Kinetics of two voltage-gated K+ conductances in substantia nigra dopaminergic neurons

BR A IN RE S E A RCH 1 1 73 ( 20 0 7 ) 2 7 –3 5

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

Kinetics of two voltage-gated K + conductances in substantia nigra dopaminergic neurons Dekel Segev, Alon Korngreen⁎ The Mina & Everard Goodman Faculty of Life Sciences and the Susan & Leslie Gonda Multidiciplinary Brain Research Center, Bar-Ilan University, Ramat-Gan, Israel

A R T I C LE I N FO

AB S T R A C T

Article history:

The substantia nigra (SN) is part of the basal ganglia which is a section in the movement circuit in

Accepted 2 August 2007

the brain. Dopaminergic neurons (DA) constitute the bulk of substantia nigra neurons and are

Available online 9 August 2007

related to diseases such as Parkinson's disease. Aiming at describing the mechanism of action potential firing in these cells, the current research examined the biophysical characteristics of

Keywords:

voltage-gated K+ conductances in the dopaminergic neurons of the SN. The outside-out

K+ channel

configuration of the patch-clamp technique was used to measure from dopaminergic neurons

Substantia nigra

in acute brain slices. Two types of K+ voltage-gated conductances, a fast-inactivating A-type-like

Dopaminergic neuron

K+ conductance (Kfast) and a slow-inactivating delayed rectifier-like K+ conductance (Kslow), were

Patch-clamp

isolated in these neurons using kinetic separation protocols. The data suggested that a fast-

Kinetic modeling

inactivating conductance was due to 69% of the total voltage-gated K+ conductances, while the

Action potential

remainder of the voltage-gated K+ conductance was due to the activation of a slow-inactivating K+ conductance. The two voltage-gated K+ conductances were analyzed assuming a Hodgkin– Huxley model with two activation and one inactivation gate. The kinetic models obtained from this analysis were used in a numerical simulation of the action potential. This simulation suggested that Kfast may be involved in the modulation of the height and width of action potentials initiated at different resting membrane potentials while Kslow may participate in action potential repolarization. This mechanism may indicate that SN dopaminergic neurons may perform analog coding by modulation of action potential shape. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

The substantia nigra (SN) is a nucleus in the basal ganglia that is part of the movement circuit (Brodal, 2004; Häusser et al., 1995; Lin et al., 2003). The dopaminergic neurons of the SN are implicated in many different brain disorders. Thus, they are related to the symptoms of Parkinson's disease and schizophrenia, and lately their involvement in attentiveness disorders and hyperactivity has been suggested (Marsden, 2006). The DA neurons of the SN produce both spontaneous pacemaker action

potentials and respond to rapid synaptic input by generating action potential bursts. The control of these two different modes is important and is possibly part of the SN role in the movement circuit (Komendantov et al., 2004; Overton and Clark, 1997). Furthermore, the width of the action potential of the DA neurons has been shown to be sensitive to changes in the resting membrane potential (Nedergaard, 1999). In order to produce these complex firing modes a cellular array of voltagegated ion channels is required. Indeed, many voltage-gated ion channels have been identified in SN dopaminergic neurons

⁎ Corresponding author. Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 52900, Israel. Fax: +972 3 5352184. E-mail address: [email protected] (A. Korngreen). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.08.006

28

BR A IN RE S EA RCH 1 1 73 ( 20 0 7 ) 2 7 –35

(Komendantov et al., 2004; Nedergaard, 2004; Neuhoff et al., 2002; Sarpal et al., 2004; Scroggs et al., 2001; Silva et al., 1990; Wolfart et al., 2001; Wolfart and Roeper, 2002; Yung et al., 1991). A partial list of these conductances includes several voltagegated Ca2+ conductances (Cardozo and Bean, 1995; Durante et al., 2004; Mercuri et al., 1994; Nedergaard et al., 1993) that have been recently shown to be involved in the spontaneous firing of DA neurons (Puopolo et al., 2007), two calcium-activated K+ conductances (Silva et al., 1990), two voltage-gated K+ conductances (Silva et al., 1990) and a hyperpolarization activated cation conductance (Ih) (Mercuri et al., 1995; Silva et al., 1990). Interestingly, unlike in other pacemaker neurons, in DA neurons Ih is not responsible for spontaneous firing that appears to be regulated by K+ conductances (Liss et al., 2001) and subthreshold activity of Na+ and Ca2+ conductances (Puopolo et al., 2007). While several suggestions of the underlying mechanism of spontaneous and evoked firing of DA neurons have been made the mechanism remains unclear, probably due to the involvement of many voltage-gated ion channels. Arriving at a cellular mechanism requires providing a kinetic model for all the voltage-gated conductances expressed in DA neurons and combining these kinetics into a numerical model (Hodgkin and Huxley, 1952). Here we applied the outside-out configuration of the patch-clamp technique (Hamill et al., 1981) to record voltage-gated K+ currents from DA neurons. These currents were subjected to kinetic analysis using Hodgkin–Huxley formalism (Hodgkin and Huxley, 1952) and the resulting models were used to explain the function of the K+ conductances in the AP of DA neurons.

2.

Vectastatin ABC kit and DAB staining to reveal their morphology (Fig. 1C). Visual inspection verified that the origin of the axon was from the dendrites (Fig. 1C), a typical characteristic of SN dopaminergic neurons (Häusser et al., 1995; Tepper et al., 1987). It has been reported that SN dopaminergic neurons express several voltage- and calcium-gated K+ conductances (Komendantov et al., 2004; Nedergaard, 2004; Neuhoff et al., 2002; Sarpal et al., 2004; Scroggs et al., 2001; Silva et al., 1990; Wolfart et al., 2001; Wolfart and Roeper, 2002; Yung et al., 1991). In the research reported here we focused on the kinetic analysis of voltage-gated K+ conductances. Due to the no spherical shape

Results

While the similar morphological characteristics of dopaminergic and GABAergic neurons in the acute brain slice preparation of the SN preclude visual differentiation between the two populations it is possible to distinguish between them using electrophysiological properties (Häusser et al., 1995; Häusser and Yung, 1994). The dopaminergic neurons comprise 85% of the neurons in the SN (Yung et al., 1991) and have a wide action potential (∼1.3 ms) (Nedergaard, 2004; Yung et al., 1991), an afterhyperpolarization with a large time constant and a prominent sag (Häusser et al., 1995; Yung et al., 1991). This is contrary to the GABAergic neurons that display a sharper AP and less sag (Häusser et al., 1995). Therefore, we performed several tests for each cell recorded in this study in order to identify dopaminergic neurons. In each experiment, the SN was imaged using IR-DIC and large cells, which could have been either dopaminergic or GABAergic, were identified (Fig. 1A). A patch-clamp pipette was attached to these cells in the whole-cell configuration of the patch-clamp technique and the response of the cell to current injection was recorded (Fig. 1B). Cells that displayed prominent sag following hyperpolarizing current injection and relatively broad action potentials (Häusser et al., 1995; Häusser and Yung, 1994; Richards et al., 1997; Yung et al., 1991) were selected (Fig. 1B) and outside-out patches were extracted from the soma of these cells. In addition, some cells were filled, using the whole-cell configuration, with biocytin and fixated post experimentally in paraformaldehyde. These cells were later stained using the

Fig. 1 – Identification of dopaminergic neurons in the SN. (A) A dopaminergic neuron from the SN with a patch-pipette attached in the whole-cell mode viewed through an infrared differential interference contrast microscope. (B) Response of a dopaminergic neuron to current injection. The prominent sag and relatively broad APs indicate that this neuron was indeed a dopaminergic neuron. (C) The same neuron after filling and staining with biocitin and fixing the brain slice with moviol. The origin of the axon is indicated by an arrow. The picture was taken using a light microscope.

BR A IN RE S E A RCH 1 1 73 ( 20 0 7 ) 2 7 –3 5

29

of DA neurons that contain a large dendritic tree any wholecell voltage-clamp experiment will suffer from severe lack of space clamp (Häusser, 2003; Schaefer et al., 2003, 2007). Thus, all experiments were carried out using excised patches. Following patch extraction, voltage-gated Na+ and Ca2+ conductances were blocked by bath application 120 nM TTX and 200 μM Cd2+ respectively. A representative experiment displaying the effect of Cd2+ on the recorded currents is shown in Fig. 2A. As expected, blocking voltage-gated Ca2+ conductances reduced the outward current induced by a 300 ms voltage-clamp command from −100 mV to +60 mV by 32± 10% (n = 4) probably due to the reduction in the activity of calcium activated K+ conductances (Fig. 2A). Thus, Cd2+ blocks voltage-gated Ca2+ currents that generate inward Ca2+ currents. This block reduces the inward Ca2+ current and therefore reduces the opening probability of Ca2+ activated K+ currents. Following this procedure the remaining current, displaying voltage-dependent activation and inac-

Fig. 3 – Steady-state inactivation of outward K+ current in outside-out patches. (A) Steady-state inactivation of voltage-gated K+ currents. Membrane potential was held for 2700 ms at voltages ranging from − 100 to 0 mV in 5 mV increments (the pre-pulse was truncated to facilitate the display of the outward currents of which only every second trace is displayed for clarity) and then depolarized to +60 mV. Sampled at 5 kHz and filtered at 2 kHz. Leak was subtracted on-line using a P/6 protocol. (B) The peak current at +60 mV following a given voltage step was normalized to the maximal current at +60 mV following a step from −100 mV. The steady-state inactivation curve was calculated from 7 experiments similar to that displayed in panel A. The smooth line is a curve fit of a sum of two Boltzmann functions (Eq. (2)). Error bars are SEM. Fig. 2 – Voltage-dependent K+ currents in outside-out patches. (A) Outward currents recorded following a voltage-clamp step from − 100 to +50 mV in the presence of 120 nM TTX (top trace). The amplitude of the current decreased after bath application of 200 μM Cd2+ to block voltage-gated Ca2+ conductances and subsequently calcium dependent K+ conductances (lower trace). (B) Voltage-dependent K+ current measured in response to a series of voltage steps from −100 to +50 mV in 10 mV increments following block of voltage-gated Ca2+ and Na+ currents. Sampled at 10 kHz and filtered at 5 kHz.

tivation, was surmised to result from the activation of voltagegated K+ conductances alone (Fig. 2B). To avoid using channel blockers that may affect more than one K+ conductance we first tested whether it was possible to identify and isolate components of the total K+ current using voltage-clamp protocols (Gurkiewicz and Korngreen, 2006; Korngreen et al., 2005; Korngreen and Sakmann, 2000). The steadystate inactivation of the K+ current was measured using a voltageclamp protocol in which the voltage was held for 2700 ms at various levels and then stepped to +60 mV to record the K+ current

30

BR A IN RE S EA RCH 1 1 73 ( 20 0 7 ) 2 7 –35

Fig. 4 – Kinetic separation of two voltage-gated K+ conductances. (A) Outward currents recorded from an outside-out patch. The voltage protocol of a 400 ms pulse to −110 mV followed by a 90 ms pulse to voltages between −80 and + 80 mV at 10 mV increments is shown below the current traces. The −110 mV pre-pulse was truncated to facilitate the display of the outward current. Sampled at 20 kHz and filtered at 5 kHz. Leak was subtracted on-line using a P/6 protocol. The scale bars apply also to panels B and C. (B) Outward currents from the same nucleated patch as in panel A. The voltage protocol was similar to that in A except for a 60 ms pulse to −50 mV inserted after the −110 mV step to inactivate the fast K+ current. The voltage protocol is shown below the current traces. (C) The difference current between the recording shown in panel A and that shown in panel B. (D) Activation curves of the slow K+ current isolated in panel B ( ) and the fast K+ current isolated in panel C ( ). The conductance was calculated from the maximal current amplitude that was divided by the K+ ion driving force calculated from the Nernst equation and normalized to the maximal conductance in a given series of voltages. The smooth lines are the fits to a Boltzmann function describing a Hodgkin–Huxley-like model with two gates (n = 6). Error bars are ± SEM.

•

(Fig. 3A). The K+ current activated immediately after the onset of depolarization was first to inactivate followed by the inactivation of a slower current component (Fig. 3A). The steady-state inactivation curve (Fig. 3B), constructed by measuring the peak of the activated current, displayed a dependence on the membrane potential that could be fit to a sum of two Boltzmann functions assuming a single inactivation gate (Eq. (2)). One Boltzmann distribution had a V1/2 of −80.1±0.6 mV and k=6.8±0.5 mV with a relative contribution of 69±3% to the total current and the other curve had a V1/2 of −19.±4 mV and k=16±1 mV (n=7). The steady-state inactivation curve predicted that the recorded K+ current was the result of the activation of two voltage-gated conductances. The large difference between the inactivation V1/2 of the two conductances enabled the design of a voltage-clamp protocol that separated between the two conductances. The total K+ current was recorded using a series of depolarizing voltage-clamp commands (Fig. 4A). Following a

○

60 ms pre-pulse to −50 mV only a sustained K+ current was activated (Fig. 4B). This delayed-rectifier-like current will hereafter be referred to as Kslow. Subtraction of this current from the total current (Fig. 4A) revealed a fast-inactivating K+ voltagegated current (Fig. 4C). This A-type like current will be referred to as Kfast. The steady-state activation curves for Kslow and Kfast were calculated by dividing the maximal current recorded during each depolarizing voltage-clamp command by the driving force. We observed, both during this stage of the analysis and later during the kinetic analysis, that the activation of the two currents was best described by a two-gate Hodgkin–Huxley type model (Hodgkin and Huxley, 1952). Therefore, the steady-state activation curves were fit using a Boltzmann function (Eq. (2)) raised to the power of two. This procedure revealed the voltage of halfactivation (V1/2) and inverse slope (k) of the two K+ conductances. For Kslow we obtained V1/2 = −31± 3 mV and k =23± 1 mV (n =6) and for Kfast V1/2 = −39 ± 3 mV and k= 24± 2 mV (n= 6).

BR A IN RE S E A RCH 1 1 73 ( 20 0 7 ) 2 7 –3 5

In parallel, we used the Hodgkin–Huxley formalism to analyze the kinetics of the two voltage-gated K+ conductances (Fig. 5). We tested several models and found the two-gate Hodgkin–Huxley-like model to fit the current traces best. The fit between the recorded current and the kinetic model is shown for two current traces of Kslow activated by voltageclamp commands to +20 and + 70 mV in Fig. 5A. This analysis was repeated on 6 more outside-out patches and the average voltage dependence of the activation time constant of Kslow, displaying a distorted bell shape curve, is shown in Fig. 5B. Similar kinetic analysis was performed for traces of Kfast (Fig. 5C) producing the average voltage dependence of the activation time constant of Kfast which is plotted in Fig. 5D. Due to the relatively small currents typically encountered in this study it was not possible to accurately measure the deactivation time constant for both Kslow and Kfast by tail current protocols. Finally, we analyzed the inactivation kinetics of both Kslow and Kfast (Fig. 6). The inactivation time constant was extracted by mono-exponential curve fitting to the decay phase of the current activated by a depolarizing voltage-clamp

31

command (analysis not shown). For Kfast, the inactivation time constant was measured also at potentials below the threshold of channel activation using recovery-from-inactivation protocols (Fig. 6A). Interestingly, the inactivation time constant was not dependent on voltage both for Kfast (Fig. 6B) and for Kslow (Fig. 6C). To try and understand the role of the two K+ conductances we carried out numerical simulations. SN dopaminergic neurons contain many voltage-gated ion channels. For many of these channels a good kinetic description is lacking. Moreover, the dendrites of these neurons display active propagation of the action potential and the action potential initiation zone is located in the dendrites due to the dendritic origin of the axon (Häusser et al., 1995). Thus, it is currently not possible to provide a full compartmental model of these neurons without risking introducing a bias into the model. Instead of building a full compartmental model we investigated the activation of the two K+ conductances isolated in this study by simulating the activation of these conductances by an action potential voltageclamp command. The activation and inactivation kinetics described in the previous figures were converted to a computer code

Fig. 5 – Activation kinetics of the two voltage-dependent K+ conductances. (A) Representative traces generated by voltage-clamp commands to + 20 and + 70 mV showing the activation and kinetics of Kslow. Currents were filtered at 10 kHz and sampled at 50 kHz and the leak was subtracted on-line using a P/6 protocol. The activation of Kslow was fit to a two-gate Hodgkin–Huxley model (smooth lines). (B) The activation time constant calculated as displayed in A from 6 patches was fit to the equation t = 0.65 + 4.3 / (2.7exp(45V) + 0.018exp(620V)). (C) Similar to A only for Kfast. (D) Similar to B only for Kfast. The activation time constant was fit to the equation t = 0.55 + 0.52exp(−40.6V).

32

BR A IN RE S EA RCH 1 1 73 ( 20 0 7 ) 2 7 –35

using the NMODL extension of the simulation environment NEURON (Hines and Carnevale, 1997, 2000) and the models of the two channels were inserted into a single compartment neuron. This point neuron was voltage-clamped using an action potential train recorded from the soma of SN dopaminergic neurons (Fig. 7A). The activation of Kfast during the AP train is displayed in Fig. 7B. The conductance was partially active at the

resting membrane potential and increased during each AP. Due to inactivation the fraction of conductance that was activated gradually decreased during the depolarization leading to smaller activation of the conductance during each AP in the train (Fig. 7B). Contrary to this, the activation of Kslow increased during the AP train (Fig. 7C) corresponding to the increase in the width of the AP (Fig. 7A).

3.

Discussion

In this study we have used pharmacological and kinetic isolation protocols in order to dissect the kinetics of two voltage-gated K+ conductances in SN dopaminergic neurons. Using the large difference between the inactivation V1/2 (Fig. 3) it was possible to design a voltage-clamp protocol for the separation of the two conductances (Fig. 4). Kinetic analysis assuming a Hodgkin– Huxley-like model with two gates provided a quantitative description of the activation process of the two conductances (Fig. 5). The inactivation process of the two conductances was not dependent on the membrane potential (Fig. 6). Finally, numerical simulations, using the extracted kinetics, revealed the activation pattern of the two isolated conductances during a train of action potentials (Fig. 7). Electrophysiological investigations have identified many voltage-gated ion channels in SN dopaminergic neurons (Cardozo and Bean, 1995; Durante et al., 2004; Komendantov et al., 2004; Mercuri et al., 1994; Nedergaard, 2004; Nedergaard et al., 1993; Neuhoff et al., 2002; Sarpal et al., 2004; Scroggs et al., 2001; Silva et al., 1990; Wolfart et al., 2001; Wolfart and Roeper, 2002; Yung et al., 1991). In the current research, we focused on voltage-gated K+ channels since they influence, among others, action potential shape, burst firing caused by synaptic input and spontaneous pacemaker firing. After blocking sodium currents and calcium currents and as a result also calcium-dependent K+ currents, we identified two voltage-gated K+ conductances. It is important to note that the pacemaker activity of SN dopaminergic neurons is also highly influenced by Ca2+ dependent K+ conductances (Bennett et al., 2000; Kang and Kitai, 1993a,b; Kita et al., 1986; Wilson and Callaway, 2000). It would be of interest to combine the kinetics of the conductances we characterized here with those of other Fig. 6 – Voltage dependence of the inactivation of the voltage-gated K+ conductances. (A) Recovery from inactivation of K+ current in outside-out patches at −100 mV. The double-pulse protocol was used. Kfast was inactivated by depolarizing the patch for 400 ms to +60 mV following a 500 ms pulse to −110 mV (truncated). The patch was then hyperpolarized to − 110 mV for varying durations, and the relative recovery from inactivation was measured by a second step to +60 mV. Data sampled at 5 kHz and filtered at 2 kHz. Leak and capacitative currents were subtracted on-line using a P/6 protocol. (B) Time constant of inactivation of Kfast from recovery protocols ( , n = 5), and mono-exponential fits of voltage traces similar to that displayed in Fig. 4C (▲, n = 8) plotted as a function of voltage. (C) Time constant of inactivation of Kslow from mono-exponential fits of voltage traces similar to that displayed in Fig. 4B ( , n = 6) plotted as a function of voltage.

•

•

BR A IN RE S E A RCH 1 1 73 ( 20 0 7 ) 2 7 –3 5

Fig. 7 – Simulating the activation of Kslow and Kfast during an AP train. (A) An AP train recorded from the soma of a SN dopaminergic neuron in response to a 300 pA current injection. This AP train was used as a voltage-clamp command in a NEURON simulation. (B) Simulation of the activation of Kfast during the train of APs shown in A. (C) Simulation of the activation of Kslow during the train of APs shown in A.

currents that affect pacemaking in order to reach a better understanding of the physiology of SN dopaminergic neurons. Histological and immunological investigations observed a large number of different ion channels and/or subunits of those channels in SN dopaminergic neurons. Of these a significant fraction are subunits of Kv channels including Kv4.3 (Liss et al.,

33

2001; Yang et al., 2001), Kv1.4, (Chung et al., 2000; Lujan et al., 2003; Rhodes et al., 1997), Kv1.1, Kv1.2, Kv1.3, Kv1.5, Kv1.6 (Chung et al., 2000; Rhodes et al., 1997) and Kvβ1, Kvβ2 (Rhodes et al., 1997). This variety of channel subunits leads to the conclusion that native K+ channels are heteromers, containing probably both α and β subunits. The subunit composition greatly affects the properties of K+ channels. Therefore, it is not possible, at present, to derive the subunit composition of native K+ channels by comparison of their functional properties with those of cloned and heterologously expressed K+ channels. Using numerical simulations we were able to calculate the activation pattern of the two conductances by a train of action potentials (Fig. 7). In a typical AP train generated in SN dopaminergic neurons by current injection the amplitude and width of the APs increase during the train. Moreover, the waveform of the AP has been shown to be sensitive to the resting membrane potential (Nedergaard, 1999). The simulations have shown that the activation of Kfast was bigger when the APs were small and sharp and decreased as the APs grew taller and wider (Fig. 7B). Conversely, activation of Kslow was smaller when the APs were small and sharp and increased as the APs grew taller and wider (Fig. 7C). This suggested a possible functional role for the two conductances in modulation of the AP in SN dopaminergic neurons. Thus, Kfast modulates the height and participates in the repolarization phase of the AP whereas Kslow participates mainly in the repolarization phase of the AP. Currently, it is not possible to provide a more accurate description of the various phases of the AP since much kinetic information is missing especially on Ca2+ activated K+ conductances known to be expressed in these neurons. This has been nicely noted in the study carried out by Nedergaard (1999) where it has been shown that the AP waveform was modulated by block of voltage-gated Ca2+ conductances which probably reduced the activity of Ca2+ dependent K+ conductances. The modulation of the AP waveform in SN dopaminergic neurons by Kfast may have functional implication to the physiology of these neurons. It has been shown that the AP robustly back-propagates into the dendritic tree (Gentet and Williams, 2007; Häusser et al., 1995) and that Kfast is expressed in the dendrites although in a smaller density than at the soma (Gentet and Williams, 2007). Moreover, Kfast has been shown to be a target for biochemical modulation in SN dopaminergic neurons (Yang et al., 2001). Finally, it has been shown that dopamine is released from somatic and dendritic sites in SN dopaminergic neurons (Geffen et al., 1976). Therefore, it may be possible to suggest that the modulation of the AP waveform by Kfast may propagate into the dendrite and these alter the level of dendritic neurotransmitter release. Recent evidence has highlighted the role of A-type-like K+ conductances in the modulation of the AP waveform (Ali et al., 2007; Alle and Geiger, 2006; Geiger and Jonas, 2000; Korngreen et al., 2005; Shu et al., 2006) and subsequently in the release of neurotransmitters (Ali et al., 2007; Alle and Geiger, 2006; Geiger and Jonas, 2000). This additionally analog coding has been hypothesized to enhance the computational properties of neurons in several areas of the CNS (Clark and Häusser, 2006; Marder, 2006). It is tempting to speculate that the modulation of the AP waveform by Kfast in dopaminergic neurons may impact dopamine release at both dendritic and axonal locations leading to modulation of the coding of DA neurons. In this conjecture it is interesting to note

34

BR A IN RE S EA RCH 1 1 73 ( 20 0 7 ) 2 7 –35

that DA neurons have been implicated in the coding of reward probability and uncertainty (Fiorillo et al., 2003; Morris et al., 2004; Tobler et al., 2005). Therefore, additional analog coding of synaptic input by DA neurons may greatly enhance their ability to code such complex input patterns.

4.

Experimental procedures

4.1.

Slice preparation

Slices (coronal, 300 μm thick) were prepared from 18- to 22-dayold Wistar rats that were killed by rapid decapitation using previously described techniques (Häusser et al., 1995). Slices were perfused throughout the experiment with an oxygenated artificial cerebrospinal fluid (ACSF) containing: (mM) 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 25 Glucose (pH 7.4 with 5% CO2, 310 mosM/kg). All experiments were carried out at room temperature (22–25 °C). Dopaminergic neurons in the SN were visually identified using infrared differential interference contrast (IR-DIC) videomicroscopy (Stuart et al., 1993). The standard pipette solution used for outside-out recordings contained (mM): 125 K-gluconate, 20 KCl, 10 HEPES, 4 MgATP, 10 Na-phosphocreatine, 0.5 EGTA, 0.3 GTP (pH 7.2 with KOH, 312 mosM/kg). In all experiments, following the establishment of the outside-out configuration, 120 nM Tetrodotoxin and 200 μM Cd2+ were added to the bath solution to block voltage-gated Na+ and Ca2+ channels respectively. In some experiments 0.2% biocytin was added to the pipette solution. At the end of the experiment the slices were fixed in cold 100 mM phosphate buffer (PBS, pH 7.4) containing 4% paraformaldehyde. After fixation the slices were incubated for 2 h in avidin-biotinilated horseradish peroxidase (ABCElite, Vector-Labs, Peterborough, UK) and the stain was developed using diaminobenzidine.

4.2.

Analysis and simulations

All off-line data analysis including curve fitting was carried out with IGOR (WaveMetrics, Lake Oswego, USA) on a PC computer. Experimental results were observed in cells from two or more animals. Therefore, all the results for a particular experiment were pooled and displayed as means ± SEM. Groups were compared with two-way ANOVA. Current traces were analyzed assuming a Hodgkin–Huxley model (Hodgkin and Huxley, 1952). The activation and deactivation current traces were fit to the general equation: IðtÞ ¼

pffiffiffiffiffiffi pffiffiffiffiffiffi pffiffiffiffi n Il  n Il  n Io Þet=τ Þn

ð1Þ

where t is time, I∞ is the steady-state current, Io is the current at t=0, τ is the time constant of the exponential relaxation, and n is the number of gates in the model. Since Io is close to zero at the holding potential the above equation simplifies to: I(t)=I∞(1−e−t /τ )n. The normalized conductance was fit to a Boltzmann equation: G=Gmax ¼ 1=ð1 þ eðVV1=2 Þ=k Þn

ð2Þ

where G/Gmax is the conductance normalized to its maximal value, V is membrane potential, V1/2 is the voltage at which the conductance is half-maximal (for a single gate, n=1), and k is the slope factor.

All numerical simulations were carried out using NEURON (Hines and Carnevale, 1997). The kinetics of the voltage-gated K+ channels was expressed using the NMODL extension of NEURON (Hines and Carnevale, 2000). To avoid making assumptions regarding unknown properties of the axonal initiation zone of the AP we used experimentally recorded APs as voltage-clamp commands in a simulated single-compartment neuron to follow the conductance changes of each K+ conductance.

REFERENCES

Ali, A.B., Bannister, P.A., Thomson, A.M., 2007. Robust correlations between action potential duration and the properties of synaptic connections in layer 4 interneurones in juvenile and adult neocortical slices. J. Physiol. 580, 149–169. Alle, H., Geiger, J.R., 2006. Combined analog and action potential coding in hippocampal mossy fibers. Science 311, 1290–1293. Bennett, B.D., Callaway, J.C., Wilson, C.J., 2000. Intrinsic membrane properties underlying spontaneous tonic firing in neostriatal cholinergic interneurons. J. Neurosci. 20, 8493–8503. Brodal, P., 2004. THe Central Nervous System—Structure and Function, Third edn. Oxford University Press, New York. Cardozo, D.L., Bean, B.P., 1995. Voltage-dependent calcium channels in rat midbrain dopamine neurons: modulation by dopamine and GABAB receptors. J. Neurophysiol. 74, 1137–1148. Chung, Y.H., Shin, C.M., Kim, M.J., Cha, C.I., 2000. Immunohistochemical study on the distribution of six members of the Kv1 channel subunits in the rat basal ganglia. Brain Res. 875, 164–170. Clark, B., Häusser, M., 2006. Neural coding: hybrid analog and digital signalling in axons. Curr. Biol. 16, R585–R588. Durante, P., Cardenas, C.G., Whittaker, J.A., Kitai, S.T., Scroggs, R.S., 2004. Low-threshold L-type calcium channels in rat dopamine neurons. J. Neurophysiol. 91, 1450–1454. Fiorillo, C.D., Tobler, P.N., Schultz, W., 2003. Discrete coding of reward probability and uncertainty by dopamine neurons. Science 299, 1898–1902. Geffen, L.B., Jessell, T.M., Cuello, A.C., Iversen, L.L., 1976. Release of dopamine from dendrites in rat substantia nigra. Nature 260, 258–260. Geiger, J.R.P., Jonas, P., 2000. Dynamic control of presynaptic Ca2+ inflow by fast-inactivating K+ channels in hippocampal mossy fiber boutons. Neuron 28, 927–939. Gentet, L.J., Williams, S.R., 2007. Dopamine gates action potential backpropagation in midbrain dopaminergic neurons. J. Neurosci. 27, 1892–1901. Gurkiewicz, M., Korngreen, A., 2006. Recording, analysis, and function of dendritic voltage-gated channels. Pflugers Arch. 453, 283–292. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J., 1981. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers. Arch.-Eur. J. Physiol. 391, 85–100. Häusser, M., 2003. Revealing the properties of dendritic voltage-gated channels: a new approach to the space clamp problem. Biophys. J. 84, 3497–3498. Häusser, M.A., Yung, W.H., 1994. Inhibitory synaptic potentials in guinea-pig substantia nigra dopamine neurones in vitro. J. Physiol. 479 (Pt 3), 401–422. Häusser, M., Stuart, G., Racca, C., Sakmann, B., 1995. Axonal initiation and active dendritic propagation of action potentials in substantia nigra neurons. Neuron 15, 637–647.

BR A IN RE S E A RCH 1 1 73 ( 20 0 7 ) 2 7 –3 5

Hines, M.L., Carnevale, N.T., 1997. The NEURON simulation environment. Neural Comput. 9, 1179–1209. Hines, M.L., Carnevale, N.T., 2000. Expanding NEURON's repertoire of mechanisms with NMODL. Neural Comput. 12, 995–1007. Hodgkin, A.L., Huxley, A.F., 1952. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117, 500–544. Kang, Y., Kitai, S.T., 1993a. Calcium spike underlying rhythmic firing in dopaminergic neurons of the rat substantia nigra. Neurosci. Res. 18, 195–207. Kang, Y., Kitai, S.T., 1993b. A whole cell patch-clamp study on the pacemaker potential in dopaminergic neurons of rat substantia nigra compacta. Neurosci. Res. 18, 209–221. Kita, T., Kita, H., Kitai, S.T., 1986. Electrical membrane properties of rat substantia nigra compacta neurons in an in vitro slice preparation. Brain Res. 372, 21–30. Komendantov, A.O., Komendantova, O.G., Johnson, S.W., Canavier, C.C., 2004. A modeling study suggests complementary roles for GABAA and NMDA receptors and the SK channel in regulating the firing pattern in midbrain dopamine neurons. J. Neurophysiol. 91, 346–357. Korngreen, A., Sakmann, B., 2000. Voltage-gated K+ channels in layer 5 neocortical pyramidal neurones from young rats: subtypes and gradients. J. Physiol. 525, 621–639. Korngreen, A., Kaiser, K.M., Zilberter, Y., 2005. Subthreshold inactivation of voltage-gated K+ channels modulates action potentials in neocortical bitufted interneurones from rats. J. Physiol. 562, 421–437. Lin, J.Y., van Wyk, M., Bowala, T.K., Teo, M.Y., Lipski, J., 2003. Dendritic projections and dye-coupling in dopaminergic neurons of the substantia nigra examined in horizontal brain slices from young rats. J. Neurophysiol. 90, 2531–2535. Liss, B., Franz, O., Sewing, S., Bruns, R., Neuhoff, H., Roeper, J., 2001. Tuning pacemaker frequency of individual dopaminergic neurons by Kv4.3L and KChip3.1 transcription. EMBO J. 20, 5715–5724. Lujan, R., de Cabo de la Vega, C., Dominguez del Toro, E., Ballesta, J.J., Criado, M., Juiz, J.M., 2003. Immunohistochemical localization of the voltage-gated potassium channel subunit Kv1.4 in the central nervous system of the adult rat. J. Chem. Neuroanat 26, 209–224. Marder, E., 2006. Neurobiology: extending influence. Nature 441, 702–703. Marsden, C.A., 2006. Dopamine: the rewarding years. Br. J. Pharmacol. 147 (Suppl 1), S136–S144. Mercuri, N.B., Bonci, A., Calabresi, P., Stratta, F., Stefani, A., Bernardi, G., 1994. Effects of dihydropyridine calcium antagonists on rat midbrain dopaminergic neurones. Br. J. Pharmacol. 113, 831–838. Mercuri, N.B., Bonci, A., Calabresi, P., Stefani, A., Bernardi, G., 1995. Properties of the hyperpolarization-activated cation current Ih in rat midbrain dopaminergic neurons. Eur. J. Neurosci. 7, 462–469. Morris, G., Arkadir, D., Nevet, A., Vaadia, E., Bergman, H., 2004. Coincident but distinct messages of midbrain dopamine and striatal tonically active neurons. Neuron 43, 133–143. Nedergaard, S., 1999. Regulation of action potential size and excitability in substantia nigra compacta neurons: sensitivity to 4-aminopyridine. J. Neurophysiol. 82, 2903–2913. Nedergaard, S., 2004. A Ca2+-independent slow afterhyperpolarization in substantia nigra compacta neurons. Neuroscience 125, 841–852. Nedergaard, S., Flatman, J.A., Engberg, I., 1993. Nifedipine- and omega-conotoxin-sensitive Ca2+ conductances in guinea-pig substantia nigra pars compacta neurones. J. Physiol. 466, 727–747. Neuhoff, H., Neu, A., Liss, B., Roeper, J., 2002. Ih channels contribute to the different functional properties of identified dopaminergic subpopulations in the midbrain. J. Neurosci. 22, 1290–1302.

35

Overton, P.G., Clark, D., 1997. Burst firing in midbrain dopaminergic neurons. Brain Res. Brain Res. Rev. 25, 312–334. Puopolo, M., Raviola, E., Bean, B.P., 2007. Roles of subthreshold calcium current and sodium current in spontaneous firing of mouse midbrain dopamine neurons. J. Neurosci. 27, 645–656. Rhodes, K.J., Strassle, B.W., Monaghan, M.M., Bekele-Arcuri, Z., Matos, M.F., Trimmer, J.S., 1997. Association and colocalization of the Kvβ1 and Kvβ2 beta-subunits with Kv1 alpha-subunits in mammalian brain K+ channel complexes. J. Neurosci. 17, 8246–8258. Richards, C.D., Shiroyama, T., Kitai, S.T., 1997. Electrophysiological and immunocytochemical characterization of GABA and dopamine neurons in the substantia nigra of the rat. Neuroscience 80, 545–557. Sarpal, D., Koenig, J.I., Adelman, J.P., Brady, D., Prendeville, L.C., Shepard, P.D., 2004. Regional distribution of SK3 mRNA-containing neurons in the adult and adolescent rat ventral midbrain and their relationship to dopamine-containing cells. Synapse 53, 104–113. Schaefer, A.T., Helmstaedter, M., Sakmann, B., Korngreen, A., 2003. Correction of conductance measurements in non-space-clamped structures: 1. Voltage-gated K+ channels. Biophys. J. 84, 3508–3528. Schaefer, A.T., Helmstaedter, M., Schmitt, A.C., Bar-Yehuda, D., Almog, M., Ben-Porat, H., Sakmann, B., Korngreen, A., 2007. Dendritic voltage-gated K+ conductance gradient in pyramidal neurones of neocortical layer 5B from rats. J. Physiol. 579, 737–752. Scroggs, R.S., Cardenas, C.G., Whittaker, J.A., Kitai, S.T., 2001. Muscarine reduces calcium-dependent electrical activity in substantia nigra dopaminergic neurons. J. Neurophysiol. 86, 2966–2972. Shu, Y., Hasenstaub, A., Duque, A., Yu, Y., McCormick, D.A., 2006. Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential. Nature 441, 761–765. Silva, N.L., Pechura, C.M., Barker, J.L., 1990. Postnatal rat nigrostriatal dopaminergic neurons exhibit five types of potassium conductances. J. Neurophysiol. 64, 262–272. Stuart, G.J., Dodt, H.U., Sakmann, B., 1993. Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflügers. Arch.-Eur. J. Physiol. 423, 511–518. Tepper, J.M., Sawyer, S.F., Groves, P.M., 1987. Electrophysiologically identified nigral dopaminergic neurons intracellularly labeled with HRP: light-microscopic analysis. J. Neurosci. 7, 2794–2806. Tobler, P.N., Fiorillo, C.D., Schultz, W., 2005. Adaptive coding of reward value by dopamine neurons. Science 307, 1642–1645. Wilson, C.J., Callaway, J.C., 2000. Coupled oscillator model of the dopaminergic neuron of the substantia nigra. J. Neurophysiol. 83, 3084–3100. Wolfart, J., Roeper, J., 2002. Selective coupling of T-type calcium channels to SK potassium channels prevents intrinsic bursting in dopaminergic midbrain neurons. J. Neurosci. 22, 3404–3413. Wolfart, J., Neuhoff, H., Franz, O., Roeper, J., 2001. Differential expression of the small-conductance, calcium-activated potassium channel SK3 is critical for pacemaker control in dopaminergic midbrain neurons. J. Neurosci. 21, 3443–3456. Yang, F., Feng, L., Zheng, F., Johnson, S.W., Du, J., Shen, L., Wu, C.P., Lu, B., 2001. GDNF acutely modulates excitability and A-type K+ channels in midbrain dopaminergic neurons. Nat. Neurosci. 4, 1071–1078. Yung, W.H., Häusser, M.A., Jack, J.J., 1991. Electrophysiology of dopaminergic and non-dopaminergic neurones of the guinea-pig substantia nigra pars compacta in vitro. J. Physiol. 436, 643–667.