Subthalamic nucleus exclusively evokes dopamine release in the tail of the striatum.

A distinct population of dopamine neurons in the substantia nigra pars lateralis (SNL) has a unique projection to the most caudolateral (tail) region of the striatum. Here, using two electrochemical techniques to measure basal dopamine and electrically evoked dopamine release in anesthetized rats, we characterized this pathway, and compared it with the 'classic' nigrostriatal pathway from neighboring substantia nigra pars compacta (SNc) dopamine neurons to the dorsolateral striatum. We found that the tail striatum constitutes a distinct dopamine domain compared with the dorsolateral striatum, with consistently lower basal and evoked dopamine, and diverse dopamine release kinetics. Importantly, electrical stimulation of the SNL and SNc evoked dopamine release in entirely separate striatal regions; the tail and dorsolateral striatum, respectively. Furthermore, we showed that stimulation of the subthalamic nucleus (STN) evoked dopamine release exclusively in the tail striatum, likely via the SNL, consistent with previous anatomical evidence of STN afferents to SNL dopamine neurons. Our work identifies the STN as an important modulator of dopamine release in a novel dopamine pathway to the tail striatum, largely independent of the classic nigrostriatal pathway, which necessitates a revision of the basal ganglia circuitry with the STN positioned as a central integrator of striatal information.

Action potential and calcium dependence of tonic somatodendritic dopamine release in the Substantia Nigra pars compacta.

Despite the importance of somatodendritic dopamine (DA) release in the Substantia Nigra pars compacta (SNc), its mechanism remains poorly understood. Using a novel approach combining fast-scan controlled-adsorption voltammetry (FSCAV) and single-unit electrophysiology, we have investigated the mechanism of somatodendritic release by directly correlating basal (non-stimulated) extracellular DA concentration ([DA]out ), with pharmacologically-induced changes of firing of nigral dopaminergic neurons in rat brain slices. FSCAV measurements indicated that basal [DA]out in the SNc was 40.7 ± 2.0 nM (at 34 ± 0.5°C), which was enhanced by amphetamine, cocaine, and L-DOPA, and reduced by VMAT2 inhibitor, Ro4-1284. Complete inhibition of firing by TTX decreased basal [DA]out , but this reduction was smaller than the effect of D2 receptor agonist, quinpirole. Despite similar effects on neuronal firing, the larger decrease in [DA]out evoked by quinpirole was attributed to cell membrane hyperpolarization and greater reduction in cytosolic free Ca2+ ([Ca2+ ]in ). Decreasing extracellular Ca2+ also reduced basal [DA]out , despite increasing firing frequency. Furthermore, inhibiting L-type Ca2+ channels decreased basal [DA]out , although specific Cav 1.3 channel inhibition did not affect firing rate. Inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase (SERCA) also decreased [DA]out , demonstrating the importance of intracellular Ca2+ stores for somatodendritic release. Finally, in vivo FSCAV measurements showed that basal [DA]out in the SNc was 79.8 ± 10.9 nM in urethane-anesthetized rats, which was enhanced by amphetamine. Overall, our findings indicate that although tonic somatodendritic DA release is largely independent of action potentials, basal [DA]out is strongly regulated by voltage-dependent Ca2+ influx and release of intracellular Ca2+ . OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Crosstalk between mitochondria, calcium channels and actin cytoskeleton modulates noradrenergic activity of locus coeruleus neurons.

Locus coeruleus (LC) is the name of a group of large sized neurons located at the brain stem, which provides the main source of noradrenaline to the central nervous system, virtually, innervating the whole brain. All noradrenergic signalling provided by this nucleus is dependent on an intrinsic pacemaker process. Our study aims to understand how noradrenergic neurons finely tune their pacemaker processes and regulate their activities. Here we present that mitochondrial perturbation in the LC from mice, inhibits spontaneous firing by a hyperpolarizing response that involves Ca2+ entry via L-type Ca2+ channels and the actin cytoskeleton. We found that pharmacological perturbation of mitochondria from LC neurons using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a dominant hyperpolarizing response when electrophysiological approaches were performed. Surprisingly, the CCCP-induced hyperpolarizing response was dependent on L-type Ca2+ channel-mediated Ca2+ entry, as it was inhibited by: the removal of extracellular Ca2+ ; the addition of Cd2+ ; nifedipine or nicardipine; but not by the intracellular dialysis with the Ca2+ chelator 1,2-Bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the latter indicating that the response was not because of a global change in [Ca2+ ]c but does not exclude action at intracellular microdomains. Further to this, the incubation of slices with cytochalasin D, an agent that depolymerises the actin cytoskeleton, inhibited the hyperpolarizing response indicating an involvement of the actin cytoskeleton. The data are consistent with the hypothesis that there is a crosstalk between mitochondria and L-type Ca2+ channels leading to modulation of noradrenergic neuronal activity mediated by the actin cytoskeleton. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Dopamine Dysregulation and Altered Responses to Drugs Affecting Dopaminergic Transmission in a New Dopamine Transporter Knockout (DAT-KO) Rat Model.

Under normal conditions, dopamine (DA) clearance after release largely depends on uptake by the DA transporter (DAT). DAT expression/activity is reduced in some neuropsychiatric and neurological disorders. Our aim was to characterize the behavioral, neurochemical and electrophysiological effects of eliminating DAT in a novel knockout rat model we generated using CRISPR/Cas9. Consistent with existing DAT-KO models, our DAT-KO rats displayed increased locomotion, paradoxical calming by amphetamine, and reduced kinetics of DA clearance after stimulated release. Reduced DA kinetics were demonstrated using fast-scan cyclic voltammetry in brain slices containing the striatum or substantia nigra pars compacta (SNc) and in the dorsal striatum in vivo. Cocaine enhanced DA release in wild-type (WT) but not DAT-KO rats. Basal extracellular DA concentration measured with fast-scan controlled-adsorption voltammetry was higher in DAT-KO rats both in the striatum and SNc and was enhanced by L-DOPA (particularly after pharmacological block of monoamine oxidase), confirming that DA release after L-DOPA is not due to DAT reversal. The baseline firing frequency of SNc neurons was similar in both genotypes. However, D2 receptor-mediated inhibition of firing (by quinpirole or L-DOPA) was blunted in DAT-KO rats, while GABAB-mediated inhibition was preserved. We have also provided new data for the DAT-KO rat regarding the effects of slowing DA diffusion with dextran and blocking organic cation transporter 3 with corticosterone. Together, our results validate our DAT-KO rat and provide new insights into the mechanisms of chronic dysregulation of the DA system by addressing several unresolved issues in previous studies with other DAT-KO models.

Expression and functional properties of TRPM2 channels in dopaminergic neurons of the substantia nigra of the rat.

Transient receptor potential melastatin 2 (TRPM2) channels are sensitive to oxidative stress, and their activation can lead to cell death. Although these channels have been extensively studied in expression systems, their role in the brain, particularly in the substantia nigra pars compacta (SNc), remains unknown. In this study, we assessed the expression and functional properties of TRPM2 channels in rat dopaminergic SNc neurons, using acute brain slices. RT-PCR analysis revealed TRPM2 mRNA expression in the SNc region. Immunohistochemistry demonstrated expression of TRPM2 protein in tyrosine hydroxylase-positive neurons. Channel function was tested with whole cell patch-clamp recordings and calcium (fura-2) imaging. Intracellular application of ADP-ribose (50-400 µM) evoked a dose-dependent, desensitizing inward current and intracellular free calcium concentration ([Ca(2+)](i)) rise. These responses were strongly inhibited by the nonselective TRPM2 channel blockers clotrimazole and flufenamic acid. Exogenous application of H(2)O(2) (1-5 mM) evoked a rise in [Ca(2+)](i) and an outward current mainly due to activation of ATP-sensitive potassium (K(ATP)) channels. Inhibition of K(+) conductance with Cs(+) and tetraethylammonium unmasked an inward current. The inward current and/or [Ca(2+)](i) rise were partially blocked by clotrimazole and N-(p-amylcinnamoyl)anthranilic acid (ACA). The H(2)O(2)-induced [Ca(2+)](i) rise was abolished in "zero" extracellular Ca(2+) concentration and was enhanced at higher baseline [Ca(2+)](i), consistent with activation of TRPM2 channels in the cell membrane. These results provide evidence for the functional expression of TRPM2 channels in dopaminergic SNc neurons. Given the involvement of oxidative stress in degeneration of SNc neurons in Parkinson's disease, further studies are needed to determine the pathophysiological role of these channels in the disease process.

Properties of dopaminergic neurons in organotypic mesencephalic-striatal co-cultures--evidence for a facilitatory effect of dopamine on the glutamatergic input mediated by α-1 adrenergic receptors.

Organotypic cultures (OCs) have been widely used to investigate the midbrain dopaminergic system, but only a few studies focused on the functional properties of dopaminergic neurons and their synaptic inputs from dopaminergic and non-dopaminergic neurons also contained in such cultures. In addition, it is not clear whether the culturing process affects the intrinsic neuronal properties and the expression of specific receptors and transporters. We performed patch-clamp recordings from dopaminergic neurons in mesencephalic-striatal co-cultures obtained from transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. Some (10/44) GFP+ neurons displayed a bursting activity that renders the firing of these cells similar to that of the dopaminergic neurons in vivo. The culturing process reduced the hyperpolarization-activated current (I(h) ) and the expression of D2 receptors. Downregulation of D2 receptor mRNA and protein was confirmed with reverse transcriptase polymerase chain reaction and Western blotting. Immunocytochemistry revealed that many synaptic terminals, most likely originating from dopaminergic neurons, co-expressed the dopamine (DA) transporter and the vesicular glutamate transporter-2, suggesting a co-release of DA and glutamate. Interestingly, exogenous DA decreased glutamate release in young cultures [days in vitro (DIV)<20] by acting on pre-synaptic D2 receptors, while in older cultures (DIV>26) DA increased glutamate release by acting on α-1 adrenoreceptors. The facilitatory effect of DA on glutamatergic transmission to midbrain dopaminergic neurons may be important in conditions when the expression of D2 receptors is compromised, such as long-term treatment with antipsychotic drugs. Our data show that midbrain OCs at DIV>26 may provide a suitable model of such conditions.

Effect of low Mg2+ and bicuculline on cell survival in hippocampal slice cultures.

A reliable model system of epileptiform insult would facilitate investigation into the underlying biological mechanisms. Epileptiform insult was induced in hippocampal slice cultures by lowering extracellular Mg(2+), (+)-bicuculline, or (-)-bicuculline methochloride, a stable salt form of bicuculline (both forms block GABA(A) receptors). Cell death was assessed by propidium iodide uptake. Low Mg(2+) or (+)-bicuculline did not produce cell death regardless of dose or incubation period. Exposure to 100 microM (-)-bicuculline methochloride for 48 hr resulted in prominent CA1 cell death. These findings demonstrate that not all pro-epileptic drugs/ion changes used routinely for electrophysiological recording of seizure activity lead to cell death in hippocampal slice cultures and that treatment with bicuculline methochloride can be used as a reliable model for epileptiform insult.

IH current generates the afterhyperpolarisation following activation of subthreshold cortical synaptic inputs to striatal cholinergic interneurons.

Pauses in the tonic firing of striatal cholinergic interneurons emerge during reward-related learning and are triggered by neutral cues which develop behavioural significance. In a previous in vivo study we have proposed that these pauses in firing may be due to intrinsically generated afterhyperpolarisations (AHPs) evoked by excitatory synaptic inputs, including those below the threshold for action potential firing. The aim of this study was to investigate the mechanism of the AHPs using a brain slice preparation which preserved both cerebral hemispheres. Augmenting cortically evoked postsynaptic potentials (PSPs) by repetitive stimulation of cortical afferents evoked AHPs that were unaffected by blocking either GABA(A) receptors with bicuculline, or GABA(B) receptors with saclofen or CGP55845. Apamin (a blocker of small conductance Ca(2+)-activated K(+) channels) had minimal effects, while chelation of intracellular Ca(2+) with BAPTA reduced the AHP by about 30%. In contrast, blocking hyperpolarisation and cyclic nucleotide activated (HCN) cation current (I(H)) with ZD7288 or Cs(+) diminished the size of the AHPs by 60% and reduced the proportion of episodes that contained this hyperpolarisation. The reversal potential (20 mV) and voltage dependence of the AHPs were consistent with the hypothesis that a transient deactivation of I(H) caused most of the AHP at hyperpolarised potentials, while the slow AHP-type Ca(2+)-activated K(+) channels increasingly contributed at more depolarised membrane potentials. Subthreshold somatic current injections yielded similar AHPs with a median duration of approximately 700 ms that were not affected by firing of a single action potential. These results indicate that transient deactivation of HCN channels evokes pauses in tonic firing of cholinergic interneurons, an event likely to be elicited by augmentation of afferent synaptic inputs during learning.

Acute action of rotenone on nigral dopaminergic neurons--involvement of reactive oxygen species and disruption of Ca2+ homeostasis.

Rotenone is a toxin used to generate animal models of Parkinson's disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05-1 microm) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 +/- 15 pA) associated with increases in intracellular [Ca(2+)] ([Ca(2+)](i)) (73.8 +/- 7.7 nm) and intracellular [Na(+)] (3.1 +/- 0.6 mm) (all with 1 microm). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca(2+)](i) rise was abolished by removing extracellular Ca(2+), and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N-(p-amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca(2+)](i) rise resulted in a large (46.6 +/- 25.3 nm) Ca(2+) response when baseline [Ca(2+)](i) was increased by a 'priming' protocol that activated voltage-gated Ca(2+) channels. There was also a positive correlation between 'naturally' occurring variations in baseline [Ca(2+)](i) and the rotenone-induced [Ca(2+)](i) rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K(+) channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca(2+)](i) rise by a small increase in baseline [Ca(2+)](i).

Role of homeostatic feedback mechanisms in modulating methylphenidate actions on phasic dopamine signaling in the striatum of awake behaving rats.

Methylphenidate is an established treatment for attention-deficit hyperactivity disorder that also has abuse potential. Both properties may relate to blocking dopamine and norepinephrine reuptake. We measured the effects of methylphenidate on dopamine dynamics in freely moving rats. Methylphenidate alone had no effect on the amplitude of phasic responses to cues or reward. However, when administered with the D2 receptor antagonist raclopride, methylphenidate increased dopamine responses, while raclopride alone had no effect. Using brain slices of substantia nigra or striatum, we confirmed that methylphenidate effects on firing rate of nigral dopamine neurons and dopamine release from terminals are constrained by negative feedback. A computational model using physiologically relevant parameters revealed that actions of methylphenidate on norepinephrine and dopamine transporters, and the effects of changes in tonic dopamine levels on D2 receptors, are necessary and sufficient to account for the experimental findings. In addition, non-linear fitting of the model to the data from freely moving animals revealed that methylphenidate significantly slowed the initial cue response dynamics. These results show that homeostatic regulation of dopamine release in the face of changing tonic levels of extracellular dopamine should be taken into account to understand the therapeutic benefits and abuse potential of methylphenidate.

Electrophysiological Characterization of Novel Effects of the Uptake-2 Blocker Decynium-22 (D-22) on Dopaminergic Neurons in the Substantia Nigra Pars Compacta.

Extracellular levels of dopamine (DA) and other monoamines in the brain depend not only on the classic transporters encoded by SLC6A gene family such as DAT, NET and SERT, but also a more recently identified group of low-affinity/high-capacity 'Uptake-2' transporters, mainly OCT3 and PMAT. The most frequently used pharmacological tool in functional studies of Uptake-2 is decynium-22 (D-22) known to block these transporters. However, the effectiveness of this drug in enhancing extracellular DA remains uncertain. Our aim was to test the hypothesis that D-22 increases extracellular levels of DA released from the somatodendritic region of dopaminergic neurons in the substantia nigra pars compacta (SNc) by reducing the OCT3/PMAT-dependent component of DA uptake. Extracellular DA was assessed indirectly, by evoking D2-IPSCs in SNc neurons following stimulated release of this neurotransmitter in midbrain slices obtained from mice. Recordings were conducted after partial inhibition of DAT with nomifensine, and after application of L-DOPA which increased the releasable DA pool. Contrary to our expectations, D-22 reduced, rather than increased, the amplitude of D2-IPSCs. Other effects included inhibition of GABAB-IPSCs and Ih current, and a reduction in firing frequency of nigral neurons. These results show that in addition to the previously known non-specific inhibitory action on α1 adrenoceptors, D-22 exerts additional off-target effects by inhibiting dopaminergic and GABAergic synaptic transmission in the SNc and the spontaneous (pacemaker) activity of nigral neurons. It remains to be established if these novel effects contribute to a reduction in spontaneous locomotor activity reported in previous studies after systemic drug administration.

P2Y1 receptor modulation of the pre-Bötzinger complex inspiratory rhythm generating network in vitro.

ATP is released during hypoxia from the ventrolateral medulla (VLM) and activates purinergic P2 receptors (P2Rs) at unknown loci to offset the secondary hypoxic depression of breathing. In this study, we used rhythmically active medullary slices from neonatal rat to map, in relation to anatomical and molecular markers of the pre-Bötzinger complex (preBötC) (a proposed site of rhythm generation), the effects of ATP on respiratory rhythm and identify the P2R subtypes responsible for these actions. Unilateral microinjections of ATP in a three-dimensional grid within the VLM revealed a "hotspot" where ATP (0.1 mM) evoked a rapid 2.2 +/- 0.1-fold increase in inspiratory frequency followed by a brief reduction to 0.83 +/- 0.02 of baseline. The hotspot was identified as the preBötC based on histology, overlap of injection sites with NK1R immunolabeling, and potentiation or inhibition of respiratory frequency by SP ([Sar9-Met(O2)11]-substance P) or DAMGO ([D-Ala2,N-MePhe4,Gly-ol5]-enkephalin), respectively. The relative potency of P2R agonists [2MeSADP (2-methylthioadenosine 5'-diphosphate) approximately = 2MeSATP (2-methylthioadenosine 5'-triphosphate) approximately = ATPgammas (adenosine 5'-[gamma-thio]triphosphate tetralithium salt) approximately = ATP >> UTP approximately = alphabeta meATP (alpha,beta-methylene-adenosine 5'-triphosphate)] and attenuation of the ATP response by MRS2179 (2'-deoxy-N6-methyladenosine-3',5'-bisphosphate) (P2Y1 antagonist) indicate that the excitation is mediated by P2Y1Rs. The post-ATP inhibition, which was never observed in response to ATPgammas, is dependent on ATP hydrolysis. These data establish in neonatal rats that respiratory rhythm generating networks in the preBötC are exquisitely sensitive to P2Y1R activation, and suggest a role for P2Y1Rs in respiratory motor control, particularly in the P2R excitation of rhythm that occurs during hypoxia.

Contribution of calpain activation to early stages of hippocampal damage during oxygen-glucose deprivation.

Calpains are Ca(2+)-activated enzymes which cleave cytoskeletal and other proteins, contributing to neuronal damage in conditions of pathological intracellular Ca(2+) elevation, including stroke. However, the consequences of calpain overactivation have typically been observed hours after insult. To identify the earliest events attributable to calpain activation, and thus potentially isolate calpain substrates involved in acute neuronal damage, we dynamically recorded the effects of calpain inhibition in an in vitro model of stroke. Extracellular DC potentials and fEPSPs were monitored together with changes of light transmittance (as a measure of cell and mitochondrial swelling) and Rh 123 fluorescence (to monitor mitochondrial membrane potential; DeltaPsi(m)) in hippocampal slices obtained from P12-P17 rats. No differences were observed in the latencies of fEPSP disruption or onset of extracellular DC shifts associated with hypoxic spreading depression (HSD) evoked by oxygen-glucose deprivation (OGD) under control conditions or in the presence of calpain inhibitor III (MDL 28170). However, a significant difference was observed in transmitted light signals during OGD with calpain inhibition. Given the potential contribution of mitochondrial swelling to changes in light transmittance, these experiments were also conducted in the presence of cyclosporin A to block opening of the mitochondrial permeability transition pore (MPTP). Our results indicate that differences in OGD-induced changes of light transmittance in the presence of MDL 28170 are not likely the result of MPTP blockade or changes in dendritic beading. We propose that calpain inhibition may alter changes in light transmittance by limiting conformational changes of mitochondria.

Acute sensitivity of astrocytes in the Substantia Nigra to oxygen and glucose deprivation (OGD) compared with hippocampal astrocytes in brain slices.

The Substantia Nigra is a brainstem nucleus critical for movement control. Although its dopamine-producing neurons degenerate in Parkinsons disease, little is known of the acute effects of ischemia in this region. We recently reported that oxygen and glucose deprivation (OGD) in brain slices, an in vitro ischemia model, evokes a profound depolarization and swelling of GABAergic neurons in the Substantia Nigra pars reticulata (SNr), but not dopaminergic neurons in the Substantia Nigra pars compacta (SNc). The current study characterised the effects of OGD on nigral astrocytes, and compared these with the established responses of astrocytes in the CA1 hippocampal region. Intracellular recordings were made from astrocytes at the border between SNc and SNr subregions, in midbrain slices from postnatal day 21-23 rats. Immunoreactivity for astrocyte-specific proteins was also assessed. OGD evoked a slow, then fast depolarization of nigral astrocytes. The fast phase developed during the anoxic depolarization (indicated by a fast negative shift of extracellular DC potential and increase in light transmittance) and rapid increase in extracellular K+ concentration in the SNr. This biphasic response resembled the OGD-evoked depolarization of hippocampal astrocytes. However, unlike the partial repolarization seen in hippocampal cells after reperfusion with O2 and glucose, nigral astrocytes remained depolarized near 0 mV. In addition, immunoreactivity for glial fibrillary acidic protein-positive astrocytes markedly decreased in the Substantia Nigra after OGD, while in the hippocampus remained unchanged. These data indicate an acute post-ischemic withdrawal of astrocytic support in the Substantia Nigra, but not in the hippocampus.

Paradoxical lower sensitivity of Locus Coeruleus than Substantia Nigra pars compacta neurons to acute actions of rotenone.

Parkinson's disease (PD) is not only associated with degeneration of dopaminergic (DAergic) neurons in the Substantia Nigra, but also with profound loss of noradrenergic neurons in the Locus Coeruleus (LC). Remarkably, LC degeneration may exceed, or even precede the loss of nigral DAergic neurons, suggesting that LC neurons may be more susceptible to damage by various insults. Using a combination of electrophysiology, fluorescence imaging and electrochemistry, we directly compared the responses of LC, nigral DAergic and nigral non-dopaminergic (non-DAergic) neurons in rat brain slices to acute application of rotenone, a mitochondrial toxin used to create animal and in vitro models of PD. Rotenone (0.01-5.0µM) dose-dependently inhibited the firing of all three groups of neurons, primarily by activating KATP channels. The toxin also depolarised mitochondrial potential (Ψm) and released reactive oxygen species (H2O2). When KATP channels were blocked, rotenone (1µM) increased the firing of LC neurons by activating an inward current associated with dose-dependent increase of cytosolic free Ca2+ ([Ca2+]i). This effect was attenuated by blocking oxidative stress-sensitive TRPM2 channels, and by pre-treatment of slices with anti-oxidants. These results demonstrate that rotenone inhibits the activity of LC neurons mainly by activating KATP channels, and increases [Ca2+]ivia TRPM2 channels. Since the responses of LC neurons were smaller than those of nigral DAergic neurons, our study shows that LC neurons are paradoxically less sensitive to acute effects of this parkinsonian toxin.

Differential spread of anoxic depolarization contributes to the pattern of neuronal injury after oxygen and glucose deprivation (OGD) in the Substantia Nigra in rat brain slices.

Anoxic depolarization (AD) is an acute event evoked by brain ischemia, involving a profound loss of cell membrane potential and swelling that spreads over susceptible parts of the gray matter. Its occurrence is a strong predictor of the severity of neuronal injury. Little is known about this event in the Substantia Nigra, a midbrain nucleus critical for motor control. We tested the effects of oxygen and glucose deprivation (OGD), an in vitro model of brain ischemia, in rat midbrain slices. AD developed within 4min from OGD onset and spread in the Substantia Nigra pars reticulata (SNr), but not through the Substantia Nigra pars compacta (SNc). This differential effect involved a contrasting pattern of changes in membrane potential between dopamine-producing SNc and non-dopaminergic SNr neurons. A fast depolarization in SNr neurons was not followed by repolarization after the end of OGD, and was associated with swollen somata and beaded dendrites. In contrast, slowly developing depolarization of SNc neurons led to repolarization after OGD ended, and no changes in neuronal morphology were observed. The AD-resistance of the SNc involved smaller dysregulations of K+ and Ca2+ ions, and a slower loss of energy metabolites. Our results show that acute ischemia profoundly impairs the function and morphology of SNr neurons but not adjacent SNc neurons, and that the surprising higher tolerance of SNc neurons correlates with the resistance of the SNc region to AD. This differential response may affect the pattern of early neuronal injury that develops in the brainstem after acute ischemic insults.

Involvement of TRP-like channels in the acute ischemic response of hippocampal CA1 neurons in brain slices.

During a period of acute ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro, CA1 neurons depolarize, swell and become overloaded with calcium. Our aim was to test the hypothesis that the initial responses to OGD are at least partly due to transient receptor potential (TRP) channel activation. As some TRP channels are temperature-sensitive, we also compared the effects of pharmacological blockade of the channels with the effects of reducing temperature. Acute hippocampal slices (350 mum) obtained from Wistar rats were submerged in ACSF at 36 degrees C. CA1 neurons were monitored electrophysiologically using extracellular, intracellular or whole-cell patch-clamp recordings. Cell swelling was assessed by recording changes in relative tissue resistance, and changes in intracellular calcium were measured after loading neurons with fura-2 dextran. Blockers of TRP channels (ruthenium red, La3+, Gd3+, 2-APB) or lowering temperature by 3 degrees C reduced responses to OGD. This included: (a) an increased delay to negative shifts of extracellular DC potential; (b) reduction in rate of the initial slow membrane depolarization, slower development of OGD-induced increase in cell input resistance and slower development of whole-cell inward current; (c) reduced tissue swelling; and (d) a smaller rise in intracellular calcium. Mild hypothermia (33 degrees C) and La3+ or Gd3+ (100 microM) showed an occlusion effect when delay to extracellular DC shifts was measured. Expression of TRPM2/TRPM7 (oxidative stress-sensitive) and TRPV3/TRPV4 (temperature-sensitive) channels was demonstrated in the CA1 subfield with RT-PCR. These results indicate that TRP or TRP-like channels are activated by cellular stress and contribute to ischemia-induced membrane depolarization, intracellular calcium accumulation and cell swelling. We also hypothesize that closing of some TRP channels (TRPV3 and/or TRPV4) by lowering temperature may be partly responsible for the neuroprotective effect of hypothermia.

Acute effects of 6-hydroxydopamine on dopaminergic neurons of the rat substantia nigra pars compacta in vitro.

6-Hydroxydopamine (6-OHDA) is a neurotoxin which has been implicated in the degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc) in Parkinson's disease (PD), and is frequently used to produce animal models of the disease. The aim of our study, conducted on midbrain slices obtained from young Wistar rats, was to determine the little known acute effects of this toxin (0.2-2.0 mM; 10-20 min exposure; 34 degrees C) on electrophysiological properties, intracellular Ca2+ levels and dendritic morphology of SNc neurons. Four experimental approaches were used: extracellular recording of firing frequency, whole-cell patch-clamping, ratiometric fura-2 imaging, and cell labeling with lucifer yellow (LY) or dextran-rhodamine. Extracellular recording revealed a concentration-dependent decrease in the tonic, pacemaker-like firing. In whole-cell recordings in voltage-clamp (V(hold) -60 mV), smaller doses (0.2-0.5 mM) induced an outward current (or cell membrane hyperpolarization in current-clamp), which could in some cells be reversed with tolbutamide (blocker of ATP-dependent K+ channels). A higher dose (1.0-2.0 mM) caused rapid reductions of cell membrane capacitance and membrane resistance. Toxin exposure gradually increased the intracellular Ca2+ level, which did not subsequently return to control. The increase in Ca2+ signal was not prevented by depletion of intracellular Ca2+ stores with thapsigargin (10 microM) or cyclopiazonic acid (30 microM), nor by removing extracellular Ca2+. Cell membrane current and Ca2+ responses were not prevented by blocking dopamine transporter (DAT). Cells loaded with LY or dextran-rhodamine showed signs of damage (cell membrane blebbing) in dendrites following toxin exposure (1 mM; 10-20 min). These results demonstrate that the oxidative and metabolic stress induced in SNc neurons by 6-OHDA results in rapid dose-dependent changes of cell membrane properties with morphological evidence of dendritic damage, as well as in disturbance of intracellular Ca2+ homeostasis.

A novel electrochemical approach for prolonged measurement of absolute levels of extracellular dopamine in brain slices.

Tonic dopamine (DA) levels influence the activity of dopaminergic neurons and the dynamics of fast dopaminergic transmission. Although carbon fiber microelectrodes and fast-scan cyclic voltammetry (FSCV) have been extensively used to quantify stimulus-induced release and uptake of DA in vivo and in vitro, this technique relies on background subtraction and thus cannot provide information about absolute extracellular concentrations. It is also generally not suitable for prolonged (>90 s) recordings due to drift of the background current. A recently reported, modified FSCV approach called fast-scan controlled-adsorption voltammetry (FSCAV) has been used to assess tonic DA levels in solution and in the anesthetized mouse brain. Here we describe a novel extension of FSCAV to investigate pharmacologically induced, slowly occurring changes in tonic (background) extracellular DA concentration, and phasic (stimulated) DA release in brain slices. FSCAV was used to measure adsorption dynamics and changes in DA concentration (for up to 1.5 h, sampling interval 30 s, detection threshold < 10 nM) evoked by drugs affecting DA release and uptake (amphetamine, l-DOPA, pargyline, cocaine, Ro4-1284) in submerged striatal slices obtained from rats. We also show that combined FSCAV-FSCV recordings can be used for concurrent study of stimulated release and changes in tonic DA concentration. Our results demonstrate that FSCAV can be effectively used in brain slices to measure prolonged changes in extracellular level of endogenous DA expressed as absolute values, complementing studies conducted in vivo with microdialysis.

Excitatory drive from the Subthalamic nucleus attenuates GABAergic transmission in the Substantia Nigra pars compacta via endocannabinoids.

Endocannabinoids (eCBs) are cannabis-like substances produced in the brain where their primary function is to regulate synaptic transmission by inhibiting neurotransmitter release in a retrograde fashion. We have recently demonstrated a novel mechanism regulating GABAergic transmission from neurons in the Substantia Nigra pars reticulata (SNr) to dopaminergic neurons in the Substantia Nigra pars compacta (SNc) mediated by eCBs. Production of eCBs was initiated by spillover of glutamate, yet the source of the glutamate was not determined (Freestone et al., 2014; Neuropharmacology 79 p467). The present study aimed at elucidating the potential role of glutamatergic terminals arising from neurons in the Subthalamic nucleus (STN) in driving the eCB-mediated modulation of this inhibitory transmission. GABAergic IPSCs or IPSPs evoked in SNc neurons by electrical stimuli delivered to the SNr region were transiently inhibited by electrical or pharmacological (U-tube application of muscarinic agonist carbachol [100 µM]) stimulation of the STN (to 74±5% and 69±4% respectively). In both stimulation protocols, the attenuation of GABAergic transmission was abolished by cannabinoid receptor 1 antagonist rimonabant (3 µM), and reduced by group 1 metabotropic glutamate receptor antagonist CPCCOEt (100 µM), consistent with a glutamate-initiated and eCB-mediated mechanism. The carbachol-induced attenuation of GABAergic transmission was abolished by M3 muscarinic receptor antagonist 4-DAMP (10 µM), confirming a specific activation of STN neurons. These results demonstrate that glutamatergic projection from the STN to dopaminergic SNc neurons underlies an eCB-mediated inhibition of GABAergic input to these neurons.