Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine co-varies with risk factors for Parkinson's.

Ca2+ entry into nigrostriatal dopamine (DA) neurons and axons via L-type voltage-gated Ca2+ channels (LTCCs) contributes, respectively, to pacemaker activity and DA release and has long been thought to contribute to vulnerability to degeneration in Parkinson's disease. LTCC function is greater in DA axons and neurons from substantia nigra pars compacta than from ventral tegmental area, but this is not explained by channel expression level. We tested the hypothesis that LTCC control of DA release is governed rather by local mechanisms, focussing on candidate biological factors known to operate differently between types of DA neurons and/or be associated with their differing vulnerability to parkinsonism, including biological sex, α-synuclein, DA transporters (DATs) and calbindin-D28k (Calb1). We detected evoked DA release ex vivo in mouse striatal slices using fast-scan cyclic voltammetry and assessed LTCC support of DA release by detecting the inhibition of DA release by the LTCC inhibitors isradipine or CP8. Using genetic knockouts or pharmacological manipulations, we identified that striatal LTCC support of DA release depended on multiple intersecting factors, in a regionally and sexually divergent manner. LTCC function was promoted by factors associated with Parkinsonian risk, including male sex, α-synuclein, DAT and a dorsolateral co-ordinate, but limited by factors associated with protection, that is, female sex, glucocerebrosidase activity, Calb1 and ventromedial co-ordinate. Together, these data show that LTCC function in DA axons and isradipine effect are locally governed and suggest they vary in a manner that in turn might impact on, or reflect, the cellular stress that leads to parkinsonian degeneration.

Axo-glial interactions between midbrain dopamine neurons and oligodendrocyte lineage cells in the anterior corpus callosum.

Oligodendrocyte progenitor cells (OPCs) receive synaptic innervation from glutamatergic and GABAergic axons and can be dynamically regulated by neural activity, resulting in activity-dependent changes in patterns of axon myelination. However, it remains unclear to what extent other types of neurons may innervate OPCs. Here, we provide evidence implicating midbrain dopamine neurons in the innervation of oligodendrocyte lineage cells in the anterior corpus callosum and nearby white matter tracts of male and female adult mice. Dopaminergic axon terminals were identified in the corpus callosum of DAT-Cre mice after injection of an eYFP reporter virus into the midbrain. Furthermore, fast-scan cyclic voltammetry revealed monoaminergic transients in the anterior corpus callosum, consistent with the anatomical findings. Using RNAscope, we further demonstrate that ~ 40% of Olig2 + /Pdfgra + cells and ~ 20% of Olig2 + /Pdgfra- cells in the anterior corpus callosum express Drd1 and Drd2 transcripts. These results suggest that oligodendrocyte lineage cells may respond to dopamine released from midbrain dopamine axons, which could affect myelination. Together, this work broadens our understanding of neuron-glia interactions with important implications for myelin plasticity by identifying midbrain dopamine axons as a potential regulator of corpus callosal oligodendrocyte lineage cells.

Striatal Dopamine Transporter Function Is Facilitated by Converging Biology of α-Synuclein and Cholesterol.

Striatal dopamine transporters (DAT) powerfully regulate dopamine signaling, and can contribute risk to degeneration in Parkinson's disease (PD). DATs can interact with the neuronal protein α-synuclein, which is associated with the etiology and molecular pathology of idiopathic and familial PD. Here, we tested whether DAT function in governing dopamine (DA) uptake and release is modified in a human-α-synuclein-overexpressing (SNCA-OVX) transgenic mouse model of early PD. Using fast-scan cyclic voltammetry (FCV) in ex vivo acute striatal slices to detect DA release, and biochemical assays, we show that several aspects of DAT function are promoted in SNCA-OVX mice. Compared to background control α-synuclein-null mice (Snca-null), the SNCA-OVX mice have elevated DA uptake rates, and more pronounced effects of DAT inhibitors on evoked extracellular DA concentrations ([DA]o) and on short-term plasticity (STP) in DA release, indicating DATs play a greater role in limiting DA release and in driving STP. We found that DAT membrane levels and radioligand binding sites correlated with α-synuclein level. Furthermore, DAT function in Snca-null and SNCA-OVX mice could also be promoted by applying cholesterol, and using Tof-SIMS we found genotype-differences in striatal lipids, with lower striatal cholesterol in SNCA-OVX mice. An inhibitor of cholesterol efflux transporter ABCA1 or a cholesterol chelator in SNCA-OVX mice reduced the effects of DAT-inhibitors on evoked [DA]o. Together these data indicate that human α-synuclein in a mouse model of PD promotes striatal DAT function, in a manner supported by extracellular cholesterol, suggesting converging biology of α-synuclein and cholesterol that regulates DAT function and could impact DA function and PD pathophysiology.

GABA uptake transporters support dopamine release in dorsal striatum with maladaptive downregulation in a parkinsonism model.

Striatal dopamine (DA) is critical for action and learning. Recent data show that DA release is under tonic inhibition by striatal GABA. Ambient striatal GABA tone on striatal projection neurons can be determined by plasma membrane GABA uptake transporters (GATs) located on astrocytes and neurons. However, whether striatal GATs and astrocytes determine DA output are unknown. We reveal that DA release in mouse dorsolateral striatum, but not nucleus accumbens core, is governed by GAT-1 and GAT-3. These GATs are partly localized to astrocytes, and are enriched in dorsolateral striatum compared to accumbens core. In a mouse model of early parkinsonism, GATs are downregulated, tonic GABAergic inhibition of DA release augmented, and nigrostriatal GABA co-release attenuated. These data define previously unappreciated and important roles for GATs and astrocytes in supporting DA release in striatum, and reveal a maladaptive plasticity in early parkinsonism that impairs DA output in vulnerable striatal regions.

Plasticity in striatal dopamine release is governed by release-independent depression and the dopamine transporter.

Mesostriatal dopaminergic neurons possess extensively branched axonal arbours. Whether action potentials are converted to dopamine output in the striatum will be influenced dynamically and critically by axonal properties and mechanisms that are poorly understood. Here, we address the roles for mechanisms governing release probability and axonal activity in determining short-term plasticity of dopamine release, using fast-scan cyclic voltammetry in the ex vivo mouse striatum. We show that brief short-term facilitation and longer short term depression are only weakly dependent on the level of initial release, i.e. are release insensitive. Rather, short-term plasticity is strongly determined by mechanisms which govern axonal activation, including K+-gated excitability and the dopamine transporter, particularly in the dorsal striatum. We identify the dopamine transporter as a master regulator of dopamine short-term plasticity, governing the balance between release-dependent and independent mechanisms that also show region-specific gating.

Calbindin-D28K Limits Dopamine Release in Ventral but Not Dorsal Striatum by Regulating Ca2+ Availability and Dopamine Transporter Function.

The calcium-binding protein calbindin-D28K, or calb1, is expressed at higher levels by dopamine (DA) neurons originating in the ventral tegmental area (VTA) than in the adjacent substantia nigra pars compacta (SNc). Calb1 has received attention for a potential role in neuroprotection in Parkinson's disease. The underlying physiological roles for calb1 are incompletely understood. We used cre-loxP technology to knock down calb1 in mouse DA neurons to test whether calb1 governs axonal release of DA in the striatum, detected using fast-scan cyclic voltammetry ex vivo. In the ventral but not dorsal striatum, calb1 knockdown elevated DA release and modified the spatiotemporal coupling of Ca2+ entry to DA release. Furthermore, calb1 knockdown enhanced DA uptake but attenuated the impact of DA transporter (DAT) inhibition by cocaine on underlying DA release. These data reveal that calb1 acts through a range of mechanisms underpinning both DA release and uptake to limit DA transmission in the ventral but not dorsal striatum.

Selective vulnerability in α-synucleinopathies.

Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy are neurodegenerative disorders resulting in progressive motor/cognitive deficits among other symptoms. They are characterised by stereotypical brain cell loss accompanied by the formation of proteinaceous aggregations of the protein α-synuclein (α-syn), being, therefore, termed α-synucleinopathies. Although the presence of α-syn inclusions is a common hallmark of these disorders, the exact nature of the deposited protein is specific to each disease. Different neuroanatomical regions and cellular populations manifest a differential vulnerability to the appearance of protein deposits, cell dysfunction, and cell death, leading to phenotypic diversity. The present review describes the multiple factors that contribute to the selective vulnerability in α-synucleinopathies. We explore the intrinsic cellular properties in the affected regions, including the physiological and pathophysiological roles of endogenous α-syn, the metabolic and genetic build-up of the cells and their connectivity. These factors converge with the variability of the α-syn conformational strains and their spreading capacity to dictate the phenotypic diversity and regional vulnerability of each disease. Finally, we describe the exogenous and environmental factors that potentially contribute by igniting and modulating the differential pathology in α-synucleinopathies. In conclusion, we think that it is the confluence of this disruption of the cellular metabolic state and α-syn structural equilibrium through the anatomical connectivity which appears to initiate cascades of pathological processes triggered by genetic, environmental, or stochastic events that result in the "death by a thousand cuts" profile of α-synucleinopathies.

Targeted Activation of Cholinergic Interneurons Accounts for the Modulation of Dopamine by Striatal Nicotinic Receptors.

Striatal dopamine (DA) is a major player in action selection and reinforcement. DA release is under strong local control by striatal ACh acting at axonal nicotinic ACh receptors (nAChRs) on DA axons. Striatal nAChRs have been shown to control how DA is released in response to ascending activity from DA neurons, and they also directly drive DA release following synchronized activity in a small local cholinergic network. The source of striatal ACh has been thought to arise solely from intrinsic cholinergic interneurons (ChIs), but recent findings have identified a source of cholinergic inputs to striatum from brainstem nuclei, the pedunculopontine nucleus (PPN) and laterodorsal tegmentum (LDT). Here, we used targeted optogenetic activation alongside DA detection with fast-scan cyclic voltammetry to test whether ChIs alone and/or brainstem afferents to the striatum can account for how ACh drives and modulates DA release in rat striatum. We demonstrate that targeted transient light activation of rat striatal ChIs drives striatal DA release, corroborating and extending previous observations in mouse to rat. However, the same light stimulation targeted to cholinergic brainstem afferents did not drive DA release, and nor did it modulate DA release activated subsequently by electrical stimulation, whereas targeted activation of ChIs did so. We were unable to obtain any evidence for DA modulation by PPN/LDT stimulation. By contrast, we could readily identify that striatal ChIs alone are sufficient to provide a source of ACh that powerfully regulates DA via nAChRs.

The Striosome and Matrix Compartments of the Striatum: A Path through the Labyrinth from Neurochemistry toward Function.

The striatum is a heterogeneous structure with a diverse range of neuron types and neuromodulators. Three decades of anatomical and biochemical studies have established that the neurochemical organization of striatum is not uniformly heterogeneous, but rather, can be differentiated into neurochemically discrete compartments known as striosomes (also known as patches) and matrix. These compartments are well understood to differ in their expression of neurochemical markers, with some differences in afferent and efferent connectivity and have also been suggested to have different involvement in a range of neurological diseases. However, the functional outcomes of striosome-matrix organization are poorly understood. Now, recent findings and new experimental tools are beginning to reveal that the distinctions between striosomes and matrix have distinct consequences for striatal synapse function. Here, we review recent findings that suggest there can be distinct regulation of neural function in striosome versus matrix compartments, particularly compartment-specific neurochemical interactions. We highlight that new transgenic and viral tools are becoming available that should now accelerate the pace of advances in understanding of these long-mysterious striatal compartments.

LRRK2 BAC transgenic rats develop progressive, L-DOPA-responsive motor impairment, and deficits in dopamine circuit function.

Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18-21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics.

Transcription factors FOXA1 and FOXA2 maintain dopaminergic neuronal properties and control feeding behavior in adult mice.

Midbrain dopaminergic (mDA) neurons are implicated in cognitive functions, neuropsychiatric disorders, and pathological conditions; hence understanding genes regulating their homeostasis has medical relevance. Transcription factors FOXA1 and FOXA2 (FOXA1/2) are key determinants of mDA neuronal identity during development, but their roles in adult mDA neurons are unknown. We used a conditional knockout strategy to specifically ablate FOXA1/2 in mDA neurons of adult mice. We show that deletion of Foxa1/2 results in down-regulation of tyrosine hydroxylase, the rate-limiting enzyme of dopamine (DA) biosynthesis, specifically in dopaminergic neurons of the substantia nigra pars compacta (SNc). In addition, DA synthesis and striatal DA transmission were reduced after Foxa1/2 deletion. Furthermore, the burst-firing activity characteristic of SNc mDA neurons was drastically reduced in the absence of FOXA1/2. These molecular and functional alterations lead to a severe feeding deficit in adult Foxa1/2 mutant mice, independently of motor control, which could be rescued by L-DOPA treatment. FOXA1/2 therefore control the maintenance of molecular and physiological properties of SNc mDA neurons and impact on feeding behavior in adult mice.

Substance P Weights Striatal Dopamine Transmission Differently within the Striosome-Matrix Axis.

The mammalian striatum has a topographical organization of input-output connectivity, but a complex internal, nonlaminar neuronal architecture comprising projection neurons of two types interspersed among multiple interneuron types and potential local neuromodulators. From this cellular melange arises a biochemical compartmentalization of areas termed striosomes and extrastriosomal matrix. The functions of these compartments are poorly understood but might confer distinct features to striatal signal processing and be discretely governed. Dopamine transmission occurs throughout striosomes and matrix, and is reported to be modulated by the striosomally enriched neuromodulator substance P. However, reported effects are conflicting, ranging from facilitation to inhibition. We addressed whether dopamine transmission is modulated differently in striosome-matrix compartments by substance P.We paired detection of evoked dopamine release at carbon-fiber microelectrodes in mouse striatal slices with subsequent identification of the location of recording sites with respect to µ-opioid receptor-rich striosomes. Substance P had bidirectional effects on dopamine release that varied between recording sites and were prevented by inhibition of neurokinin-1 receptors. The direction of modulation was determined by location within the striosomal-matrix axis: dopamine release was boosted in striosome centers, diminished in striosomal-matrix border regions, and unaffected in the matrix. In turn, this different weighting of dopamine transmission by substance P modified the apparent center-surround contrast of striosomal dopamine signals. These data reveal that dopamine transmission can be differentially modulated within the striosomal-matrix axis, and furthermore, indicate a functionally distinct zone at the striosome-matrix interface, which may have key impacts on striatal integration.

Gating of dopamine transmission by calcium and axonal N-, Q-, T- and L-type voltage-gated calcium channels differs between striatal domains.

KEY POINTS: The voltage-gated Ca(2+) channels (VGCCs) that catalyse striatal dopamine transmission are critical to dopamine function and might prime subpopulations of neurons for parkinsonian degeneration. However, the VGCCs that operate on mesostriatal axons are incompletely defined; previous studies encompassed channels on striatal cholinergic interneurons that strongly influence dopamine transmission. We define that multiple types of axonal VGCCs operate that extend beyond classic presynaptic N/P/Q channels to include T- and L-types. We reveal differences in VGCC function between mouse axon types that in humans are vulnerable versus resistant to Parkinson's disease. We show for the first time that this is underpinned by different sensitivity of dopamine transmission to extracellular Ca(2+) and by different spatiotemporal intracellular Ca(2+) microdomains. These data define key principles of how Ca(2+) and VGCCs govern dopamine transmission in the healthy brain and reveal differences between neuron types that might contribute to vulnerability in disease. ABSTRACT: The axonal voltage-gated Ca(2+) channels (VGCCs) that catalyse dopamine (DA) transmission are incompletely defined. Yet, they are critical to DA function and might prime subpopulations of DA neurons for parkinsonian degeneration. Previous studies of VGCCs will have encompassed those on striatal cholinergic interneurons, which strongly influence DA transmission. We identify which VGCCs on DA axons govern DA transmission, we determine their dynamic properties and reveal an underlying basis for differences between the caudate putamen (CPu) and nucleus accumbens (NAc). We detected DA release evoked electrically during nicotinic receptor blockade or optogenetically by light activation of channel rhodopsin-expressing DA axons in mouse striatal slices. Subtype-specific VGCC blockers indicated that N-, Q-, T- and L-VGCCs govern DA release in CPu, but in NAc, T and L-channels are relatively silent. The roles of the most dominant channels were inversely frequency-dependent, due to low-pass filtering of DA release by Ca(2+)-dependent relationships between initial release probability and short-term plasticity. Ca(2+) concentration-response curves revealed that differences between CPu and NAc were due to greater underlying Ca(2+) sensitivity of DA transmission from CPu axons. Functions for 'silent' L- and T-channels in NAc could be unmasked by elevating extracellular [Ca(2+)]. Furthermore, we identified a greater coupling between BAPTA-sensitive, fast Ca(2+) transients and DA transmission in CPu axons, and evidence for endogenous fast buffering of Ca(2+) in NAc. These data reveal that a range of VGCCs operate dynamically on DA axons, depending on local driving forces. Furthermore, they reveal dramatic differences in Ca(2+) handling between axonal subpopulations that show different vulnerability to parkinsonian degeneration.

Ni(2+) affects dopamine uptake which limits suitability as inhibitor of T-type voltage-gated Ca(2+) channels.

Neuronal T-type voltage-gated Ca(2+) channels are reported to have physiological roles that include regulation of burst firing, Ca(2+) oscillations, and neurotransmitter release. These roles are often exposed experimentally by blocking T-type channels with micromolar Ni(2+). We used Ni(2+) to explore the role of axonal T-type channels in dopamine (DA) release in mouse striatum, but identified significant off-target effects on DA uptake. Ni(2+) (100 µM) reversibly increased electrically evoked DA release and markedly extended its extracellular lifetime, detected using fast-scan cyclic voltammetry. Prior inhibition of the DA transporter (DAT) by cocaine (5 µM) occluded the facilitatory action of Ni(2+) on DA release and conversely, allowed Ni(2+) to inhibit release, presumably through T-channel inhibition. Ni(2+) further prolonged the timecourse of DA clearance suggesting further inhibition of DA uptake. In summary, Ni(2+) has major effects on DA transmission besides those due to T-channels that likely involve inhibition of the DAT.