirect pathway SPNs at postnatal time points as evidenced by co-localization with EGFP in both Drd1-EGFP and Drd2-EGFP BAC transgenic mice. Foxo1 was not detected in striatal interneurons as marked by the transcription factor Nkx2.1. Conditional knockout of Foxo1 using Dlx5/6-CIE mice results in reduced expression of the SPN marker Darpp-32, as well as in the direct pathway SPN markers Ebf1 and Zfp521 within the embryonic striatum at E15.5. However, this phenotype improves in the conditional mutants by E18.5. Interestingly, the Foxo family members, Foxo3 and Foxo6, remain expressed at late embryonic stages in the Foxo1 cKOs unlike the Isl1 cKOs where Foxo1/3/6 as well as the Foxo1/3 target Bach2 are all reduced. Taken together, these findings suggest that Foxo-regulated pathways are downstream of Isl1 in the survival and/or differentiation of direct pathway SPNs.

Modulation of direct pathway striatal projection neurons by muscarinic M4-type receptors

Models of basal ganglia (BG) function posit a dynamic balance between two classes of striatal projection neurons (SPNs): direct pathway neurons (dSPNs) that facilitate movements, and indirect pathway neurons (iSPNs) that repress movement execution. Two main modulatory transmitters regulate the output of these neurons: dopamine (DA) and acetylcholine (ACh). dSPNs express D1-type DA, M1-and M4-type ACh receptors, while iSPNs express D2-type DA and M1-type ACh receptors. Actions of M1-, D1-, and D2-receptors have been extensively reported, but we still ignore most actions of muscarinic M4-type receptors. Here, we used whole-cell recordings in acutely dissociated neurons, pharmacological tools such as mamba-toxins, and BAC D1or2-eGFP transgenic mice to show that activation of M4-type receptors with bath applied muscarine enhances Ca2+-currents through CaV1-channels in dSPNs and not in iSPNs. This action increases excitability of dSPNs after both direct current injection and synaptically driven stimulation. The increases in Ca2+-current and excitability were blocked specifically by mamba toxin-3, suggesting mediation via M4-type receptors. M4-receptor activation also increased network activity of dSPNs but not of iSPNs as seen with calcium-imaging techniques. Moreover, actions of D1-type and M4-type receptors may add to produce a larger enhancement of excitability of dSPNs or, paradoxically, oppose each other depending on the order of their activation. Possible implications of these findings are discussed.

Duration differences of corticostriatal responses in striatal projection neurons depend on calcium activated potassium currents

The firing of striatal projection neurons (SPNs) exhibits afterhyperpolarizing potentials (AHPs) that determine discharge frequency. They are in part generated by Ca2+-activated K+-currents involving BK and SK components. It has previously been shown that suprathreshold corticostriatal responses are more prolonged and evoke more action potentials in direct pathway SPNs (dSPNs) than in indirect pathway SPNs (iSPNs). In contrast, iSPNs generate dendritic autoregenerative responses. Using whole cell recordings in brain slices, we asked whether the participation of Ca2+-activated K+-currents plays a role in these responses. Secondly, we asked if these currents may explain some differences in synaptic integration between dSPNs and iSPNs. Neurons obtained from BAC D1 and D2 GFP mice were recorded. We used charybdotoxin and apamin to block BK and SK channels, respectively. Both antagonists increased the depolarization and delayed the repolarization of suprathreshold corticostriatal responses in both neuron classes. We also used NS 1619 and NS 309 (CyPPA), to enhance BK and SK channels, respectively. Current enhancers hyperpolarized and accelerated the repolarization of corticostriatal responses in both neuron classes. Nevertheless, these drugs made evident that the contribution of Ca2+-activated K+-currents was different in dSPNs as compared to iSPNs: in dSPNs their activation was slower as though calcium took a diffusion delay to activate them. In contrast, their activation was fast and then sustained in iSPNs as though calcium flux activates them at the moment of entry. The blockade of Ca2+-activated K+-currents made iSPNs to look as dSPNs. Conversely, their enhancement made dSPNs to look as iSPNs. It is concluded that Ca2+-activated K+-currents are a main intrinsic determinant causing the differences in synaptic integration between corticostriatal polysynaptic responses between dSPNs and iSPNs.