BackgroundStriatal fast-spiking interneurons (FSI) are a subset of GABAergic cells that express calcium-binding protein parvalbumin (PV). They provide feed-forward inhibition to striatal projection neurons (SPNs), receive cortical, thalamic and dopaminergic inputs and are coupled together by electrical and chemical synapses, being important components of the striatal circuitry. It is known that dopamine (DA) depolarizes FSI via D1-class DA receptors, but no studies about the ionic mechanism of this action have been reported. Here we ask about the ion channels that are the effectors of DA actions. This work studies their Ca2+ currents.ResultsWhole-cell recordings in acutely dissociated and identified FSI from PV-Cre transgenic mice were used to show that FSI express an array of voltage gated Ca2+ channel classes: CaV1, CaV2.1, CaV2.2, CaV2.3 and CaV3. However, CaV1 Ca2+ channel carries most of the whole-cell Ca2+ current in FSI. Activation of D1-like class of DA receptors by the D1-receptor selective agonist SKF-81297 (SKF) enhances whole-cell Ca2+ currents through CaV1 channels modulation. A previous block of CaV1 channels with nicardipine occludes the action of the DA-agonist, suggesting that no other Ca2+ channel is modulated by D1-receptor activation. Bath application of SKF in brain slices increases the firing rate and activity of FSI as measured with both whole-cell and Ca2+ imaging recordings. These actions are reduced by nicardipine.ConclusionsThe present work discloses one final effector of DA modulation in FSI. We conclude that the facilitatory action of DA in FSI is in part due to CaV1 Ca2+ channels positive modulation.

Acute dopamine receptor blockade in substantia nigra pars reticulata: a possible model for drug-induced Parkinsonism. Dopamine (DA) depletion modifies the firing pattern of neurons in the substantia nigra pars reticulata (SNr), shifting their mostly tonic firing toward irregularity and bursting, traits of pathological firing underlying rigidity and postural instability in Parkinson's disease (PD) patients and animal models of Parkinsonism (PS). Drug-induced Parkinsonism (DIP) represents 20 -40% of clinical cases of PS, becoming a problem for differential diagnosis, and is still not well studied with physiological tools. It may co-occur with tardive dyskinesia. Here we use in vitro slice preparations including the SNr to observe drug-induced pathological firing by using drugs that most likely produce it, DA-receptor antagonists (SCH23390 plus sulpiride), to compare with firing patterns found in DA-depleted tissue. The hypothesis is that SNr firing would be similar under both conditions, a prerequisite to the proposal of a similar preparation to test other DIP-producing drugs. Firing was analyzed with three complementary metrics, showing similarities between DA depletion and acute DAreceptor blockade. Moreover, blockade of either nonselective cationic channels or Ca v 3 T-type calcium channels hyperpolarized the membrane and abolished bursting and irregular firing, silencing SNr neurons in both conditions. Therefore, currents generating firing in control conditions are in part responsible for pathological firing. Haloperidol, a DIP-producing drug, reproduced DA-receptor antagonist firing modifications. Since acute DA-receptor blockade induces SNr neuron firing similar to that found in the 6-hydroxydopamine model of PS, output basal ganglia neurons may play a role in generating DIP. Therefore, this study opens the way to test other DIP-producing drugs.NEW & NOTEWORTHY Dopamine (DA) depletion enhances substantia nigra pars reticulata (SNr) neuron bursting and irregular firing, hallmarks of Parkinsonism. Several drugs, including antipsychotics, antidepressants, and calcium channel antagonists, among others, produce drug-induced Parkinsonism. Here we show the first comparison between SNr neuron firing after DA depletion vs. firing found after acute blockade of DA receptors. It was found that firing in both conditions is similar, implying that pathological SNr neuron firing is also a physiological correlate of drug-induced Parkinsonism.

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AbstractHuman embryonic stem cells (hESCs) differentiate into specialized cells, including midbrain dopaminergic neurons (DAN). Non-human primates (NHPs) injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine develop some alterations observed in Parkinson’s disease (PD) patients. We obtained DAN from hESCs and confirmed that they express dopaminergic markers, generate action potentials, and release dopamine (DA) in vitro. DAN were transplanted bilaterally in the putamen of parkinsonian NHPs. After grafting, animals improved motor behavior, evaluated by the HALLWAY task, and in agreement with this recovery, DA release was detected by microdialysis. Imaging techniques revealed changes in fractional anisotropy and mean diffusivity in magnetic resonance imaging and higher 11C-DTBZ binding in positron-emission tomography scans, associated with grafts. Postmortem analysis showed that transplanted DAN survived over ten months in the putamen, without developing tumors, in the immunosuppressed NHPs. These results indicate that cell replacement therapy with hESCs-derived DAN causes long-term biochemical, anatomical, and physiological improvements in this model of PD.

Patch clamp recording performed in vitro using brain slice preparations is a standard technique used in cellular biophysics and neurophysiology to study the electrical activity of neurons. In particular, our research group is interested in obtaining patch clamp recordings from neurons in the CA1, CA3, and dentate gyrus regions of the hippocampal formation to investigate how the excitability of neurons change during development and aging. To carry out these experiments, we must first dissect out the brain and obtain slices, all while keeping the brain healthy so that the neurons survive and can later be recorded. Here we outline our procedures for anesthetizing, perfusing, dissecting out the brain, and finally obtaining slices. This protocol can also be used as a teaching tool to train students in the handling and dissection of rodents, and the preparation of brain tissue. The slices obtained can also be used for neuroanatomical studies or in training students to identify different brain structures. Our goals in sharing this protocol are to be transparent about our scientific methodology and to help other researchers performing similar experiments.