Christian R Lee

Publications of Christian R Lee

• Regulation of Substantia Nigra Pars Reticulata GABAergic Neuron Activity by H₂O₂ via Flufenamic Acid-Sensitive Channels and K(ATP) Channels.

  Authors: Christian R Lee, Paul Witkovsky, Margaret E Rice

  Frontiers in systems neuroscience. 01/2011; 5:14.

  Substantia nigra pars reticulata (SNr) GABAergic neurons are key output neurons of the basal ganglia. Given the role of these neurons in motor control, it is important to understand factors thatSubstantia nigra pars reticulata (SNr) GABAergic neurons are key output neurons of the basal ganglia. Given the role of these neurons in motor control, it is important to understand factors that regulate their firing rate and pattern. One potential regulator is hydrogen peroxide (H₂O₂), a reactive oxygen species that is increasingly recognized as a neuromodulator. We used whole-cell current clamp recordings of SNr GABAergic neurons in guinea-pig midbrain slices to determine how H₂O₂ affects the activity of these neurons and to explore the classes of ion channels underlying those effects. Elevation of H₂O₂ levels caused an increase in the spontaneous firing rate of SNr GABAergic neurons, whether by application of exogenous H₂O₂ or amplification of endogenous H₂O₂ through inhibition of glutathione peroxidase with mercaptosuccinate. This effect was reversed by flufenamic acid (FFA), implicating transient receptor potential (TRP) channels. Conversely, depletion of endogenous H₂O₂ by catalase, a peroxidase enzyme, decreased spontaneous firing rate and firing precision of SNr neurons, demonstrating tonic control of firing rate by H₂O₂. Elevation of H₂O₂ in the presence of FFA revealed an inhibition of tonic firing that was prevented by blockade of ATP-sensitive K(+) (K(ATP)) channels with glibenclamide. In contrast to guinea-pig SNr neurons, the dominant effect of H₂O₂ elevation in mouse SNr GABAergic neurons was hyperpolarization, indicating a species difference in H₂O₂-dependent regulation. Thus, H₂O₂ is an endogenous modulator of SNr GABAergic neurons, acting primarily through presumed TRP channels in guinea-pig SNr, with additional modulation via K(ATP) channels to regulate SNr output.

• Immunocytochemical identification of proteins involved in dopamine release from the somatodendritic compartment of nigral dopaminergic neurons.

  Authors: Paul Witkovsky, Jyoti C Patel, Christian R Lee, Margaret E Rice

  Neuroscience. 09/2009;

  We examined the somatodendritic compartment of nigral dopaminergic neurons by immunocytochemistry and confocal microscopy, with the aim of identifying proteins that participate in dopamine packagingWe examined the somatodendritic compartment of nigral dopaminergic neurons by immunocytochemistry and confocal microscopy, with the aim of identifying proteins that participate in dopamine packaging and release. Nigral dopaminergic neurons were identified by location, cellular features and tyrosine hydroxylase immunoreactivity. Immunoreactive puncta of vesicular monoamine transporter type 2 and proton ATPase, both involved in the packaging of dopamine for release, were located primarily in dopaminergic cell bodies, but were absent in distal dopaminergic dendrites. Many presynaptic proteins associated with transmitter release at fast synapses were absent in nigral dopaminergic neurons, including synaptotagmin 1, syntaxin1, synaptic vesicle proteins 2a and 2b, synaptophysin and synaptobrevin 1 (VAMP 1). On the other hand, syntaxin 3, synaptobrevin 2 (VAMP 2) and SNAP-25-immunoreactivities were found in dopaminergic somata and dendrites Our data imply that the storage and exocytosis of dopamine from the somatodendritic compartment of nigral dopaminergic neurons is mechanistically distinct from transmitter release at axon terminals utilizing amino acid neurotransmitters.

• Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling.

  Authors: Li Bao, Marat V Avshalumov, Jyoti C Patel, Christian R Lee, Evan W Miller, Christopher J Chang, Margaret E Rice

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 08/2009; 29(28):9002-10.

  Hydrogen peroxide (H(2)O(2)) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier forHydrogen peroxide (H(2)O(2)) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H(2)O(2) signaling, which activates ATP-sensitive potassium (K(ATP)) channels to inhibit dopamine release. However, the origin of this modulatory H(2)O(2) has been elusive. Here we addressed three possible sources of H(2)O(2) produced for rapid neuronal signaling in striatum: mitochondrial respiration, monoamine oxidase (MAO), and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H(2)O(2) and tissue analysis of ATP, we found that coapplication of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H(2)O(2) production, but maintained tissue ATP content. Strikingly, coapplication of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H(2)O(2) in release modulation. In contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H(2)O(2) production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H(2)O(2) generation for dynamic neuronal signaling.

• Basal ganglia control of substantia nigra dopaminergic neurons.

  Authors: Christian R Lee, James M Tepper

  Journal of neural transmission. Supplementum. 01/2009;

  Although substantia nigra dopaminergic neurons are spontaneously active both in vivo and in vitro, this activity does not depend on afferent input as these neurons express an endogenousAlthough substantia nigra dopaminergic neurons are spontaneously active both in vivo and in vitro, this activity does not depend on afferent input as these neurons express an endogenous calcium-dependent oscillatory mechanism sufficient to drive action potential generation. However, afferents to these neurons, a large proportion of them GABAergic and arising from other nuclei in the basal ganglia, play a crucial role in modulating the activity of dopaminergic neurons. In the absence of afferent activity or when in brain slices, dopaminergic neurons fire in a very regular, pacemaker-like mode. Phasic activity in GABAergic, glutamatergic, and cholinergic inputs modulates the pacemaker activity into two other modes. The most common is a random firing pattern in which interspike intervals assume a Poisson-like distribution, and a less common pattern, often in response to a conditioned stimulus or a reward in which the neurons fire bursts of 2-8 spikes time-locked to the stimulus. Typically in vivo, all three firing patterns are observed, intermixed, in single nigrostriatal neurons varying over time. Although the precise mechanism(s) underlying the burst are currently the focus of intensive study, it is obvious that bursting must be triggered by afferent inputs. Most of the afferents to substantia nigra pars compacta dopaminergic neurons comprise monosynaptic inputs from GABAergic projection neurons in the ipsilateral neostriatum, the globus pallidus, and the substantia nigra pars reticulata. A smaller fraction of the basal ganglia inputs, something less than 30%, are glutamatergic and arise principally from the ipsilateral subthalamic nucleus and pedunculopontine nucleus. The pedunculopontine nucleus also sends a cholinergic input to nigral dopaminergic neurons. The GABAergic pars reticulata projection neurons also receive inputs from all of these sources, in some cases relaying them disynaptically to the dopaminergic neurons, thereby playing a particularly significant role in setting and/or modulating the firing pattern of the nigrostriatal neurons.

• A calcium-activated nonselective cation conductance underlies the plateau potential in rat substantia nigra GABAergic neurons.

  Authors: Christian R Lee, James M Tepper

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 07/2007; 27(24):6531-41.

  Plateau potentials can be elicited in nigral GABAergic neurons by injection of 500 ms depolarizing current pulses from hyperpolarized holding potentials in whole-cell recordings in vitro. InPlateau potentials can be elicited in nigral GABAergic neurons by injection of 500 ms depolarizing current pulses from hyperpolarized holding potentials in whole-cell recordings in vitro. In approximately one-third of these neurons, plateau potentials were observed under control conditions and could be elicited in the remaining neurons after blocking potassium conductances. Application of the L-type calcium channel agonist Bay K 8644 or activation of NMDA receptors enhanced plateau potentials observed under control conditions and caused a plateau to be elicited in neurons not exhibiting it previously. The plateau potential was abolished in calcium-free buffer, as well as by nickel or cadmium. The L-type calcium channel blockers nimodipine and nifedipine abolished the plateau potential observed under control conditions but did not affect plateaus unmasked by tetraethylammonium. Plateau potentials observed under control conditions as well as those observed in the presence of Bay K 8644, NMDA, or tetraethylammonium were abolished in low-sodium buffer and by the calcium-activated nonselective cation conductance blocker flufenamic acid. These data suggest that nigral plateau potentials are mediated by a calcium-activated nonselective cation conductance (I(CAN)) that is activated by calcium entry predominantly through L-type calcium channels. In many nigral neurons, I(CAN) is masked by tetraethylammonium-sensitive potassium conductances, but plateaus can be evoked after increasing calcium conductances. The I(CAN)-mediated plateau potential in nigral GABAergic neurons likely affects the way these neurons integrate input and may represent a mechanism contributing to the rhythmic firing of these neurons seen in pathological conditions such as Parkinson's disease.

• Morphological and physiological properties of parvalbumin- and calretinin-containing gamma-aminobutyric acidergic neurons in the substantia nigra.

  Authors: Christian R Lee, James M Tepper

  The Journal of comparative neurology. 03/2007; 500(5):958-72.

  Evidence for the existence of different populations of gamma-aminobutyric acid (GABA)-ergic neurons in the substantia nigra comes partially from anatomical studies, which have shown there to beEvidence for the existence of different populations of gamma-aminobutyric acid (GABA)-ergic neurons in the substantia nigra comes partially from anatomical studies, which have shown there to be little if any overlap between the calcium-binding proteins parvalbumin and calretinin in individual neurons, suggesting that these may represent neuronal subtypes with distinct electrophysiological and/or anatomical properties. We obtained whole-cell recordings from neurons in the substantia nigra pars reticulata in rat brain slices and labeled them with biocytin, followed by immunocytochemical staining for parvalbumin and calretinin. In other cases, neurons were retrogradely labeled from the thalamus or tectum and immunocytochemically identified to determine their projection sites. Intracellularly stained neurons were found to have a variety of somatic sizes and shapes. Reconstructions revealed that all parvalbumin- and calretinin-positive neurons issued at least one axon collateral, which ramified within the substantia nigra pars reticulata and/or pars compacta. Local collaterals were of medium caliber and branched modestly, expressing many long, smooth segments that then issued numerous en passant or terminal boutons, consistent with previous in vivo studies. There were no clear differences in the electrophysiological or morphological properties of neurons expressing parvalbumin or calretinin. Retrograde tracing experiments revealed that both parvalbumin- and calretinin-containing neurons project nonpreferentially to the thalamus or tectum. In sum, the parvalbumin- and calretinin-containing GABAergic neurons of the substantia nigra pars reticulata cannot be differentiated on the basis of their electrophysiological properties, morphological properties, or target nuclei, and both parvalbumin- and calretinin-containing projection neurons issue local axon collaterals that arborize within the substantia nigra.

• GABAergic control of substantia nigra dopaminergic neurons.

  Authors: James M Tepper, Christian R Lee

  Progress in brain research. 02/2007; 160:189-208.

  At least 70% of the afferents to substantia nigra dopaminergic neurons are GABAergic. The vast majority of these arise from the neostriatum, the external globus pallidus and the substantia nigra parsAt least 70% of the afferents to substantia nigra dopaminergic neurons are GABAergic. The vast majority of these arise from the neostriatum, the external globus pallidus and the substantia nigra pars reticulata. Nigral dopaminergic neurons express both GABA(A) and GABA(B) receptors, and are inhibited by local application of GABA(A) or GABA(B) agonists in vivo and in vitro. However, in vivo, synaptic responses elicited by stimulation of neostriatal or pallidal afferents, or antidromic activation of nigral pars reticulata GABAergic projection neurons are mediated predominantly or exclusively by GABA(A) receptors. The clearest and most consistent role for the nigral GABA(B) receptor in vivo is as an inhibitory autoreceptor that presynaptically modulates GABA(A) synaptic responses that originate from all three principal GABAergic inputs. The firing pattern of dopaminergic neurons is also effectively modulated by GABAergic inputs in vivo. Local blockade of nigral GABA(A) receptors causes dopaminergic neurons to shift to a burst firing pattern regardless of the original firing pattern. This is accompanied by a modest increase in spontaneous firing rate. The GABAergic inputs from the axon collaterals of the pars reticulata projection neurons seem to be a particularly important source of a GABA(A) tone to the dopaminergic neurons, inhibition of which leads to burst firing. The globus pallidus exerts powerful control over the pars reticulata input, and through the latter, disynaptically over the dopaminergic neurons. Inhibition of pallidal output leads to a slight decrease in firing of the dopaminergic neurons due to disinhibition of the pars reticulata neurons whereas increased firing of pallidal neurons leads to burst firing in dopaminergic neurons that is associated with a modest increase in spontaneous firing rate and a significant increase in extracellular levels of dopamine in the neostriatum. The pallidal disynaptic disinhibitory control of the dopaminergic neurons dominates the monosynaptic inhibitory influence because of a differential sensitivity to GABA of the two nigral neuron types. Nigral GABAergic neurons are more sensitive to GABA(A)-mediated inhibition than dopaminergic neurons, in part due to a more hyperpolarized GABA(A) reversal potential. The more depolarized GABA(A) reversal potential in the dopaminergic neurons is due to the absence of KCC2, the chloride transporter responsible for setting up a hyperpolarizing Cl(-) gradient in most mature CNS neurons. The data reviewed in this chapter have made it increasingly clear that in addition to the effects that nigral GABAergic output neurons have on their target nuclei outside of the basal ganglia, local interactions between GABAergic projection neurons and dopaminergic neurons are crucially important to the functioning of the nigral dopaminergic neurons.

• Cell type-specific differences in chloride-regulatory mechanisms and GABA(A) receptor-mediated inhibition in rat substantia nigra.

  Authors: Alexandra Gulácsi, Christian R Lee, Attila Sík, Tero Viitanen, Kai Kaila, James M Tepper, Tamás F Freund

  The Journal of neuroscience : the official journal of the Society for Neuroscience. 10/2003; 23(23):8237-46.

  The regulation of intracellular chloride has important roles in neuronal function, especially by setting the magnitude and direction of the Cl- flux gated by GABA(A) receptors. Previous studies haveThe regulation of intracellular chloride has important roles in neuronal function, especially by setting the magnitude and direction of the Cl- flux gated by GABA(A) receptors. Previous studies have shown that GABA(A)-mediated inhibition is less effective in dopaminergic than in GABAergic neurons in substantia nigra. We studied whether this phenomenon may be related to a difference in Cl-regulatory mechanisms. Light-microscopic immunocytochemistry revealed that the potassium-chloride cotransporter 2 (KCC2) was localized only in the dendrites of nondopaminergic (primarily GABAergic) neurons in the substantia nigra, whereas the voltage-sensitive chloride channel 2 (ClC-2) was observed only in the dopaminergic neurons of the pars compacta. Electron-microscopic immunogold labeling confirmed that KCC2 is localized in the dendritic plasma membrane of GABAergic neurons close to inhibitory synapses. Confocal microscopy showed that ClC-2 was selectively expressed in the somatic and dendritic cell membranes of the dopaminergic neurons. Gramicidin-perforated-patch recordings revealed that the GABA(A) IPSP reversal potential was significantly less negative and had a much smaller hyperpolarizing driving force in dopaminergic than in GABAergic neurons. The GABA(A) reversal potential was significantly less negative in bicarbonate-free buffer in dopaminergic but not in GABAergic neurons. The present study suggests that KCC2 is responsible for maintaining the low intracellular Cl- concentration in nigral GABAergic neurons, whereas a sodium-dependent anion (Cl--HCO3-) exchanger and ClC-2 are likely to serve this role in dopaminergic neurons. The relatively low efficacy of GABAA-mediated inhibition in nigral dopaminergic neurons compared with nigral GABAergic neurons may be related to their lack of KCC2.