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Laboratoire de Neurophysiologie et Nouvelles Microscopies, Institut National de la Santé et de la Recherche Médicale U603; Centre Nationale de la Recherche Scientifique FRE2500; Ecole Supérieure de Physique et de Chimie Industrielles; and Université Paris Descartes, Paris, France
Submitted 2 September 2005; accepted in final form 13 January 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). The expression of the Ih is correlated with the expression of the types 2 and 4 hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective (HCN2 and HCN4) mRNAs (Santoro et al. 2000). The relay neurons of the thalamic somatosensory nucleus ventralis basalis (nVB) receive an inhibitory GABAergic input from the adjacent nucleus reticularis thalami (nRT), contributing to an inhibitory feedback loop responsible for the spindle waves recorded during the early transition from waking to sleep (McCormick and Bal 1997; Steriade et al. 1993). The nRT neurons generate rhythmic bursts (Contreras et al. 1993), which involve low-threshold calcium (Ca2+) spikes and apamin-sensitive Ca2+-activated K+ currents (Bal and McCormick 1993). The nRT neurons express high levels of HCN2 mRNA, but surprisingly, unlike their nVB counterparts, they were shown to lack the Ih (Santoro et al. 2000).
Two reasons may explain the lack of a functional Ih in the nRT neurons. First, nonuniform and remote dendritically (Magee 1998) or axonally (Southan et al. 2000) expressed HCN2 subunits could generate a Ih undetected in leaky somatic recordings (Cathala and Paupardin-Tritsch 1999; Watts et al. 1996). Second, the association of a HCN subunit with an accessory 
subunit could modify the channel properties, for example, the co-expression of the HCN2 subunit with a MinK-related peptide 1 (MiRP1) subunit could generate an instantaneous Ih (Proenza et al. 2002) or a Ih with faster kinetics (Qu et al. 2004).
To test these possibilities, we performed recordings in conditions that reduce the leak conductance of the nRT neurons. In the presence of Ba2+ to inactivate inward rectifiers, we find that the nRT neurons express a hyperpolarization-activated slowly activating inward current that is sensitive to 2 mM cesium (Cs+) and to 50 µM ZD7288. Also the observed current is carried by K+ and Na+ ions and sensitive to the presence of 20 µM 8-bromo-cAMP (8-Br-cAMP) in the pipette. We also show that blocking the hyperpolarization-activated current with 2 mM Cs+, induce a hyperpolarization and an increase of the input resistance (Rin) of the nRT neurons. We conclude that the nRT neurons express a functional Ih that has hitherto been undetected and that contributes to the control of the nRT neuron excitability. Its slow kinetics indicate that the Ih is carried by an homomeric HCN2 channel.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The experiments were performed with newborn NMRI mice (P7–P14, Charles River, L’Arbresle, France), P0 being the day of birth in the local animal house. Some controls, where indicated, used older juvenile mice (P14–P23). All protocols followed the European Union and institutional guidelines. The animals were anesthetized by injection of pentobarbital (15 mg/kg ip) and decapitated. The brain was rapidly removed and placed in oxygenated (5% CO2-95% O2), cold (4°C), artificial cerebrospinal fluid (ACSF) with 10 mM lactate. The TC slices were cut (400 µm thickness at P7–P14; 350 µm at P16–P19) using a vibratome (VT1000S, Leica, Nussloch, Germany) following a procedure that maintained an intact TC connection (Agmon and Connors 1991) as described before (Laurent et al. 2002). The slices were maintained 30 min at 34°C and later at room temperature (RT, 22–24°C) in oxygenated standard ACSF.
Electrophysiological recording
The slices were placed in a small (1 ml) recording chamber, and perfused at 2 ml/min with ACSF at RT (22–24°C). Several control experiments were made near physiological temperature (32–34°C). The temperature was switched from RT to physiological temperature by circulating the perfusion tube through a tube filled with temperature-controlled water. Reaching the new temperature took near 3 min. The slices were maintained stable using a platinum ring. The recordings were performed in nVB and nRT under visual control on an upright fixed-stage microscope (Axioskop FS, Zeiss, Göttingen, Germany) with Nomarski optics and an infrared video camera (Newvicon C2400; Hamamatsu, Shizuoka, Japan). Somatic whole cell patch recordings were established using borosilicate pipettes having 4–5 M
resistance, and an Axopatch 200B amplifier (Axon Instruments, Sunnyvalle, CA). We compensated the series resistance and membrane capacitance during recordings (range: 40–60%). The voltage and current signals were filtered (1 kHz), digitized (0.53 or 1 kHz), and stored directly on the computer by a Labmaster TL-1 DMA acquisition board (Axon Instruments). The current- and voltage- protocols were generated using the program Acquis1 (Biologic, Claix, France). The Ih was generated in the voltage-clamp mode, maintaining the cell at –60 mV and applying negative voltage steps for 5 or 10 s between –70 and –130 mV with increments of –10 or –5 mV.
Data analysis
The data were analyzed off-line using Acquis1 and Igor Pro 4.1 (WaveMetrics, Portland, OR). The input resistance (Rin) of the neurons at resting membrane potential (RMP) was estimated in the current-clamp mode by injection of a small negative (–30 pA) current (I) of short-duration (500 ms) and measurement of the voltage change (U) at the end of the step and by using the Ohm’s law Rin = U/I.
The amplitude of the hyperpolarization-activated Ih reached a steady state at the end of the more negative (–130 mV) voltage steps, and it was measured as the difference between the amplitude of the so-called steady-state current (Iss) measured at the end of the voltage step, and the amplitude of the instantaneous current (Iinst) measured at the beginning of the voltage inward relaxation (Fig. 1). The current/voltage (I-V) curves of the Ih were obtained by plotting the amplitude of the Ih against the membrane potential (Vm) during the negative voltage step. The I-V curves were fitted with a sigmoid function: Amin + Amax/{1 + exp[(Vm – V
)/s]}, Amin being the minimal current amplitude, Amax the maximal current amplitude, V the half-activation voltage (in mV), and s the slope (in mV/pA). The time constants of the Ih activation were obtained by fitting the current response during the hyperpolarizing voltage step with a double-exponential function: Afast x exp(–t/
fast) + Aslow x exp(–t/slow), Afast and Aslow being the amplitude of the fast and the slow components, respectively, and fast and slow the fast and the slow time constants, respectively. The relative contribution of the fast component (rel Afast) was calculated as: Afast/Afast + Aslow. The weighted time constant of activation () was calculated as: fast x rel Afast + slow x (1 –rel Afast).
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Solutions and pharmacological compounds
Standard ACSF contained (in mM): 125 NaCl, 2.85 KCl, 1.25 KH2PO4, 1.5 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose, 298 ± 5 mosM. All drugs were supplied by Sigma (St. Louis, MO) unless otherwise specified. The following drugs were bath-applied : barium chloride (BaCl2, 1 mM), tetrodotoxin (TTX, 1 µM; Latoxan, Valence, France), cesium chloride (CsCl, 2 mM), nickel chloride (NiCl2, 100 µM), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 µM), 4-amino-pyridine (4-AP, 2 mM), 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288, 50 µM, Tocris Cookson, UK), 8-bromo-cAMP (8-Br-cAMP, 20 µM). KH2PO4 and MgSO4 were removed from the Ba2+-containing solutions and replaced by equimolar concentrations of KCl and MgCl2 respectively. The high K+ ACSF (15 mM) was obtained by replacing 11 mM NaCl by the equimolar concentration of KCl. The low Na+ ACSF (26 mM) was obtained by replacing 125 mM Na+ by N-methyl-D-glucamine (NMDG). The intracellular pipette solution contained (in mM): 132 Kgluconate, 5 NaCl, 2 MgCl2, 10 HEPES, 0.5 EGTA, 2 K2-ATP, and 0.2 Na-GTP; pH 7.35 adjusted with KOH. Potentials were corrected for –10-mV liquid junction potential.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Contreras et al. 1993; McCormick and Bal 1997; McCormick and Pape 1990). The high-frequency bursts of action potentials generated at the end of the hyperpolarizing current steps were superimposed on slower Ca2+ spikes. The tonic discharge of action potentials was induced by injection of depolarizing currents. The characteristics of the action potentials generated by depolarizing currents differed in nRT and nVB (Table 1): the action potential threshold was more negative in the nRT, its amplitude was larger, and its half-amplitude duration was shorter than in the nVB.
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To identify the voltage-gated currents that contribute to the anomalous rectification, we recorded the currents generated by hyperpolarizing voltage steps from –70 to –130 mV in voltage-clamp mode and in the presence of 1 µM TTX (Fig. 1, B and D). The nRT and nVB neurons expressed very different current responses in standard ACSF. In nVB neurons (Fig. 1D), the voltage steps to potentials below –80 mV generated a slow hyperpolarization-activated inward current and a tail current (Itail) due to the activation of the Ih found in the adult thalamic relay neurons (McCormick and Pape 1990). The amplitude of the Ih calculated as the difference between the instantaneous current (Iinst) measured at the beginning of the negative voltage step and the steady-state current (Iss) measured at the end of the negative voltage step was –404 ± 177 pA at –130 mV (n = 23).
The same voltage protocol applied to the nRT neurons failed to produce a significant slow inward relaxation in standard ACSF (Fig. 1B), confirming previous results (Santoro et al. 2000). The difference between the Iinst and the Iss currents at –130 mV was 7.23 ± 52.17 pA (n = 28) in the nRT. However, the nRT neurons express an instantaneous inward rectification at potentials below –90 mV (Fig. 1B), suggesting the expression of an instantaneous inwardly rectifying K+ (Kir) current.
Effect of Ba2+ in the nRT and the nVB neurons
The possible nonuniform distribution of Ih channels expressed along the dendrites (Berger et al. 2001; Lorincz et al. 2002; Magee 1998; Santoro et al. 1997; Stuart and Spruston 1998; Williams and Stuart 2000), and the axons (Beaumont and Zucker 2000; Cuttle et al. 2001; Fletcher and Chiappinelli 1992; Southan et al. 2000), can hamper their recording from the soma especially when a substantial somatic leak current is present. Among the currents that potentially contribute to the leak, the inwardly rectifying potassium (Kir) current is a good candidate. We therefore tested the effect of the Kir blocker Ba2+ (Hagiwara et al. 1978) in nRT and nVB in the presence of 1 µM TTX (Fig. 2). Applications of 1 mM Ba2+ induced an inward current at –60 mV in both cell types. It also reduced significantly the Iinst current at –130 mV in the nRT (Fig. 2, A and C1) and the nVB (Fig. 2, B and D1) to 40 and 32% of their respective control values.
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). The block of a voltage-dependent Im current (Halliwell and Adams 1982) could also contribute because the KCNQ2 subunit is strongly expressed in the nRT (Cooper et al. 2001). Our results indicate that the nRT and the nVB neurons of young mice express Kir currents as found in the nVB of the adult cat (Williams et al. 1997), and that the Kir current contributes strongly to the leak below –90 mV in the nRT.
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10 times smaller than the Iinst current recorded in the same nRT neurons. The amplitude of the hyperpolarization-activated current in nRT is also 10 times smaller than the Ih in nVB. The same results were obtained in the nRT of the older juvenile mice (P14–P23) where the amplitude of the hyperpolarization-activated current at –130 mV was –34.72 ± 26.43 pA (n = 8) in 1 mM Ba2+ (Supplementary Material Fig. 2).1 Most recordings were made at RT (22–24°C). To estimate the importance of the hyperpolarization-activated current in more physiological conditions, the effect of higher temperature (32–34°C) was tested in the nRT (Supplementary Material Fig. 1). Using the same voltage protocol and in presence of 1 mM Ba2+, it was found that the amplitude of the Iss was significantly enhanced from –120.54 ± 46.64 pA at 23°C to –185.89 ± 67.45 pA at 33°C (P < 0.01, n = 5). In the same neurons, the amplitude of the hyperpolarization-activated current at –130 mV was not significantly changed, being –62.98 ± 31.87 pA at 23°C and –67.58 ± 32.37 pA at 33°C (n = 5).
Characterization of hyperpolarization-activated current in the nRT neurons
Four HCN (1–4) subunits have been cloned in the mouse (Ludwig et al. 1998; Santoro et al. 1997, 1998). When expressed in heterologous cells, all four HCN subunits form functional homomeric channels and express Ih carried by K+ and Na+ ions, and sensitive to Cs+ and to the organic compound ZD7288. Their voltage sensitivity, their kinetics, and their sensitivity to cAMP vary with their subunit composition (Kaupp and Seifert 2001; Robinson and Siegelbaum 2003).
The slow hyperpolarization-activated current recorded in nRT in presence of Ba2+ displayed a voltage sensitivity similar to that seen in the nVB (Fig. 3). Their I-V curves were fitted by a sigmoid function (Fig. 3, C2 and F2) with similar parameters in nRT (V = –106 ± 1 mV; s = 9 ± 1 mV/pA; n = 14) and in nVB (V = –104 ± 1 mV; s = 10 ± 1 mV/pA; n = 13), indicating that the Ba2+-insensitive slow hyperpolarization-activated current found in the nRT is similar to the Ih observed in the nVB.
The HCN2 subunits assemble as homomeric channels with slow kinetics (Chen et al. 2001; Santoro et al. 2000). It has been suggested that their association with a MiRP1 accessory subunit gives rise to Ih with faster kinetics (Proenza et al. 2002; Qu et al. 2004). To test this possibility, we compared the activation kinetics of the hyperpolarization-activated current in nRT and nVB in presence of 1 µM TTX and 1 mM Ba2+ (Fig. 4). The hyperpolarization-activated responses generated by voltage steps below –100 mV in nRT and below –90 mV in nVB were fitted by the sum of two exponentials. We found that the currents in nRT and in VB had similar relatively slow kinetics that were accelerated by hyperpolarization. The weighted time constant of activation at –130 mV was slightly larger in nRT ( = 861 ± 226 ms, n = 9) than in nVB ( = 559 ± 156 ms, n = 7), and it was reduced by hyperpolarization in both areas (Fig. 4G). The fast and the slow time constants of activation (fast and slow) were significantly (P < 0.05) smaller at –130 mV than at –110 mV in nRT and in nVB (Fig. 4, B and E, respectively). Finally the relative contribution of the fast component was also increased by hyperpolarization in nRT and in nVB (Fig. 4, C and F, respectively).
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fast = 565.26 and 286.95 ms; slow = 4,111.67 and 1,752.82 ms at 23 and 33°C, respectively); the relative amplitude of the fast component being similar at both temperatures (Afast/Afast + Aslow = 0.88 and 0.89 at 23 and 33°C, respectively). Our results show that the hyperpolarization-activated current we found in nRT has slow kinetics very similar to those found in nVB. Their slow kinetics are also similar to these found for the recombinant homomeric HCN2 channels.
If the slow Ba2+-insensitive hyperpolarization-activated current of nRT neurons is identical to the Ih, then it should be sensitive to Cs+ and to the organic compound ZD7288. Figure 5 illustrates the effect of 2 mM Cs+ and 50 µM ZD7288 tested in nRT in presence of 1 µM TTX and 1 mM Ba2+. The application of Cs+ (n = 6) reduced significantly (P < 0.0001) the amplitude of the hyperpolarization-activated current from –49 ± 16 pA in control to 7 ± 4 pA in Cs+ (n = 6). The same effect was obtained with the application of ZD7288 (n = 3), reducing the hyperpolarization-activated current from –55 ± 34 pA in control to –6 ± 7 in ZD7288 (P = 0.07).
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). To test whether the hyperpolarization-activated current seen in the nRT was carried by K+ ions, we tested the effect of an extracellular ACSF solution with high (15 mM) K+ concentration, in presence of 1 µM TTX and 1 mM Ba2+ (Fig. 6). CNQX (10 µM) was also present to block the spontaneous AMPA receptor-mediated miniature excitatory postsynaptic currents (EPSCs) and 200 µM Ni2+ to block the low-threshold T-type Ca2+ channels. In some experiments, 2 mM 4-AP was also added to block the transient K+ current (IA). In these conditions, high K+ induced an inward current at –60 mV and increased significantly (P < 0.01) the amplitude of the hyperpolarization-activated current seen below –80 mV. Its amplitude at –130 mV was increased 2.85 times when the K+ concentration increased from 4.5 to 15 mM (n = 5) and 6.92 times when the K+ concentration increased from 1.5 to 15 mM (n = 4). Simultaneously the amplitude of a tail current with slow kinetics and voltage dependence typical of the Ih became visible in high 15 mM K+. Both the slow inward relaxation during the negative voltage steps and the tail current seen in 15 mM K+ were also blocked by 50 µM ZD7288. Similarly, in older juvenile mice (P14–P23), the amplitude of the hyperpolarization-activated current was enhanced over the whole voltage range, being at –130 mV, –34.72 ± 26.43 pA (n = 8) in 4.5 m K+ and –109.07 ± 100.61 pA (n = 4) in 15 mM K+ (supplementary Fig. 2). To test whether the Ih recorded in the nRT was also carried by the Na+ ions, we studied the effect of reducing the external Na+ concentration, replacing 125 mM Na+ by NMDG in presence of 1 µM TTX, 1 mM Ba2+, 200 µM Ni2+, and 10 µM CNQX. The experiments were performed with 15 mM K+ in the external solution to enhance the hyperpolarization-activated current in control (Fig. 7). In these conditions, the amplitude of the hyperpolarization-activated current at –130 mV was significantly (P = 0.0004) reduced by the partial substitution of the Na+ ions by NMDG being –127.97 ± 72.03 pA in control Na+ concentration and –113.16 ± 70.74 pA in NMDG (n = 6 cells). At the same time, the amplitude of the tail current was also reduced. These results show that the slow hyperpolarization-activated inward current seen in the nRT is carried both by K+ and Na+ ions.
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), the effect of cAMP being particular strong for the HCN2 subunit (Chen et al. 2001; Wainger et al. 2001). We tested the effect of cAMP on the hyperpolarization-activated current in the nRT using 20 µM 8-Br-cAMP, a nonhydrolysable form of cAMP (Fig. 8). The experiments were performed in the presence of 1 µM TTX, 1 mM Ba2+, 200 µM Ni2+, 100 µM CNQX, and 15 mM K+ in the external medium. The controls were obtained from a first group of nRT neurons (n = 8) using the standard pipette solution. The effect of cAMP was tested by adding 20 µM 8-Br-cAMP in the pipette solution of a second group of neurons (n = 8). Fitting the I-V curves with a sigmoid function, we found that the activation curves were displaced toward more positive potentials when 8-Br-cAMP was present in the pipette solution (Fig. 8A): the V was significantly (P < 0.001) more negative in control (–112 ± 1 mV) than with 20 µM 8-Br-cAMP (–108 ± 1 mV). We also found that the rate of activation of the hyperpolarization-activated current in nRT was significantly faster during the first 2 s of the negative voltage step when 8-Br-cAMP was present in the pipette solution (Fig. 8, B–E). The responses during the voltage steps at –130 mV were normalized to their value at the end of the step. The superimposition of the average data (Fig. 8B) and of the individual cells (Fig. 8C) in control and in 8-Br-cAMP shows that the delay of opening the channels is reduced when 8-Br-cAMP is present in the pipette solution, the difference between the average curves in control and in 8-Br-cAMP (Fig. 8D) being maximal and significant (P < 0.05) during the first 2 s (Fig. 8E). These results show that, like the Ih, the hyperpolarization-activated current we found in the nRT, is sensitive to cAMP.
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During our voltage-clamp experiments in the nRT, 1 mM Ba2+ was present in the external solution and relatively negative membrane potentials (less than –90 mV) were necessary to activate the hyperpolarization-activated current, raising the question of the possibility for this current to be activated in physiological conditions.
To investigate whether the hyperpolarization-activated current could be activated in more physiological conditions, we tested the effect of the Ih blocker (Cs+ 2 mM, n = 7, Fig. 9) in the current-clamp mode in standard ACSF. The application of 2 mM Cs+ induced a hyperpolarization of most neurons (5/7) as expected if a cationic K+/Na+-permeable channel is being blocked by Cs+. The hyperpolarization was associated with an increase of the Rin. The hyperpolarization was significant (P < 0.0001) being 2.27 ± 1.55 mV in three neurons that were not manually clamped during the application of Cs+. In control, as shown before (Fig. 1), the I-V curve between –70 and –120 mV was not linear, and the Rin was significantly (P < 0.05, n = 7) smaller at membrane potential below –90 mV and larger at membrane potential above –90 mV. The application of Cs+ induced a larger increase of the Rin when the membrane potential was more negative than –90 mV. Below –90 mV, the Rin was significantly increased from 162.61 ± 92.20 M in control to 398.25 ± 217.15 M in Cs+ (paired t-test, P = 0.02; n = 7). Above –90 mV, the effect of Cs on the Rin was smaller, but a significant change was also seen (Rin = 265.66 ± 75.00 M in control; 301.76 ± 88.99 M in Cs; P = 0.02, n = 7). Similar effects of 50 µM ZD7288 were seen on the Rin of the nRT neurons. A significant increase of the Rin above –90 mV from 434.49 ± 145.86 M in control to 725.46 ± 169.50 M in ZD7288. The Rin below –90 mV was also significantly increased from 282.83 ± 23.85 M in control to 662.23 ± 172.95 M in ZD7288 (P = 0.02, n = 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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nRT neurons express the Ih
Previous work has shown that the Ih is insensitive to Ba2+ (Ishii et al. 1999; Ludwig et al. 1998; Munsch and Pape 1999; Santoro et al. 1998; Xu et al. 2004). Therefore reducing the leak by Ba2+ has probably an indirect effect on the Ih, allowing a better electrical access to its remote site of expression (Cathala and Paupardin-Tritsch 1999; Watts et al. 1996). Using Ba2+ to reduce the leak conductance, we demonstrate the expression of the Ih in the nRT. In support of this conclusion, we show that the current was insensitive to Ba2+ (Figs. 2 and 3), known to block the Kir currents without affecting the Ih (Pape 1996). It is also blocked by specific blockers of the Ih, Cs+, and ZD7288 (Fig. 5). It activates at potentials more negative than the RMP (Fig. 3) with slow kinetics (Fig. 4) similar to those described for the homomeric HCN2 channels (Chen et al. 2001; Santoro et al. 2000). It is carried both by the K+ and Na+ ions (Figs. 6 and 7). Finally, it is modulated by intracellular cAMP (Fig. 8). The recombinant HCN2 channels are very sensitive to the intracellular cAMP. In the inside-out patches, the binding of cAMP to the intracellular cyclic nucleotide-binding domain induces a +17 mV shift of the activation curve. The kinetics of activation is also greatly accelerated by cAMP (Chen et al. 2001; Wainger et al. 2001). In the nRT, we found that the effect of 8-Br-cAMP on the V is relatively smaller on the order of 6 mV, suggesting that the cyclic nucleotide binding domain of the HCN2 channel is already partly occupied by cAMP. The fact that the V was relatively negative (–112 mV) may be due the remote expression of the HCN2 channels in the nRT as discussed after.
Santoro and his collaborators were puzzled by the lack of Ih in the nRT because the HCN2 subunit mRNA, which translates into functional homomeric Ih channels, is expressed by the neurons of nRT (Robinson and Siegelbaum 2003; Santoro et al. 2000). Several possibilities could explain the lack of Ih in the nRT. First the mRNA found in the nRT is not translated into a functional protein, or it is translated but the protein is not efficiently targeted to the plasma membrane. The location of the HCN2 subunit protein found in the nRT by immunolabeling (Notomi and Shigemoto 2004) has not yet been studied at the subcellular level, therefore it is unclear whether the immunolabeling is due to the nVB axon terminals or to the nRT neurons themselves. Second its has been suggested that the HCN2 subunit may interact with a MiRP1 accessory subunit and express a Ih with instantaneous or faster kinetics (Proenza et al. 2002; Qu et al. 2004). Third the expression of the Ih by remote dendrites (Berger et al. 2001; Lorincz et al. 2002; Magee 1998; Santoro et al. 1997; Stuart and Spruston 1998; Williams and Stuart 2000), and axon terminals (Beaumont and Zucker 2000; Cuttle et al. 2001; Fletcher and Chiappinelli 1992; Southan et al. 2000), and the presence of a relatively leaky somatic recording site may hamper the recording of the Ih.
Our results support the hypothesis that a leak conductance may hamper the recording if a slow Ih with a nonuniform distribution. As reported before (Destexhe et al. 1996), good voltage-clamp control is difficult to achieve in the nRT neurons. We found that the nRT neurons display a substantial inward rectification especially when the membrane potential is more negative than –90 mV. This current is mostly carried by a Ba2+-sensitive Kir current. Importantly, the amplitude of the leak current at –130 mV is 10 times larger than the amplitude of the Ih we recorded in nRT in presence of Ba2+. This is in contrast with the situation in the nVB where the leak current and of the Ih are of comparable amplitude (Fig. 2). The amplitude of the Ih recorded at the soma was also enhanced by the application of Ba2+ in the nVB (Fig. 2), suggesting that the HCN subunits are probably nonuniformly distributed with along the dendrites or the axons of the relay neurons as shown for example in retinal and hippocampal neurons (Fletcher and Chiappinelli 1992; Magee 1999). We have shown that in the presence of Ba2+, the instantaneous I-V curve becomes linear and is not sensitive to Cs+, suggesting that the nRT neurons do not express any instantaneous Ih that could be attributed to the HCN2 subunits forming a complex with a MiRP1 subunit (Proenza et al. 2002). Moreover our results show that the nRT neurons express an Ih with slow kinetics of activation similar to those seen with homomeric HCN2 channels (Chen et al. 2001; Santoro et al. 2000), suggesting that the accessory MiRP1 subunit is not associated with HCN2 subunits to form Ihs with faster kinetics (Qu et al. 2004).
Possible functional consequences of the expression of the Ih in the nRT
The Ih has an important contribution to the regulation of the neuronal excitability, regulating the RMP, the overshoots of the membrane potential (such as the anomalous rectification and the transient depolarization rebound, and the afterhyperpolarization, AHP), contributing to the pacemaker oscillatory activity (Pape 1996). Our results show that the Ih that we have shown in the nRT is active in standard physiological condition and contributes to the RMP and to the Rin (Fig. 9) of the neurons. Its possible contribution to the oscillatory behavior of the nRT neurons and to the control of GABA release by the axonal terminals need to be further investigated.
During the transition from wake to sleep, spindle waves at 7–14 Hz occur every 3–10 s in the TC pathways. The spindles result from reciprocal synaptic interactions between the relay neurons and the nRT GABAergic neurons and from their intrinsic oscillatory behavior (Fuentealba and Steriade 2005; McCormick and Bal 1997; Steriade et al. 1993). The nRT neurons generate intrinsic rhythmic bursts that give rise to action potentials at 7–12 Hz (Mulle et al. 1986; Steriade et al. 1986). Several voltage-gated channels contribute to the oscillatory bursting activity of relay neurons (McCormick and Pape 1990) and of nRT neurons (Bal and McCormick 1993). Among them, experimental data (Bal and McCormick 1996; Luthi et al. 1998) and simulation (Destexhe et al. 1993) indicate that the Ih favors the slow (2 Hz) oscillation of the relay neurons. It has been proposed that the intrinsic oscillatory behavior of the nRT neurons is sustained by low-threshold Ca2+ currents, by a Ca2+-activated K+ current, and by a Ca2+-activated cationic current (Bal and McCormick 1993; Destexhe et al. 1993, 1994, 1996; Huguenard and Prince 1992). Despite its relatively small amplitude, the Ih is expressed by the GABAergic neurons of the nRT and should be taken into consideration for a better understanding of the mechanisms that sustain the spindle waves and the slow waves in the TC pathways.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The Supplementary Material for this article (two figures) is available online at http://jn.physiology.org/cgi/content/full/00922.2005/DC1. 
Address for reprint requests and other correspondence: N. Ropert, INSERM U603, CNRS FRE2500, Laboratoire de Neurophysiologie et Nouvelles Microscopies, Université Descartes Paris V, 45 rue des Saints-Pères, 75270 Paris cedex 06, France (E-mail nicole.ropert{at}univ-paris5.fr)
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REFERENCES |
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; n = 19) and in nVB (
; n = 17). The voltage-current curve is nonlinear, the input resistance Rin being reduced by hyperpolarization. F: amplitude (mean ± SD) of the Iinst (
) and Ih (
). The slope of the I-V curve is not linear in control being larger below –80 mV. In presence of Ba2+, the I-V curve becomes linear and both curves cross near –90 mV. C1: hyperpolarization-activated current (Iss – Iinst) in the same nRT neuron is plotted against Vm in control (
