Intrinsic and integrative properties of substantia nigra pars reticulata neurons

Abstract

The GABA projection neurons of the substantia nigra pars reticulata (SNr) are output neurons for the basal ganglia and thus critical for movement control. Their most striking neurophysiological feature is sustained, spontaneous high frequency spike firing. A fundamental question is: what are the key ion channels supporting the remarkable firing capability in these neurons? Recent studies indicate that these neurons express tonically active type 3 transient receptor potential (TRPC3) channels that conduct a Na-dependent inward current even at hyperpolarized membrane potentials. When the membrane potential reaches -60 mV, a voltage-gated persistent sodium current (I(NaP)) starts to activate, further depolarizing the membrane potential. At or slightly below -50 mV, the large transient voltage-activated sodium current (I(NaT)) starts to activate and eventually triggers the rapid rising phase of action potentials. SNr GABA neurons have a higher density of I(NaT), contributing to the faster rise and larger amplitude of action potentials, compared with the slow-spiking dopamine neurons. I(NaT) also recovers from inactivation more quickly in SNr GABA neurons than in nigral dopamine neurons. In SNr GABA neurons, the rising phase of the action potential triggers the activation of high-threshold, inactivation-resistant Kv3-like channels that can rapidly repolarize the membrane. These intrinsic ion channels provide SNr GABA neurons with the ability to fire spontaneous and sustained high frequency spikes. Additionally, robust GABA inputs from direct pathway medium spiny neurons in the striatum and GABA neurons in the globus pallidus may inhibit and silence SNr GABA neurons, whereas glutamate synaptic input from the subthalamic nucleus may induce burst firing in SNr GABA neurons. Thus, afferent GABA and glutamate synaptic inputs sculpt the tonic high frequency firing of SNr GABA neurons and the consequent inhibition of their targets into an integrated motor control signal that is further fine-tuned by neuromodulators including dopamine, serotonin, endocannabinoids, and H₂O₂.

Fig. 1

The substantia nigra pars reticulata (SNr) is a key output nucleus of the basal ganglia. (A) A Nissl-stained sagittal mouse brain section showing the location of SNr and other major components of the basal ganglia. Arrows indicate information flow directions. The SNr receives information from several components of the basal ganglia and sends output to the thalamus, superior colliculus (SC), and brainstem motor structures. Unpublished data of FMZ. (B) Confocal images of double immunohistochemical staining for tyrosine hydroxylase (TH, red, a key enzyme for dopamine synthesis) and parvalbumin (PV, green, expressed only in GABA neurons). Most TH-positive dopamine neurons are in SNc with their dendrites extending into SNr where most neurons are PV-positive GABA neurons. Modified from Zhou et al. 2009 with permission. (C) Examples of retrogradely labeled SNr neurons projecting to the superior colliculus (SC). Blue is a pseudocolor in this image for easier visualization. (D) Examples of retrogradely labeled SNr neurons projecting to the thalamus. C and D are modified from Lee and Tepper, 2007a with permission. Copyright 2006 Wiley-Liss, Inc.

Fig. 2

SNr GABA neurons fire sustained spontaneous high frequency spikes. (A) In intact primates, SNr GABA neurons fire tonic high frequency spikes. Modified from Schultz 1986 with permission. (B, C) In isolated preparations with fast synaptic inputs blocked and compared with nigral dopamine neurons, SNr GABA neurons still fire sustained high frequency spikes. In SNr GABA neurons, the I_h is weak whereas it is strong in nigral DA neurons as indicated by the arrow. Modified from Ding et al. 2011 with permission. (D) Spikes in SNr GABA neurons are larger in amplitude and shorter in duration than nigral DA neurons. (E, F, G) SNr GABA neurons have a higher density of the transient voltage-activated sodium current I_NaT than nigral DA neurons. E–G are modified from Seutin and Engel, 2010 with permission.

Fig. 3

TRP channels mediate a tonic inward current and depolarization in SNr GABA neurons. (A) After blocking Na_V channels with 1 µM TTX and under current clamp recording condition, bath application of 100 µM flufenamic acid (FFA) induced a hyperpolarization of about 10 mV (open arrow), indicating an FFA-sensitive tonic depolarization (depol., filled arrow). (B) When voltage clamped at −70 mV, 100 µM FFA induced an outward current (open arrow), or reduced a potential tonic inward current, as reflected by the apparent reduction of the holding current (filled arrow). (C) A linear voltage ramp from −90 mV to 10 mV was applied under control condition (black trace) and during 100 µM FFA application (blue trace). Digital subtraction (control – FFA) revealed the current inhibited by FFA (red trace) and its I–V relationship. The decreased current also indicates an increased input resistance or decreased whole cell conductance. (D) The FFA-inhibited current in C displayed at an enhanced scale. The I–V relationship is clearly linear with no signs of voltage-dependent activation or inactivation and reversed its polarity around −35 mV on average (intercept on X-axis in linear regression analysis as indicated by the black straight line). (E) Intracellular application of a TRPC3 antibody decreases the firing rate and increases the firing irregularity in SNr GABA neurons. Within the first minute of recording when the antibody was not likely to have diffused sufficiently into the cell, the firing was fast around 10 Hz and had a regular inter-spike interval (ISI). During the fifth minute of recording when a considerable amount of the antibody was likely to have diffused into the cell, the spontaneous firing became much slower and had an irregular ISI. Modified from Zhou et al., 2008 with permission.

Fig. 4

A robust, 1 mM TEA-sensitive I_DR-fast current is critical to the sustained high frequency firing in SNr GABA neurons. (A) Representative traces of Kv currents in a nucleated membrane patch isolated from a GABA neuron under control condition (A1), in the presence of 1 mM TEA (A2) and after washing out 1 mM TEA (A3). The 1 mM TEA-sensitive I_DR-fast was obtained by subtraction and displayed in A4. Holding potential was −100 mV, the first depolarizing step was to −80 mV and the last one was to +50 mV, and each step increment was 10 mV. (B) Inhibition of Kv3 channel-mediated I_DR-fast by 1 mM TEA impairs the sustained high frequency firing in SNr GABA neurons. An example SNr GABA neuron was able to fire sustained high frequency (around 45 Hz) fast spikes upon injection of 300 pA current for 10 s (B1). In the presence of 1 mM TEA that blocks I_DR-fast, the same 10 s 300 pA injection induced spike firing only for about 3 s or less (B2). Note the spike amplitude decreased progressively. The SNr neuron regained its sustained high frequency firing capability after washing out 1 mM TEA (B3). Modified from Ding et al. 2011 with permission.

Fig. 5

SNr GABA neurons have a calcium-activated plateau potential. (A) A representative SNr GABA neuron exhibiting a plateau potential that manifests as a prolonged membrane depolarization following a depolarizing current pulse delivered while the neuron is held hyperpolarized (black trace). The plateau potential is abolished in calcium-free conditions (red trace). (B) Another SNr GABA neuron exhibits a plateau potential under control conditions (black trace) that is abolished by nimodipine (10 µM; red trace) indicating the involvement of L-type calcium channels in plateau potential generation. (C) A SNr GABA neuron without a plateau potential under control conditions (black trace) is made to exhibit a plateau potential after activation of L-type calcium channels with Bay K 8644 (5 µM; red trace). (D) The plateau potential (black trace) is abolished in low-sodium conditions (red trace) suggesting that the conductance underlying the plateau potential is a calcium-activated nonselective cation conductance. (E) The plateau potential observed under control conditions (black trace) is abolished by the TRP channel blocker flufenamic acid (FFA; 200 µM; red trace). (F) A neuron not exhibiting a plateau potential under control conditions (black trace) exhibits a plateau potential in the presence of Bay K 8644 (blue trace). The evoked plateau potential is similarly abolished by FFA (red trace). Modified from Lee and Tepper, 2007b with permission. Copyright 2007 Society for Neuroscience.

Fig. 6

D1 receptor facilitation and CB1 receptor inhibition of the striatonigral axon terminals. (A) Immunostaining for D1 receptor protein reveals an intense expression of D1 receptors on striatonigral axons. Due to the curvature of the fibers in this saggital section, only part of the projection is captured in this image. AC: anterior commissure. Unpublished data of FMZ. (B) Selective photoactivation of ChR2-expressing MSN axons in SNr evoked IPSCs in SNr GABA neurons that was enhanced by a D1-like agonist SKF83822. This enhancing effect was blocked by the D1-like antagonist SCH23390. Adapted from Chuhma et al. 2011 with permission. (C) Immunostaining for CB1 receptor protein reveals an intense expression of CB1 receptors on striatonigral axons. Adapted from Fukudome et al. 2004 with permission. (D) The cannabinoid agonist WIN55212-2 (WIN) depressed striatum stimulation-evoked IPSCs in SNr GABA neurons and this effect was largely reversed by cannabinoid antagonist SR141716A (SR). PRE indicates pre-drug control. Adapted from Wallmichrath and Szabo, 2002b with permission.

Fig. 7

Striatonigral, pallidonigral, and nigrotectal synapses have different functional properties. The striatonigral synapse is facilitating (A), the pallidonigral synapse is depressing (B), and the nigrotectal synapse is sustainable or constant (C). A and B are adapted from Connelly et al. 2010 and C is adapted from Kaneda et al. 2008 with permission.

Fig. 8

Excitatory synaptic input from STN neurons affects the firing of SNr GABA neurons. STN stimulation evokes complex polysynaptic EPSCs under voltage clamp at −70 mV (A) and burst spike firing in SNr GABA neurons (B, C). Adapted from Shen and Johnson 2006 with permission.

Fig. 9

Direct dopamine excitation in SNr GABA neurons. All recordings were made in the presence of picrotoxin, D-AP5 and CNQX to block fast synaptic transmission. Sulpiride was present to prevent potential complications from D₂ autoinhibition of DA neurons. (A) Examples of spontaneous spikes under control conditions (left) and during 10 µM DA application (right). (B) Group data of DA enhancement of spiking. (C) An example recording showing that, after blocking spikes with 1 µM TTX, DA induced a clear depolarization. (D) An example recording showing that, when voltage clamped at −70 mV, DA induced an inward current in the presence of 1 µM TTX. Note that SNr GABA neurons are normally depolarized and have a large holding current when clamped at −70 mV. Modified from Zhou et al., 2009 with permission.

Fig. 10

Direct 5-HT excitation in SNr GABA neurons. (A) Immunostaining for 5-HT transporter protein reveals a very dense 5-HT axonal network that intermingles with PV-positive GABA neurons in SNr. (B) Endogenous 5-HT induces a tonic depolarization in SNr GABA neurons that is enhanced by blocking 5-HT re-uptake with 2 µM fluoxetine. Action potentials were blocked by 1 µM TTX. Ionotropic glutamate receptors and GABA_A receptors were blocked. (C) bath applied 5-HT induced a linear, voltage-independent inward current below action potential threshold. The whole cell conductance was increased during 5-HT application, indicating an opening of ion channels (e.g. TRPC3 channels), not a closing of background K channels. Note the remarkable similarity between this 5-HT current and the TRP current shown in Fig. 3C,D.A and B are unpublished data of FMZ. C is adapted from Stanford and Lacey, 1996a, with permission.

Fig. 11

Modulation of guinea-pig SNr GABA neuron firing rate by H₂O₂. (A) Spontaneous activity of a SNr GABA neuron under control conditions, in the presence of H₂O₂ (1.5 mM), and with flufenamic acid (FFA; 20 µM) in the continued presence of H₂O₂. (B) H₂O₂ causes an increase in SNr GABA neuron firing rate that is reversed by the TRP channel blocker FFA, with the resulting firing rate falling below control levels. (C) Depletion of endogenous H₂O₂ with catalase (Cat; 500 U/mL) results in a decrease in firing rate and an increase in the coefficient of variation indicating that basal H₂O₂ levels play a role in maintaining the tonic firing of SNr GABA neurons. (D) Spontaneous activity of another SNr GABA neuron under control conditions, in the presence of FFA, and with H₂O₂ in the continued presence of FFA. (E, F) Blockade of TRP channels with FFA causes a decrease in SNr GABA neuron firing rate. Addition of H₂O₂ when TRP channels are blocked results in a suppression of firing, indicating activation of a hyperpolarizing conductance by H₂O₂ (E). The H₂O₂-induced suppression of firing is prevented by the K_ATP channel blocker glibenclamide (Glib; 3µM) (F). *p < 0.05; **p < 0.01; ***p < 0.001. Modified from Lee et al., 2011 with permission.