Yan Na Wu

Multiple mechanisms underlie burst firing in rat midbrain dopamine neurons in vitro

Both apamin and NMDA evoke burst firing in dopamine neurons recorded intracellularly in slices of rat midbrain. However, apamin-induced bursting required injection of depolarizing currents, and was mimicked by Bay K8644 and blocked by nifedipine. In contrast, NMDA-induced bursting required hyperpolarizing currents and was not blocked by nifedipine. Our results show that burst firing can be evoked in dopamine neurons via two different mechanisms.

Presynaptic inhibition preferentially reduces the NMDA receptor‐mediated component of transmission in rat midbrain dopamine neurons

We used patch pipettes to record whole‐cell currents from single dopamine neurons in slices of rat midbrain. Pharmacological methods were used to isolate EPSCs evoked by focal electrical stimulation. Baclofen was significantly more potent for inhibiting NMDA receptor‐mediated EPSCs (IC50=0.24 μM) compared with inhibition of EPSCs mediated by AMPA receptors (IC50=1.72 μM). The increased potency of baclofen for inhibiting the NMDA component persisted in superfusate that contained zero Mg2+ and when postsynaptic K+ conductances were reduced by Cs+ and QX‐314. Effects of baclofen on EPSCs were blocked by the GABAB receptor antagonist CGP‐35348. Adenosine was 20 fold more potent for reducing the NMDA component of transmission (IC50=31 μM) compared with inhibition of AMPA receptor‐mediated EPSCs (IC50=654 μM). Effects of adenosine on EPSCs were blocked by the A1 receptor antagonist DPCPX. Both baclofen and adenosine significantly increased the ratio of EPSCs in paired‐pulse studies, suggesting presynaptic sites of action. Although adenosine (1 mM) did not reduce currents evoked by exogenous NMDA (10 μM), baclofen (1 μM) reduced NMDA currents by 29%. Neither baclofen nor adenosine altered currents evoked by exogenous AMPA (1 μM). We conclude that adenosine acts at presynaptic A1 receptors to cause a preferential reduction in the NMDA component of synaptic transmission. In contrast, baclofen preferentially reduces NMDA EPSCs by acting at both pre‐ and postsynaptic GABAB receptors. By regulating NMDA receptor function, A1 and GABAB receptors may play important roles in regulating the excitability of dopamine neurons.

Rotenone reduces Mg2+-dependent block of NMDA currents in substantia nigra dopamine neurons.

Rotenone is a pesticide that has been successfully used to produce a rodent model of Parkinsons disease. We reported previously that rotenone potently augmented N-methyl-D-aspartate (NMDA)-evoked currents in rat dopamine neurons via a tyrosine kinase-dependent mechanism. In this study, we investigated the effect of rotenone on the current-voltage relationship of NMDA-induced currents in substantia nigra zona compacta neurons recorded with whole-cell patch pipettes in slices of rat brain. In a physiologic concentration of extracellular Mg(2+) (1.2mM), a 30min perfusion with rotenone (100nM) produced a marked increase in current evoked by bath application of NMDA (30microM), especially when measured at relatively hyperpolarized currents. In the presence of rotenone, NMDA currents lost the characteristic region of negative-slope conductance that is normally produced by voltage-dependent block by Mg(2+). The voltage-dependent effect of rotenone on NMDA currents was mimicked by a low extracellular concentration of Mg(2+) (0.2mM) and was antagonized by a high level of Mg(2+) (6.0mM). Moreover, we report that the tyrosine kinase inhibitor genistein blocked the ability of rotenone to augment NMDA receptor currents. These results suggest that rotenone potentiates NMDA currents by a tyrosine kinase-dependent process that attenuates voltage-dependent Mg(2+) block of NMDA-gated channels. These results support the hypothesis that an excitotoxic mechanism might participate in rotenone-induced toxicity of midbrain dopamine neurons.

Rotenone potentiates NMDA currents in substantia nigra dopamine neurons.

Rotenone is a pesticide that produces a rodent model of Parkinsons disease. Although much evidence suggests that oxidative stress mediates the toxicity of rotenone on dopamine neurons, rotenone can also potentiate glutamate excitotoxicity. We used whole-cell patch pipettes to investigate actions of rotenone on currents evoked by N-methyl-d-aspartate (NMDA) in dopamine neurons in slices of rat midbrain. After superfusing the slice for 20-30 min, rotenone (100 nM) caused a 162% increase in the average amplitude of inward current evoked by 30 microM NMDA. This effect of rotenone was mimicked by the sodium pump inhibitor strophanthidin (10 microM) and was abolished when pipettes contained an ATP regeneration solution. Although strophanthidin also significantly increased the amplitude of inward currents evoked by (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 10 microM), rotenone failed to potentiate AMPA currents. Because rotenone potentiated NMDA- but not AMPA-dependent currents, this suggests that rotenone acts selectively to augment NMDA receptor function. Furthermore, the failure of rotenone to mimic strophanthidin suggests that rotenone does not inhibit sodium pump activity. Our results suggest that an excitotoxic mechanism might contribute to rotenone neurotoxicity.

Mitochondrial uncoupling agents antagonize rotenone actions in rat substantia nigra dopamine neurons.

Mild uncoupling of oxidative phosphorylation has been reported to reduce generation of reactive oxygen species (ROS) and therefore may be neuroprotective. We reported previously that the mitochondrial poison rotenone enhanced currents evoked by N-methyl-D-aspartate (NMDA) by a ROS-dependent mechanism in rat midbrain dopamine neurons. Thus, rotenone, which produces a model of Parkinsons disease in rodents, may increase the risk of dopamine neuron excitotoxicity. The purpose of this study was to test the hypothesis that oxidative phosphorylation uncoupling agents would antagonize the effect of rotenone on NMDA current. We used patch pipettes to record whole-cell currents under voltage-clamp (-60 mV) in substantia nigra dopamine neurons in slices of rat brain. Rotenone, NMDA and uncoupling agents were added to the brain slice superfusate. Inward currents evoked by NMDA (30 μM) more than doubled in amplitude after slices were superfused for 30 min with 100 nM rotenone. Continuous superfusion with the uncoupling agent carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (1-3 nM) or 2,4-dinitrophenol (100 nM) significantly antagonized and delayed the ability of rotenone to potentiate NMDA currents. Coenzyme Q₁₀ (1-10 nM), which has been reported to facilitate uncoupling protein activity, also antagonized this action of rotenone. These results suggest that mild uncoupling of oxidative phosphorylation may protect dopamine neurons against injury from mitochondrial poisons such as rotenone.

Memantine selectively blocks extrasynaptic NMDA receptors in rat substantia nigra dopamine neurons

Recent studies suggest that selective block of extrasynaptic N-methyl-d-aspartate (NMDA) receptors might protect against neurodegeneration. We recorded whole-cell currents with patch pipettes to characterize the ability of memantine, a low-affinity NMDA channel blocker, to block synaptic and extrasynaptic NMDA receptors in substantia nigra zona compacta (SNC) dopamine neurons in slices of rat brain. Pharmacologically isolated NMDA receptor-mediated EPSCs were evoked by electrical stimulation, whereas synaptic and extrasynaptic receptors were activated by superfusing the slice with NMDA (10 µM). Memantine was 15-fold more potent for blocking currents evoked by bath-applied NMDA compared to synaptic NMDA receptors. Increased potency for blocking bath-applied NMDA currents was shared by the GluN2C/GluN2D noncompetitive antagonist DQP-1105 but not by the high-affinity channel blocker MK-801. Our data suggest that memantine causes a selective block of extrasynaptic NMDA receptors that are likely to contain GluN2C/2D subunits. Our results justify further investigations on the use of memantine as a neuroprotective agent in Parkinsons disease.

Dopamine oxidation facilitates rotenone-dependent potentiation of N-methyl-D-aspartate currents in rat substantia nigra dopamine neurons.

Rotenone is a mitochondrial poison that causes dopamine cell death and is used as a model of Parkinsons disease in rodents. Recently, we showed that rotenone augments currents evoked by N-methyl-D-aspartate (NMDA) by relieving voltage-dependent Mg(2+) block in rat substantia nigra compacta (SNC) dopamine neurons. Because rotenone is well known to generate reactive oxygen species (ROS), we conducted the present experiments to evaluate the role of ROS in mediating the effect of rotenone on NMDA current augmentation. Using patch pipettes to record whole-cell currents from SNC neurons in slices of rat brain, we found that the ability of rotenone (100 nM) to increase NMDA (3-30 μM) current was antagonized by the antioxidant agent n-acetylcysteine (1 mM). In contrast, mercaptosuccinate (1 mM), which blocks glutathione peroxidase and raises tissue levels of H(2)O(2), mimicked rotenone by augmenting inward currents evoked by NMDA. Because oxidation of dopamine can also generate ROS, we explored the role of dopamine on this action of rotenone. We prepared dopamine-depleted midbrain slices from rats that had been pretreated with reserpine (5 mg/kg ip) and alpha-methyl-para-tyrosine (AMPT, 250 mg/kg ip). Dopamine depletion blocked the ability of rotenone (100 nM) to increase inward current evoked by NMDA (30 μM). Rotenone-dependent augmentation of NMDA current was also blocked by the monoamine oxidase inhibitor pargyline (100 μM) in slices prepared from normal rats. In contrast, the dopamine precursor levodopa potentiated the action of rotenone on NMDA current. These results suggest that ROS and/or dopamine oxidation products mediate the ability of rotenone to potentiate NMDA currents. Because excessive NMDA receptor stimulation can produce excitotoxicity, our results suggest that oxidative metabolism of dopamine might facilitate the neurotoxicity of rotenone.

Rotenone enhances N-methyl-D-aspartate currents by activating a tyrosine kinase in rat dopamine neurons.

Our previous work showed that the pesticide rotenone increases the amplitude of inward currents evoked by N-methyl-D-aspartate (NMDA) in substantia nigra dopamine neurons. Using patch pipettes to record whole-cell currents in rat brain slices, we report that the rotenone-induced potentiation of NMDA current is blocked by the tyrosine kinase inhibitors genistein and PP1. This action of rotenone is mimicked by H2O2, which is also blocked by genistein. Our results suggest that the rotenone-dependent increase in NMDA current is mediated by release of reactive oxygen species that activates a protein tyrosine kinase.

NMDA alters rotenone toxicity in rat substantia nigra zona compacta and ventral tegmental area dopamine neurons

Previous patch-clamp studies by our laboratory showed that acute exposure to the pesticide rotenone augments inward currents evoked by N-methyl-d-aspartate (NMDA) in substantia nigra zona compacta (SNC) dopamine neurons in slices of rat brain. The present experiments were done to search for histological evidence of increased neurotoxicity produced by combined rotenone and NMDA treatments. In horizontal slices of rat midbrain, we found that a 30 min superfusion with 100 nM rotenone caused significant injury to tyrosine hydroxylase (TH)-positive proximal dendrites in dorsal and ventral regions of the SNC and ventral tegmental area (VTA). Moreover, treatment with 100 μM NMDA potentiated rotenone toxicity. In contrast, treatment with 30 μM NMDA protected against rotenone-induced injury to dendrites in the ventral SNC and ventral VTA. Interestingly, treatment with 30 μM NMDA-alone produced an apparent increase in proximal dendrite scores in ventral SNC and dorsal VTA. We conclude that NMDA has concentration-dependent actions on rotenone toxicity that differ according to regional subtype of dopamine neuron.

AMP kinase regulates ligand-gated K-ATP channels in substantia nigra dopamine neurons

AMP-activated protein kinase (AMPK) is a master enzyme that regulates ATP-sensitive K(+) (K-ATP) channels in pancreatic beta-cells and cardiac myocytes. We used patch pipettes to record currents and potentials to investigate effects of AMPK on K-ATP currents in substantia nigra compacta (SNC) dopamine neurons in slices of rat midbrain. When slices were superfused repeatedly with the K-ATP channel opener diazoxide, we were surprised to find that diazoxide currents gradually increased in magnitude, reaching 300% of the control value 60min after starting whole-cell recording. However, diazoxide current increased significantly more, to 472% of control, when recorded in the presence of the AMPK activator A769662. Moreover, superfusing the slice with the AMPK blocking agent dorsomorphin significantly reduced diazoxide current to 38% of control. Control experiments showed that outward currents evoked by the K-ATP channel opener NN-414 also increased over time, but not currents evoked by the GABAB agonist baclofen. Delaying the application of diazoxide after starting whole-cell recording correlated with augmentation of current. Loose-patch recording showed that diazoxide produced a 34% slowing of spontaneous firing rate that did not intensify with repeated applications of diazoxide. However, superfusion with A769662 significantly augmented the inhibitory effect of diazoxide on firing rate. We conclude that K-ATP channel function is augmented by AMPK, which is activated during the process of making whole-cell recordings. Our results suggest that AMPK and K-ATP interactions may play an important role in regulating dopamine neuronal excitability.