Pacemaker rate and depolarization block in nigral dopamine neurons: a somatic sodium channel balancing act - PubMed (original) (raw)

Pacemaker rate and depolarization block in nigral dopamine neurons: a somatic sodium channel balancing act

Kristal R Tucker et al. J Neurosci. 2012.

Abstract

Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that undergo depolarization (DP) block upon moderate stimulation. Understanding DP block is important because it has been correlated with the clinical efficacy of chronic antipsychotic drug treatment. Here we describe how voltage-gated sodium (Na(V)) channels regulate DP block and pacemaker activity in DA neurons of the substantia nigra using rat brain slices. The distribution, density, and gating of Na(V) currents were manipulated by blocking native channels with tetrodotoxin and by creating virtual channels and anti-channels with dynamic clamp. Although action potentials initiate in the axon initial segment and Na(V) channels are distributed in multiple dendrites, selective reduction of Na(V) channel activity in the soma was sufficient to decrease pacemaker frequency and increase susceptibility to DP block. Conversely, increasing somatic Na(V) current density raised pacemaker frequency and lowered susceptibility to DP block. Finally, when Na(V) currents were restricted to the soma, pacemaker activity occurred at abnormally high rates due to excessive local subthreshold Na(V) current. Together with computational simulations, these data show that both the slow pacemaker rate and the sensitivity to DP block that characterizes DA neurons result from the low density of somatic Na(V) channels. More generally, we conclude that the somatodendritic distribution of Na(V) channels is a major determinant of repetitive spiking frequency.

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Figures

Figure 1.

Sodium channel and SN DA neuron model descriptions. A, Comparison of the peak NaV current/voltage response measured from SN DA neuron nucleated patches by Seutin and Engel (2010) (circles) and the modeled NaV current/voltage response based on fitting those data (squares and line). B, Steady-state inactivation (_h_∞; solid line) and activation (_m_∞3; dashed line) curves of the model NaV current used in both dynamic clamp and modeling experiments. C, SN DA neuron compartmental model description. Left, Morphology of modeled SN DA neuron. Soma and distal dendritic recording sites are located by arrows. No axon is shown. Middle, Somatic currents flowing during the interspike interval (ISI). IKv = composite voltage-gated potassium currents, Ib = sum of constitutively active GIRK current and sodium leak current, _I_Cav,L = L-type calcium current, _I_Nav = TTX-sensitive voltage-gated sodium current, and _I_Axial = current flowing out of the soma into the central compartment of the dendrite. Right, Simulated pacing activity of the model neuron as measured from the soma (top trace) and a single AP (bottom trace). Scale indicated below each trace.

Figure 2.

Inward currents of the AIS and soma are reduced during DP block induction. Representative recordings from SN DA neurons current-clamped at −60 mV followed by (A) a 2 s, 250 pA current injection and (B) a 5 s dynamic clamp application of 30 nS of virtual NMDAR conductance to induce DP block. C, The first, fourth, and last APs (top of the example trace in A) and their corresponding dV/dt (middle) and phase plane (bottom) plots. D, The APs (top) in the first position, at 1 s of stimulation, at 2 s of stimulation, and in the last position of the example trace in B and their corresponding dV/dt (middle) and phase plane (bottom) plots. The arrows in C indicate the peak net current flowing during the AIS and somatic portions of the AP. An upward deflection indicates a net inward current and a downward deflection indicates a net outward current. Peak AIS and somatic dV/dt for (D) current injection and (F) virtual NMDA-induced DP block. Error bars indicate the mean ± SEM (for current injection n = 9; for NMDA n = 7).

Figure 3.

Reduction of AIS and somatic NaV current increases susceptibility to DP block. A, Representative APs (top) from a SN pars compacta DA neuron current-clamp recording in response to a 75 pA current injection before (control) and 20 min after 10 n

TTX application (10 n

TTX) and their corresponding dV/dt (middle) and phase plane (bottom) plots. B, Representative current-clamp recordings before (control) and after 10 n

TTX application (10 n

TTX) in response to a 5 s, 75 pA current injection during normal pacing. C, Resistance to DP block as quantified by length of activity divided by the stimulus length plotted against the stimulus amplitude before (circles; control) and after 10 n

TTX (squares) application. Symbols and error bars indicate the mean ± SEM for five cells.

Figure 4.

Addition of somatic NaV current decreases susceptibility to DP block induced by distributed submaximal NaV block. A, Representative APs (top) from an SN DA neuron current-clamp recording in response to a 75 pA current injection 20 min after 10 n

TTX application (10 n

TTX; Fig. 2_A_) and with 100 nS of virtual NaV added back to the soma with dynamic clamp (10 n

TTX + 10 nS gNaV) and their corresponding dV/dt (middle) and phase plane (bottom) plots. B, Current-clamp recording from the same cell represented in Figure 2_B_ after 10 n

TTX application with the addition of 100 nS (top) and 200 nS (bottom) of virtual NaV channel conductance applied throughout recording and responses to 5 s, 75 pA current injections. C, Resistance to DP block as quantified by length of activity divided by the stimulus length plotted against the stimulus amplitude after 10 n

TTX alone (squares; Fig. 2_C_), with the addition of 100 nS (open circles), and 200 nS (triangles) of virtual NaV channel conductance. Symbols and error bars indicate the mean ± SE of 3–5 cells.

Figure 5.

Additional somatic NaV channels reduce DP block produced by current injection and virtual NMDA conductance in adolescent and older animals. Representative recordings from SN DA neurons current-clamped at −60 mV followed by (A) a 2 s, 250 pA current injection and (B) a 5 s dynamic clamp application of 30 nS of virtual NMDAR conductance, to induce DP block under control conditions (top) and with 100 nS (middle) or 200 nS (bottom) of virtual NaV added via dynamic clamp. Resistance to DP block as quantified by length of activity divided by the stimulus length plotted against the virtual NaV conductance in response to a (C) current injection and (D) virtual NMDA conductance. Current injections were 250 pA for SN DA neurons in slices from P14–P21 rats (open circles; n = 11) and 200 pA current injection to SN DA neuron in slices from a P42 rat (black square; n = 3). NMDA conductance (30 nS) was used to stimulate SN DA neurons in slices from P14–P21 (open circles; n = 12) and P42 (black squares; n = 3) rats. Symbols and error bars indicate the mean ± SEM.

Figure 6.

Addition of virtual anti-NaV channels to the soma hastens DP block. The dynamic clamp technique was used to add 100 nS of virtual anti-NaV conductance to the soma of an SN DA neuron while being current-clamped to −60 mV followed by a 250 or 300 pA current step or 30 nS virtual NMDA conductance. A, Representative traces of the first AP evoked by 250 pA current injection (top) under control conditions (control) and 100 nS of virtual anti-NaV channel conductance (−100 nS gNaV) and their corresponding dV/dt (middle) and phase plane (bottom) plots. Representative current-clamp recordings in response to (B) a 300 pA current injection and (C) 30 nS of virtual NMDA conductance under control conditions (control) and in the presence of 100 nS virtual anti-NaV channel conductance (−100 nS gNaV).

Figure 7.

NaV current regulates the slow pacemaker rate of SN DA neurons. Representative current-clamp recordings (A–C, top) from freely pacing SN DA neurons to which (A) 200, (B) 100, and (C) −100 nS of virtual NaV conductance have been added using dynamic clamp. The bottom graphs of A–C indicate the dynamic clamp current. D, Linear regression of the pacing frequency in response to different virtual NaV conductance levels. E, Second and third AP (top) and corresponding virtual NaV current (bottom) during the 200 nS gNaV application (A) to illustrate the additional NaV current added during the ISI.

Figure 8.

Replacing the distributed native NaV current with virtual NaV current reconstitutes pacing, but at a higher frequency. A, Representative SN DA neuron current-clamp recording held at −60 mV followed by a 2 s long, 50 pA current injection before (pre-TTX; top trace) and after maximal block of TTX-sensitive sodium channels with 1 μ

TTX (second trace) followed by application of 600 nS (third trace) and 800 nS (fourth trace) of virtual NaV conductance to the soma. B, AP height and (C) pacing frequency before TTX application (Native) and after 10 min of 1 μ

TTX application with different levels of virtual NaV conductance. Error bars indicate the mean and SEM of 3–9 cells. D, Representative current-clamp recordings from a freely pacing neuron before and after 1 μ

TTX followed by reconstitution of pacing with 800 nS of virtual NaV conductance.

Figure 9.

Somatic isolation of NaV current results in abnormally fast pacing in an SN DA neuron compartmental model. The simulation paralleled the experimental protocol shown in Figure 7. A, Simulated voltage traces with distributed and somatically isolated NaV in response to an 80 pA applied current pulse measured from the soma and dendrite. B, Diagram of NaV currents flowing in a simplified somatodendritic compartment with NaV channels distributed (left) or in the soma only (right). When NaV channels are distributed, Nav currents can uniformly activate Kv channels resulting in little axial current flow (left). When all of the gNaV is in the soma, the sodium current spreads axially, depolarizing the distal regions much less than the proximal regions, resulting in a proximal to distal Kv current gradient. Large arrows indicate axial current sink. C, Somatic currents flowing during the ISI of spikes 6 and 7 in the Soma Only gNaV condition in A. IKv = composite voltage-gated potassium currents, Ib = sum of constitutively active GIRK current and sodium leak current, _I_Cav,L = L-type calcium current, _I_Nav = TTX-sensitive voltage-gated sodium current, and _I_Axial = axial current. _I_Axial and _I_Nav have been plotted separately on the right graph with an expanded y_-axis scale. For comparison, see the middle panel of Figure 1_C for the ISI somatic currents flowing during the distributed gNaV condition.

Figure 10.

Simulations predict effects of NaV window current manipulations on spiking frequency. A, Steady-state inactivation and activation curves of the model NaV current (solid lines in each graph), a 5 mV right shift in the activation curve and a 5 mV left shift in the inactivation curve (m/h shift; dotted lines; top), a 5 mV right shift in the activation curve only (m shift; dotted line; middle), and a 5 mV left shift in the inactivation curve (h shift; dotted line; bottom) used in both dynamic clamp and modeling experiments. Shaded area of each inset indicates the resulting window current for each manipulation. B, Simulated activity of a model SN DA neuron held at −60 mV and stimulated with a 2 s, 80 pA current injection with somatically isolated NaV current with the indicated gating shifts.

Figure 11.

Subthreshold NaV activation is important for slow pacemaker activity when the channel is confined to the soma. A, Representative SN DA neuron current-clamp recordings held at −60 mV followed by a 2 s, 50 pA current injection after a 10 min 1 μ

TTX application and 800 nS of virtual NaV conductance (gNaV), 2000 nS of m/h shift (100 pA current step), 1200 nS of m shift, and 1200 nS of h shift as described for Figure 10. B, AP height and (C) frequency in response to the treatments described above. Error bars indicate the mean and SEM of 4–9 cells.

Figure 12.

Simulation predictions of frequency with differential somatodendritic NaV channel distribution. A, Simulated activity of model SN DA neuron held at −60 mV and stimulated with a 2 s, 80 pA current injection, and 845 nS of gNav was distributed across the 23.75 pF of the _C_m of the soma and the 191.31 pF _C_m of the dendrites at varying ratios. The traces in A, from top to bottom, have the following somatic/dendritic NaV current densities in nS/pF: Soma Only = 35.6/0, Soma 8× > Dendrite = 17.8/2.2, Soma 2.5× > Dendrite = 8.4/3.4, Soma = Dendrite = 3.9 uniformly distributed, and Dendrite Only = 0/4.4. B, Spiking frequency as a result of changing the percentage distribution of 845 nS of gNav between the soma and dendrites in the DA neuron compartmental model as demonstrated in A. C, Simulated current-clamp recordings from a pacing model neuron to which 200 nS of gNav was added to the soma (top), uniformly distributed (middle), or added to the dendrites only (bottom).

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Pacemaker rate and depolarization block in nigral dopamine neurons: a somatic sodium channel balancing act - PubMed (original) (raw)