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CARDIOVASCULAR |
1 Department of Psychiatry, University of Cincinnati, Cincinnati, OH, USA
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Abstract |
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(Received 11 April 2007;
accepted after revision 24 May 2007;
first published online 24 May 2007)
Corresponding author J. E. Stern: Department of Psychiatry, University of Cincinnati, Genome Research Institute, 2170 E. Galbraith Rd, Cincinnati, OH 45237, USA. Email: javier.stern{at}psychiatry.uc.edu
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Introduction |
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Preautonomic PVN neurones send descending projections to sympathetic related areas in the brainstem and spinal cord (Luiten et al. 1985; Pyner & Coote, 1999), including the rostral ventrolateral medulla (RVLM) (Pyner & Coote, 1999), a key centre for the control of tonic sympathetic activity and blood pressure. Accumulating evidence supports an important role for RVLM-projecting PVN (PVN-RVLM) neurones in the regulation of sympathetic outflow (Yang & Coote, 1998; Tagawa & Dampney, 1999; Allen, 2002). Furthermore, changes in PVN neuronal function have been associated with increased sympathoexcitatory drive during prevalent cardiovascular diseases, including hypertension (Herzig et al. 1991; Takeda et al. 1991; Jung et al. 2004) and heart failure (Patel et al. 2000; Li & Patel, 2003). In fact, recent work implicates the PVN-RVLM pathway in the enhanced sympathetic vasomotor tone in spontaneously hypertensive rats (Allen, 2002).
Despite the important role of PVN-RVLM neurones in the control of autonomic function in health and disease conditions, relatively little is known about the mechanisms controlling neuronal excitability in these neurones. Similarly to other neurones in the central nervous system (CNS), their firing activity is likely to result from the combined action of intrinsic and extrinsic mechanisms. While various neurotransmitter systems, including GABA, nitric oxide and angiotensin II have been shown to modulate PVN-RVLM neuronal activity (Stern et al. 2003; Cato & Toney, 2005; Li & Pan, 2005), the role intrinsic membrane properties play in controlling neuronal excitability and firing discharge in these neurones is still incompletely understood.
Voltage-gated K+ currents, including, the transient and rapidly inactivating IA (Rudy, 1988), are known to influence neuronal excitability in most CNS neuronal populations (Serodio & Rudy, 1998; Rudy et al. 1999). IA actions are mostly mediated by modulating the properties of the (Na+) action potential, as well as interspike intervals during repetitive firing (Connor & Stevens, 1971; Rogawski, 1985; Rudy, 1988; Kim et al. 2005).
A variety of voltage-gated K+ currents have been shown to be present in PVN neurones including IA, the slowly activating, non-inactivating delayed rectifier (IKDR), as well as a slowly activating, slowly inactivating K+ current (Barrett-Jolley et al. 2000; Luther et al. 2000; Luther & Tasker, 2000). While IA has been recently characterized in PVN magnocellular and non-identified parvocellular neurons (Li & Ferguson, 1996; Luther & Tasker, 2000), it is at present unknown whether IA is also expressed in PVN-RVLM neurones, and what its role is in shaping their action potential and firing properties.
Therefore, we combined in this study patch-clamp electrophysiological recordings with neuronal tract tracing and immunohistochemistry, to characterize the general biophysical and pharmacological properties of IA in PVN-RVLM neurones, and to determine its role in controlling their membrane excitability and firing discharge. In addition, we explored the possible subunit composition of K+ channels underlying IA in this neuronal population.
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Methods |
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Retrograde labelling of PVN-RVLM neurones
PVN-RVLM neurones were identified by injecting rhodamine beads unilaterally into the brainstem region containing the RVLM, as previously described (Li et al. 2003). Rats were anaesthetized intraperitoneally with a ketamine–xylazine mixture (90 and 50 mg kg–1, respectively), the rat's head was then placed in a stereotaxic apparatus, and 200 nl of rhodamine-labelled microspheres (Lumaflor, Naples, FL, USA) were pressure injected into the RVLM (starting from Bregma: 12 mm caudal along the lamina, 2 mm medial lateral, and 8 mm ventral). The location of the tracer was verified histologically, as previously described (Li et al. 2003). In general, injection sites were contained within the caudal pole of the facial nucleus to 
1 mm more caudal, and were ventrally located with respect to the nucleus ambiguous (see example in Fig. 1A). In a few instances, injections were located either rostrally or caudally to the RVLM, in which cases, no PVN retrograde labelling was observed. If the injection site was not within the region of the RVLM, the experiment was discarded.
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Three to seven days after surgery, rats were anaesthetized with nembutol (50 mg kg–1) and perfused through the heart with a cold sucrose solution (containing (mM): 200 sucrose, 2.5 KCl, 3 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20 D-glucose, 0.4 ascorbic acid, 1 CaCl2 and 2 pyruvic acid (290–310 mosmol l–1). This method has been previously shown to improve cell viability in slices obtained from adult rats (Aghajanian & Rasmussen, 1989). Rats were then quickly decapitated, and brains dissected out. Slices were cut coronally (300 µm thick) utilizing a vibroslicer (D.S.K. Microslicer, Ted Pella, Redding, CA, USA). An oxygenated ice cold artificial cerebrospinal fluid (ACSF) was used during slicing (containing (mM): 119 NaCl, 2.5 KCl, 1 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20 D-glucose, 0.4 ascorbic acid, 2 CaCl2 and 2 pyruvic acid; pH 7.4; 290–310 mosmol l–1). After sectioning, slices were placed in a holding chamber containing ACSF and then kept at room temperature until used.
Electrophysiological recordings
Slices were placed in a submersion style recording chamber, and bathed with solutions (3.0 ml min–1) that were bubbled continuously with a gas mix of 95% O2–5% CO2, and maintained at room temperature (22°C). Thin-walled (1.5 mm o.d., 1.17 mm i.d.) borosilicate glass (G150TF-3, Warner Instruments, Sarasota, FL, USA) was used to pull patch pipettes (3–6 M
) on a horizontal Flaming/Brown micropipette puller (P-97, Sutter Instruments, Novato, CA, USA). The internal solution contained (mM): 140 potassium gluconate, 0.2 EGTA, 10 Hepes, 10 KCl, 0.9 MgCl2, 4 MgATP, 0.3 NaGTP and 20 phosphocreatine (Na+); pH 7.2–7.3. Whole-cell recordings from PVN-RVLM neurones were visually made using a combination of fluorescence illumination and infrared differential interference contrast (IR-DIC) videomicroscopy. Recordings were obtained with a Multiclamp 700A amplifier (Axon Instruments, Union City, CA, USA). The voltage output was digitized at 16-bit resolution, 10 kHz (Digidata 1320A, Axon Instruments), and saved on a computer to be analysed offline. The series resistance (13.6 ± 0.4 M, n
= 100) was monitored at the beginning and end of each experiment, and the experiment was discarded if the series resistance was not stable throughout the recording. The liquid junction potential (LJP, 6.5 mV) was experimentally determined using a 2 M KCl agar bridge. Data shown were corrected for the LJP.
Intracellular labelling
During recordings, cells were intracellularly filled with biocytin (0.2%) and then stained with the avidin–biotin complex (ABC)-diaminobenzidine tetrahydrochloride (DAB) as previously described (Stern, 2001). Briefly, after recordings were completed, slices were placed in a 4% paraformaldehyde–0.2% picric acid solution, dissolved in 0.3 M PBS (pH7.3) overnight and then thoroughly rinsed with 0.01 M phosphate buffered saline (PBS). Slices were then incubated at 4°C for 1 h in 10% normal horse serum with 0.01 M PBS and 0.5% Triton X-100. Slices were again thoroughly rinsed with 0.01 M PBS and incubated overnight in ABC (Vector Laboratories) diluted 1: 100 in 0.01 M PBS containing 0.5% Triton X-100. Slices were then reacted with DAB (60 mg/100 ml) in 0.01 M PBS containing 0.5% Triton X-100, 0.05% nickel sulphate, and 0.006% H2O2, for approximately 2–3 min. Sections were then rinsed in 0.01 M PBS, mounted, and dried for 24 h (Stern, 2001). For illustration purposes, intracellularly labelled neurons were traced using a computer-asisted tracing system (Neurolucida, Microbrightfield) (Fig. 1B).
Voltage-clamp recordings of isolated voltage-gated K+ currents
Slices were bathed in an ACSF with nominal Ca2+ (0 mM) (containing (mM): 102 NaCl, 2.5 KCl, 3 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20 D-glucose, 0.4 ascorbic acid, 2 pyruvic acid, 3 EGTA, 200 µM CdCl2, 30 TEA and 0.5 µM TTX; pH 7.4; 290–310 mosmol l–1). Series resistance was electronically compensated for at least 60% throughout the recordings. The voltage error due to uncompensated series resistance at the half-activation and half-inactivation potentials for IA were 3.3 ± 0.5 mV and 1.4 ± 0.1 mV, respectively (results were not corrected).
The quality of the space clamp was assessed as previously described (Luther & Tasker, 2000). Briefly, IA 10–90% rise time and time constant (
) of inactivation were measured following activation of the current by a test command (–10 mV), preceded by conditioning steps of varying amplitudes (–120 mV through –30 mV). Plots of 10–90% rise time and inactivation time constant () as a function of conditioning steps were then generated. Varying the conditioning step should affect only the amplitude of the current, without affecting its kinetic properties. Thus, only neurones showing unchanging 10–90% rise time and inactivation as a function of the conditioning pulse, as well as a lack of relationship between current amplitude and kinetics were included for analysis; 92% of recording neurones met these criteria, and the other neurones were not included in the analysis.
All protocols were run with an output gain of 2 and a Bessel filter of 2 kHz, and were leak subtracted using a P/4 protocol.
Voltage dependence of activation of IA
In order to isolate IA, a combination of electronic and pharmacological methods were used. Calcium channels were blocked using a 0 Ca2+ ACSF containing EGTA and CdCl2 (see above). TTX and tetraethyl ammonium (TEA) were also used to block voltage-dependent Na+ channels and delayed rectifier K+ channels (IKDR), respectively. Since in many instances some TEA insensitive IKDR remained, two separate electrophysiological protocols were run in order to further isolate IA electronically. The first utilized a hyperpolarized conditioning pulse (–90 mV), which removed inactivation from IA. This pulse was followed by depolarizing command pulses (–70 to +25 mV), which resulted in the activation of both IA and IKDR. A second protocol was then run, in which a more depolarized (–40 mV) conditioning pulse was used to completely inactivate IA. Thus, when the same command pulses as above were applied, only IKDR was activated. Currents recorded under these two protocols were then electronically subtracted offline using Clampfit 8.2 (Axon Instruments). The chord conductance was calculated by measuring the peak amplitude of the evoked current at each command potential, divided then by the difference of the command potential and the reversal potential (calculated to be –104.2 mV from the Nernst equation). The chord conductance was then normalized to the maximum chord conductance obtained at +125 mV, and plotted as a function of the command potential. The plots were then fitted with a Boltzmann function, and the half-activation potential (the Vm at which 50% of IA currents are activated) was obtained. The current density was determined by dividing the current amplitude at each command potential by the cell capacitance, obtained by integrating the area under the transient capacitive phase of a 5 mV depolarizing step pulse, in the voltage-clamp mode. The rate of activation of IA was determined by measuring the 10–90% rise time from the baseline to the peak of the current (command potential = –10 mV).
Voltage dependence of inactivation of IA
In order to determine the voltage dependence of inactivation, neurones were voltage clamped at –70 mV, and the membrane was subjected to conditioning pulses of varying amplitude (–120 to –35 mV, 50 ms), which removed varying amounts of inactivation from IA. A command pulse to –10 mV was then used to activate IA. In separate sets of experiments, the duration of the prepulses were extended to 115, 120 and 150 ms, as indicated. The mean normalized IA peak amplitude was plotted as a function of the conditioning step potentials, and the I–V plots were fitted with a Boltzmann function, to determine the half-inactivation potential (the Vm at which 50% of IA is inactivated). The inactivation of IA was determined by fitting a single exponential function to the decay phase of the current activated at –10 mV following a conditioning step to –90 mV.
Time dependence of inactivation of IA To determine the time dependence of IA inactivation, neurones were voltage clamped at –70 mV, and conditioning pulses to –45 mV or –50 mV of varying durations (10–200 ms, 10 ms increments) were followed by a command pulse to –10 mV (300 ms). Plots of the IA peak amplitude as a function of the duration of the conditioning pulse were then generated. Plots were fitted by a monoexponential function, and the time constant of the decay used for quantitative purposes.
Kinetics of recovery from inactivation
Once the A-type K+ channel is inactivated following membrane depolarization, a sufficient amount of hyperpolarizing time must elapse before the channel can recover and be fully activated again. In order to determine the kinetics of recovery from the inactivated channel state, neurones were voltage clamped at –50 mV, and a hyperpolarizing conditioning pulse (–100 mV) of increasing duration (
10 ms) was applied, followed by a depolarizing command pulse (–10 mV). The mean normalized peak amplitude was plotted against the conditioning pulse duration. A single exponential function was fitted to the plot and the time constant () of recovery from inactivation was then calculated.
Current-clamp recordings of action potential waveform and firing activity
For current-clamp experiments, the ACSF used contained (mM): 119 NaCl, 2.5 KCl, 1 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20 D-glucose, 0.4 ascorbic acid, 2 CaCl2, 2 pyruvic acid; pH 7.4; 290–310 mosmol l–1. In addition, the AMPA and NMDA glutamate receptor antagonists 6,7-dinitro-quinoxaline-2,3-dione (DNQX; 10 µM) and 2-amino-5-phosphonopentanoic acid (AP5; 100 µM), respectively, and the GABAA receptor antagonist bicuculline (40 µM), were added to this solution (see below). All protocols run used an output gain of 10 and a Bessel filter of 10 kHz.
To test for the influence of IA on PVN-RVLM firing properties, the K+ channel blocker 4-aminopyridine (4-AP) was used. Since 4-AP facilitates presynaptic release of neurotransmitter (Flores-Hernandez et al. 1994), direct effects of 4-AP on intrinsic properties could be masked by this presynaptic effect. Thus, all current-clamp experiments were performed in the presence of receptor blockers of the main excitatory and inhibitory neurotransmitters in this system: GABA and glutamate (see above). Supporting the efficacy of this approach, we found that in the absence of these receptor blockers, 4-AP induced a significant decrease in PVN-RVLM input resistance (control, 1414 ± 462.7 M; 4-AP, 829.4 ± 244.3 M; n
= 7; P
= 0.05), an effect likely to be due to overall increased neuronal conductance following robust release of neurotransmitters. Conversely, an increased input resistance was observed when 4-AP was applied in the presence of the receptor blockers listed above (control, 886.1 ± 127.0 M; 4-AP, 1167 ± 147.5 M; n
= 23; P < 0.05).
Evoked action potentials To elicit individual action potentials, PVN-RVLM neurones were current clamped at either –80 mV or –50 mV, and subjected to depolarizing pulses (5 ms; 0.5–1.0 nA). Ten sweeps of evoked action potentials were averaged, and various parameters of the mean action potential waveform (peak amplitude, half-width, and 90–10% decay time) were analysed and compared before and after addition of the A-type K+ channel blocker 4-AP, using algorithms provided by Mini Analysis software (Synaptosoft, Fort Lee, NJ, USA).
Repetitive firing activity Spontaneous or evoked (direct current (DC) injection) firing discharge was recorded from PVN-RVLM neurones in continuous mode. The mean firing frequency obtained before and after addition of 4-AP (2 min period) was calculated and compared using Mini Analysis software. Neurones were arbitrarily considered responsive to 4-AP if a change in firing rate > 5% was observed.
In addition, all action potentials from the control and treated group were averaged into a single spike waveform, and various parameters of the action potential, including peak amplitude, half-width, 90–10% decay time and spike threshold, were compared. In addition, parameters related to the hyperpolarizing afterpotential (HAP), including peak amplitude, area and kinetics, were calculated and compared before and after 4-AP addition. Action potential threshold was measured at the abrupt transition from the pre-spike depolarizing ramp to the up-stroke of the action potential, as determined by algorithms provided by Mini Analysis software. HAP properties (e.g. amplitude, area and decay time course) were also determined by algorithms provided by Mini Analysis software (Synaptosoft, Fort Lee, NJ, USA).
Chemicals and 4-AP applications
All chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA), with the exceptions of pyruvic acid (MP Biomedicals, Aurora, OH, USA) and tetrodotoxin (Alomone Laboratories, Jerusalem, Israel). 4-AP was bath applied using a peristaltic pump (Gilson, Middleton, WI, USA; flow 2 ml min–1) for a period of 5 min, and then washed out with ACSF. In most cases, in our hands, a complete washout of 4-AP and its effects was not accomplished within the period that we were able to maintain a good quality recording (see, however, Fig. 6A). Thus, values corresponding to the washout period are not reported.
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For these studies, PVN-RVLM neurones were retrogradely labelled using cholera toxin B (CTB; 1%, List Biological Laboratories), using the same stereotaxic procedure as described above. Three to seven days after surgery, rats were anaesthetized with nembutol (50 mg kg–1) and perfused transcardially in 4% paraformaldehyde in 0.01 M phosphate buffered saline (PBS). Brains were then removed, post-fixed for 2–4 h, cryoprotected in 30% sucrose in 0.01 M PBS (4°C, 3 days), and then stored at –80°C until further use.
Coronal slices (30 µm) containing the PVN were cut and collected in 0.01 M PBS. Slices were then incubated in 0.01 M PBS with 0.1% Triton X-100, 0.04% NaN3 (PBSTXNaN3), and 5% normal horse serum for 1 h at room temperature. Slices were then rinsed thoroughly with 0.01 M PBS, followed by incubation with one of the following primary antibodies for A-type K+ channel subunits (Kv1.4 1: 100; Kv4.2 1: 500; and Kv4.3 1: 10000; Alomone Laboratories, Jerusalem, Israel), along with an anti-CTB antibody (goat anti-CTB 1: 2500; List Biological Laboratories) for 2 days at 4°C in PBSTXNaN3. Slices were again thoroughly rinsed. Secondary antibodies were then applied for 4 h at 4°C in PBSTXNaN3 (donkey anti-goat Cy-5: 1: 50 and donkey antirabbit FITC: 1: 250; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Slices were then rinsed thoroughly, mounted, and visualized using confocal microscopy (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA); 63x oil immersion, zoomed x 2; single optical plane = 0.5 µm thick).
Pre-adsorption controls were run in order to test the specificity of the Kv primary antibodies. The antigens were used at concentrations 5 x those of the primary antibodies (Kv1.4 15 µg ml–1, Kv4.2 8 µg ml–1, and Kv4.3 0.4 µg ml–1; Alomone Laboratories) and incubated in PBSTXNaN3 for 2 h at room temperature, with or without the primary antibodies. The solutions were then centrifuged for 5 min at 10 000 g, and the supernatants applied to the tissue, following the same procedure described above.
Statistical analysis
All values are expressed as means ± S.E.M. Student's paired t test was used to compare differences in various physiological parameters, as indicated in the text, before and after application of 4-AP. Between group differences were compared using an unpaired t test. A one- or two-way ANOVA with Bonferroni's post hoc test were used when appropriate, as indicated in the text. Fisher's exact test was used to determine differences in the incidence of a particular effect, as indicated throughout the text. All statistical analyses were conducted using the same statistical program GraphPad Prism (GraphPad Software, San Diego, CA, USA).
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Results |
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and 35.2 ± 1.4 pF, respectively. In current-clamp mode, most PVN-RVLM neurones displayed low threshold spikes (LTSs) in response to positive current injection from hyperpolarized membrane potentials (
–90 mV). Similarly to our previous study in PVN neurones innervating the dorsal vagal complex (Stern, 2001), LTSs in PVN-RVLM neurones varied greatly in shape and amplitude, including fast spikes, small humps and long-lasting plateaus (Fig. 1C). In voltage-clamp mode, using solutions that allowed isolation of K+ currents (see Methods), membrane depolarization evoked both transient (A-type, IA) and sustained outward (IKDR) components. To pharma-cologically isolate the transient component, 30 mM TEA was used (Rudy, 1988; Locke & Nerbonne, 1997). However, since a remaining, TEA-insensitive sustained component was still present at relatively depolarized membrane potentials, IA was further isolated electronically (see Methods and Fig. 2). Since the focus of the present study was on IA, the properties of IKDR were not further studied herein.
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As previously described in other neuronal types (Huguenard et al. 1991; Beck et al. 1992), A-type K+ currents (IA) in PVN-RVLM neurones were characterized by strong voltage dependency and rapid activation and inactivation kinetics, resulting in a transient outward current.
The voltage-dependent activation properties of IA were studied in 26 PVN-RVLM neurones. Depolarizing steps of increasing amplitudes (–70 mV to +25 mV, in 5 mV increments) were used to activate IA (Fig. 2A). The mean IA peak amplitude, current density and chord conductance at +25 mV were 933.8 ± 110.8 pA, 31.7 ± 4.0 pA pF–1 and 8.3 ± 1.4 ns, respectively (see Methods). The mean IA 10–90% rise time (see Methods) was determined to be 6.1 ± 0.4 ms at a command potential of –10 mV (Fig. 2B). Plots of chord conductance versus command potential were generated (see Methods), and the voltage-dependent properties of activation of IA were calculated using a Boltzmann fit (Fig. 2C). The mean IA activation threshold was –48.9 ± 1.4 mV, and the half-activation voltage was –24.5 ± 1.6 mV, with a slope factor of 11.7 ± 0.6 mV.
Inactivation properties of IA in PVN-RVLM neurones
The voltage- and time-dependent inactivation properties of IA were studied in 35 PVN-RVLM neurones. The time dependence of inactivation of IA was studied using a command pulse to –10 mV (300 ms) from a conditioning step of either –50 mV or –45 mV of successively longer durations (10–200 ms, 10 ms increments). Plots of the evoked IA amplitude as a function of the conditioning step duration were generated (see Fig. 3A). As shown, the amplitude of the evoked IA current rapidly decreased as a function of duration of the –45 mV conditioning step, reaching steady-state within a range of 80–110 ms (n = 6). Plots were fitted with a monoexponential function, and a mean time constant of 28.3 ± 3.8 ms was obtained. Similar results were observed with conditioning steps of –50 mV (results not shown).
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The mean inactivation of IA using a command potential to –10 mV was 33.9 ± 3.0 ms (Fig. 3C inset), and was found to be independent of the command potential (F
= 0.41; P
= 0.8; one-way ANOVA, data not shown).
When the voltage-dependent activation and inactivation curves of IA were plotted together, a small region of overlap between these two curves (i.e. ‘window current’) at potentials between –50 mV to –35 mV, was observed (Fig. 3D). Despite its small amplitude (1.25% of maximal IA
12 pA at a membrane potential of –45 mV) this window current may contribute substantially to subthreshold changes in membrane potential, due to the relatively high input resistance of PVN-RVLM neurones. This is in fact supported by our results showing that pharmacological blockade of IA with 5 mM 4-AP (see below) induced a significant membrane depolarization (in 4/5 cells tested), when neurones were current clamped at
–50 mV in the presence of TTX (Vm: 5.5 ± 0.5 mV, P < 0.005, paired t test, n
= 4, see Fig. 3D inset).
Recovery of IA from Inactivation
The time course of recovery of IA from inactivation was studied in 16 PVN-RVLM neurones. To vary the amount of IA available for activation, neurons were hyperpolarized to –100 mV using conditioning steps of increasing duration (10–250 ms, in 10 ms increments). IA was then activated using a depolarizing command potential to –10 mV (Fig. 4A). The normalized IA peak amplitude at each command was plotted as a function of the duration of the respective conditioning step (Fig. 4A). The plots were best fitted by a single exponential function. As depicted in the example of Fig. 4B, IA recovery from inactivation in PVN-RVLM neurones was strongly time dependent, with a mean recovery time constant () of 65.7 ± 4.5 ms.
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The sensitivity of IA to the K+ channel blocker 4-AP was studied in 17 PVN-RVLM neurones. Similar protocols as those used to study activation of IA were used here. Currents were recorded before and after bath application of 1 or 5 mM 4-AP (Fig. 5A). Using a command step to –10 mV, we found IA to be significantly inhibited by both concentrations used (1 mM 4-AP: 20.3 ± 2.8% inhibition, n = 5; 5 mM 4-AP: 44.3 ± 2.3% inhibition, n = 12; P < 0.01 and P < 0.0001, compared to control, respectively). The larger inhibition observed with 5 mM 4-AP (P < 0.0001, when compared to 1 mM 4-AP) supports a concentration-dependent sensitivity of IA to 4-AP in PVN-RVLM neurones, as previously described in other neuronal types (Bossu et al. 1996; Song et al. 1998).
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IA shapes the Na+ action potential waveform in PVN-RVLM neurones
To determine whether IA modulates action potential waveform in PVN-RVLM neurones, recordings were obtained in the current-clamp mode (n
= 15). Action potential amplitudes in all recorded neurones were 
+50 mV. Individual action potentials were evoked using short (5 ms) depolarizing pulses, while clamping the neurones at two different membrane potentials (–80 mV and –50 mV, see Methods), in order to obtain different degrees of IA inactivation. The effects of 4-AP (5 mM) on various action potential parameters were then determined. Results are summarized in Fig. 6.
At a holding potential of –80 mV, evoked action potential half-width, and 90–10% decay time were 2.0 ± 0.1 ms and 1.9 ± 0.2 ms, respectively. Bath application of 4-AP at this holding membrane potential prolonged spike duration by 94.2 ± 16.7% (P < 0.0001 versus control ACSF), and slowed down its decaying phase by 166.7 ± 27.5% (P < 0.0001, versus control ACSF) (see Fig. 6).
Interestingly, similar changes in action potential waveform to those induced by 4-AP were observed when neurones were clamped at a more depolarized Vm. Thus, at a holding potential of –50 mV, spikes were broader (half-width: 3.2 ± 0.2 ms; P < 0.0005, vs. –80 mV), and displayed slower 90–10% decay times (2.6 ± 0.3 ms; P < 0.01, versus –80 mV). These changes were likely due to a higher degree of IA inactivation at the more depolarized Vm.
At this depolarized Vm (–50 mV), 4-AP still induced similar changes in action potential waveform as those observed when neurones were clamped at –80 mV. However, these effects were significantly reduced. Thus, at a holding potential of –50 mV, 4-AP prolonged spike duration by 54.5 ± 12.4% (P < 0.05, versus the percentage change in 4-AP at –80 mV) and slowed down the decaying phase of the action potential by 95.1 ± 16.6% (P < 0.05, versus the percentage change in 4-AP at –80 mV). Results are summarized in Fig. 6. Altogether, these results suggest that the Na+ action potential waveform in PVN-RVLM neurones is regulated by IA, an effect found likely to be dependent on its voltage-dependent availability.
IA differentially regulates repetitive firing activity of PVN-RVLM neurones
The effects of 4-AP on firing activity were studied in 21 PVN-RVLM neurones. About 62% (13 out of 21) of recorded neurones were spontaneously active. In the remainder, firing activity was induced by injecting depolarizing DC (+0.6 pA to +36.7 pA). In the majority of recorded cells (62%, 13/21), 5 mM 4-AP resulted in an increased firing discharge. In the remainder (38%, 8/21), a diminished firing discharge was observed.
Interestingly, PVN-RVLM neurones that were differentially affected by 4-AP also differed in some basic intrinsic properties. Neurones whose firing activity was enhanced by 4-AP had a more hyperpolarized resting Vm (–51.7 ± 1.7 mV versus
–42.8 ± 2.2 mV, P < 0.005) and displayed a lower incidence of spontaneous activity than those inhibited by 4-AP (38.5%
versus 100%, respectively, P < 0.01, Fisher's exact test). No differences in input resistance between the two groups were observed (842.6 ± 154.2 M
versus 896.8 ± 157.4 M in 13 and 8 neurons, respectively, P > 0.5). Furthermore, as summarized in Table 1, significant differences in the Na+ action potential waveform were observed between the two groups of PVN-RVLM neurones. For example, neurones whose firing activity was increased by 4-AP displayed narrower (32%
P < 0.0005) and faster (44%
P < 0.001) decaying action potentials than those inhibited by 4-AP.
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Among the neurones whose firing activity was enhanced by 4-AP, 69% (9/13) and 31% (4/13) displayed continuous or bursting firing patterns, respectively. The relative low incidence of the latter group precluded us from obtaining a detailed analysis of the effects of 4-AP on bursting properties. Thus, all these neurones were pooled and the effect of 4-AP on their mean firing discharge was analysed (see Methods). Results are summarized in Fig. 7. Within this group, 4-AP resulted in 90% increase in firing rate (control: 1.0 ± 0.2 Hz; 4-AP: 1.9 ± 0.4 Hz; n
= 13; Fig. 7A).
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70%) and 90–10% decay time (93%), and slightly increased action potential amplitude (9%). Importantly, action potential threshold was shifted (2 mV) to a more hyperpolarized membrane potential in the presence of 4-AP. Results are summarized in Table 1. In order to rule out that changes in action potential waveform induced by 4-AP were secondary to membrane depolarization and increased firing rate per se, we performed a subset of recordings (n = 3) in which the membrane potential in the presence of 4-AP was hyperpolarized with DC injection, in order to bring the firing frequency of the recorded cell back to control levels. As shown in Fig. 7C, the action potential half-width was still prolonged in the presence of 4-AP (92.8 ± 7.1% increase in half-width, compared to control, P < 0.05, paired t test), even when the firing rate was decreased near to control levels.
In addition, we analysed the effects of 4-AP on hyperpolarizing afterpotentials (HAPs) during repetitive firing (Fig. 7B). Neither the HAP peak amplitude (measured as the difference from threshold to peak) nor its area were affected by 4-AP (P > 0.05 in both cases). Nonetheless, the absolute HAP peak potential reached a significantly more hyperpolarized membrane potential in the presence of 4-AP (control: –55.5 ± 0.9 mV; 4-AP: –58.2 ± 1.2 mV; n
= 13, P < 0.05, paired t test), likely due to the hyperpolarizing shift in spike threshold. Finally, the HAP decay time course was found to be steeper (25%, P < 0.05) in the presence of 4-AP.
Multiple correlation analysis within this subset of PVN-RVLM neurones failed to identify any significant correlation between the degree of 4-AP-induced changes in firing activity, intrinsic membrane properties and/or action potential properties (r2 values: 0.009–0.1).
IA contributes to ongoing firing activity in a subset of PVN-RVLM neurones
Among the neurones whose firing activity was inhibited by 4-AP, 75% (6/8) fired in continuous mode, while the rest (2/8) displayed a bursting pattern. In one case, 4-AP switched the firing pattern from continuous to bursting mode (Fig. 8A). On average, 4-AP decreased the firing rate of this subgroup of PVN-RVLM neurons by 40% (control ACSF: 2.3 ± 0.5 Hz; 4-AP: 1.4 ± 0.3 Hz; Fig. 8A).
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10%), and prolonged its duration (80%) and 90–10% decay time (160%). On the other hand, action potential threshold was not affected by 4-AP (results are summarized in Table 1). In a subset of recordings (n = 4) the membrane potential in the presence of 4-AP was depolarized with DC injection, in order to bring the firing frequency of the recorded neurones back to control levels. In these cases, action potential width was still prolonged by 4-AP (78.7 ± 25.8% increase in half width, compared to control, P < 0.05 paired t test).
Interestingly, opposing effects on various HAP parameters within the subset of 4-AP inhibited PVN-RVLM neurones were observed when compared to the 4-AP-enhanced group. For example, 4-AP slowed down the HAP decay slope by 24% (P < 0.05), and despite a slight decrease in HAP peak amplitude (14%, P < 0.05), the overall HAP area was increased by 27% (P < 0.01), likely due to the slower HAP decay time course. No differences in the absolute HAP peak potential were observed between control ACSF and 5 mM 4-AP (control: –53.4 ± 1.3 mV; 4-AP: –51.0 ± 3.0 mV; n
= 8, P
= 0.2). Results are summarized in Fig. 8B.
In this subset of neurones, a significant correlation between percentage changes induced by 4-AP on firing activity and HAP decay slope was observed (r2: 0.6, P < 0.02). No other significant correlations between 4-AP-induced changes in firing activity, intrinsic membrane properties and/or action potential properties were observed (r2 values: 0.009–0.3).
Effects of TEA on action potential waveform and firing activity in PVN-RVLM neurones
In addition to preferentially blocking IA, 4-AP at low millimolar concentrations may also partially block IKDR (Rudy, 1988; Luther & Tasker, 2000). In an attempt to further explore this possibility, we tested the effects of TEA, which preferentially blocks IKDR over IA (Lien et al. 2002; Melnick et al. 2004), on action potential waveform and firing activity in PVN-RVLM neurones. Bath application of 30 mM TEA robustly diminished IKDR (94.9 ± 2.2%; n = 8) while only slightly inhibiting IA (17.6 ± 3.5%; n = 8, P < 0.0001, unpaired t test; Fig. 9A). Differently from 4-AP, long lasting plateau potentials were observed in the presence of TEA, resulting in all cases in a robust inhibition of firing discharge (control: 6.0 ± 1.2 Hz; TEA: 1.0 ± 0.2 Hz; n = 4, P < 0.05 (paired t test; Fig. 9C). Since TEA per se induced robust changes in action potential waveform and firing activity, testing the effects of 4-AP in the presence of TEA in current clamp recordings was not feasible. Thus, while we cannot completely rule out that 4-AP effects on firing activity are in part due to blockade of IKDR, the disparity in the effects observed between 4-AP and TEA treatments, along with the voltage dependency of 4-AP effects on some of the measured parameters (e.g. duration of action potential) would suggest that these compounds act through different mechanisms.
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By prolonging action potentials, blockade of IA could result in an enhanced Ca2+ entry per spike (Hoffman et al. 1997; Chen, 2005), leading in turn to activation of various Ca2+-dependent mechanisms, such as Ca2+-dependent K+ channels (Sah & Davies, 2000). Depending on the complement of voltage-gated Ca2+, and Ca2+-dependent conductances available within each particular neuronal type, 4-AP-induced changes in Ca2+ entry could either increase or decrease membrane excitability. Thus, to determine to what extent 4-AP-induced changes in firing discharge in PVN-RVLM neurones were Ca2+ dependent, experiments were repeated in the presence of the broad spectrum Ca2+ channel blocker Cd2+ (200 µM; n = 12). Results are summarized in Fig. 10. In the presence of Cd2+, after-hyperpolarizing potentials (AHPs) following trains of spikes, a well-characterized Ca2+-dependent property (Hotson & Prince, 1980; Bourque et al. 1985; Storm, 1987), were blocked, supporting the efficacy of Cd2+ to block Ca2+-dependent membrane properties in these neurones (Fig. 10A).
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Importantly, 4-AP failed to affect the firing activity of all recorded neurones in slices preincubated with Cd2+ (control: 1.2 ± 0.2 Hz; 4-AP: 1.4 ± 0.2 Hz; n = 12, P = 0.3, paired t test; Fig. 10B). Furthermore, 4-AP failed to affect the action potential peak amplitude (P = 0.3), threshold (P = 0.4), HAP area (P = 0.3), or HAP slope (P = 0.1) in the presence of Cd2+.
Possible potassium channel subunits underlying A-type potassium currents in PVN-RVLM neurones
To gain insights into the possible Kv subunits underlying IA in PVN-RVLM neurons, we combined immunohistochemical identification of Kv1.4, 4.2 and 4.3 subunits (known to underlie A-type K+ currents in other CNS neurons (Coetzee et al. 1999) with neuronal tracing techniques (see Methods). Representative confocal photomicrographs of Kv1.4, 4.2 and 4.3 immunoreactivities in retrogradely labelled PVN-RVLM neurones are shown in Fig. 11. Kv1.4 and 4.3 subunits were found to be widely expressed and distributed within the PVN (Fig. 11A). On the other hand, Kv4.2 immunoreactivity was very weak and/or undetectable in the PVN. Conversely, a robust Kv4.2 immunoreactivity was observed in ependymal cells lining the third ventricle, and in glial-like processes in the median eminence (Fig. 11A2), supporting the efficacy of our approach to detect this immunoreactivity. All Kv immunoreactive reactions were blocked following preadsorption of antibodies with their respective peptides (results not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Methodological considerations
To identify PVN-RVLM neurones, rhodamine-labelled fluorescent latex microspheres were injected in the RVLM. This retrograde tracer results in highly restricted and well-defined injection sites (Katz et al. 1984), and is commonly used to trace CNS pathways, including PVN projections to brainstem nuclei (Cato & Toney, 2005; Li & Pan, 2005, 2005; Cham et al. 2006; Zahner et al. 2007). While this tracer is not taken up by fibres in passage (Katz et al. 1984; Katz & Iarovici, 1990; Krug et al. 1998), we cannot rule out potential labelling of severed axons in the area of the injection, which could include PVN projections to the spinal cord (Luiten et al. 1985). Labeling of severed axons in our studies seems, however, unlikely, because injections that were misplaced, either rostrally, caudally or laterally to the RVLM, in areas containing PVN descending axons running towards the RVLM and/or the spinal cord (Luiten et al. 1985), failed to retrogradely label neurones in the PVN.
Voltage- and space-clamp problems are often associated with voltage-clamp recordings in brain slices. We believe, however, that these potential errors were minimized in this study by the use of rigorous selection criteria for inclusion of neurons (see Methods), along with the fact that PVN neurones are known to be relatively electrotonically compact (Luther & Tasker, 2000; Stern, 2001). The good quality of our voltage-clamp experiments is supported by several lines of evidence, including: (a) stable and relatively low series resistance throughout the recordings (10 M, compared to neuronal input resistance of 1000 M), (b) small voltage errors (1–3 mV) associated with uncompensated series resistance, and (c) lack of dependence of IA rates of activation and inactivation on varying conditioning steps (Luther & Tasker, 2000). Although voltage errors cannot be completely eliminated when recording intact neurons in a slice preparation, we believe the results reported here to be relatively accurate. This is also supported by previous studies from hypothalamic neurones in which similar IA voltage and kinetic properties were reported, including studies from acutely dissociated (Cobbett et al. 1989; Hlubek & Cobbett, 1997) or intact neurones in a brain slice preparation (Luther & Tasker, 2000).
Finally, the voltage-dependent properties of IA are known to overlap with other voltage-dependent conductances, in particular the T-type Ca2+ current, previously reported to be present in parvocellular and identified preautonomic PVN neurones (Luther & Tasker, 2000; Stern, 2001). Thus, in order to isolate IA from the low-threshold T-type and other voltage-dependent Ca2+ current, voltage-clamp recordings in the present study were obtained in the presence of nominal 0 mM extracellular Ca2+, the Ca2+ chelator EGTA and the broad spectrum Ca2+ channel blocker Cd2+. A drawback of this approach, however, is that previous studies in other neuronal populations reported that extracellular divalent cations, including Ca2+ and Cd2+, could affect the voltage dependence of IA activation and inactivation (Davidson & Kehl, 1995; Hlubek & Cobbett, 1997; Song et al. 1998; Wickenden et al. 1999). Thus, it is possible that the reported voltage-dependent activation and inactivation values of IA do not reflect the physiological condition. This becomes important when comparing data obtained from voltage-clamp and current-clamp experiments in this study, since the latter were obtained in the presence of 2 mM Ca2+ and in the absence of Cd2+.
Biophysical, pharmacological and molecular properties of IA in PVN preautonomic neurones
While the biophysical and pharmacological properties of IA in PVN-RVLM neurones reported herein are generally consistent with those previously described in other CNS neuronal types (Bouskila & Dudek, 1995; Wang & Schreurs, 2006), some interesting differences were observed. For example, the activation threshold, half-activation Vm and half-inactivation Vm of IA in PVN-RVLM neurones were all found to be more hyperpolarized than those previously reported in non-identified parvocellular PVN neurones, and very similar in fact to those reported in Type 1 PVN magnocellular neurons (Li & Ferguson, 1996; Luther & Tasker, 2000).
In agreement with previous reports on other CNS regions, including the hypothalamus (Li & Ferguson, 1996; Hlubek & Cobbett, 1997; Fisher & Bourque, 1998; Luther & Tasker, 2000), we found IA in PVN-RVLM neurones to consistently show high sensitivity to the traditional A-type blocker 4-AP, and low sensitivity to the delayed-rectifier K+ channel blocker TEA (not shown) (Rudy, 1988). The sensitivity to 5 mM 4-AP block in PVN-RVLM neurones was relatively consistent across neurons (32–54% inhibition). This differs from the previously reported large variability in type II parvocellular PVN neurones (10–69% inhibition) (Luther & Tasker, 2000), likely to be due to the diverse neuronal types (e.g, neurosecretory and/or alternative preautonomic ones) included in the type II category.
Both the biophysical and pharmacological properties of IA have been shown to be dependent upon specific A-type K+ channel subunit composition, as well as the auxiliary subunits and Kv channel-interacting proteins (KChIPs) associated with them (Coetzee et al. 1999; An et al. 2000; Beck et al. 2002). Thus, differences in A-type properties between PVN-RVLM and other PVN neuronal populations could depend on the differential expression of molecularly diverse channels. A-type K+ channels have been shown to consist of Kv1.4 or Kv4.1–3 subunits, either homomerically or heteromerically assembled with other subunits from the same subfamily (Stuhmer et al. 1989). Despite the fact that the presence and functional relevance of A-type K+ currents in various hypothalamic neuronal types has been long recognized, the subunit composition of the underlying channels remains at present unknown. Using tract-tracing techniques in combination with immunohistochemistry, we found here that PVN-RVLM neurones consistently expressed high immunoreactive levels for the Kv1.4 and 4.3 subunits, with almost no immunoreactivity for the Kv4.2 subunit. These results suggest that A-type K+ channels in these neurones likely comprise one or both of the former two subunits, arranged homomerically. As a caveat, the lack of commercially available Kv4.1 antibody prevented us from assessing its expression in PVN-RVLM neurones. Moreover, the presence of immunoreactivity for these subunits does not necessarily imply their incorporation into functional channels. In this sense, previous studies indicate that Kv1- and Kv4-containing channels recover from inactivation with time constants of several seconds or milliseconds, respectively (Serodio & Rudy, 1998; Coetzee et al. 1999). Thus, our findings, showing a recovery from inactivation time constant of 70 ms, suggest that functional channels in PVN-RVLM neurones, at least those more likely activated during our recordings (i.e. located in somatic and proximal dendritic compartments), are probably composed of Kv4.3 subunits. Further studies using subunit-selective pharmacological tools for these Kv subunits would be needed to further clarify this issue.
IA influences excitability and repetitive firing properties of PVN-RVLM neurones
In agreement with previous reports in other neuronal types (Kloppenburg et al. 1999; Wang & Schreurs, 2006), our data support the presence of a tonically active IA (i.e. ‘window’ current) in PVN-RVLM neurones. This ‘window’ current was found to be available between a narrow, though apparently physiologically relevant, range of membrane potentials. However, it is important to take into account that due to differences in our recording conditions between voltage- and current-clamp studies, and our limitation to examine the divalent cation sensitivity of IA in these neurones (see above), the voltage-dependent availability of the ‘window’ current may not represent the physiological condition. Nonetheless, our results showing membrane depolarization and increased input resistance following pharmacological blockade of IA suggest that IA may play a role in setting resting membrane potential and/or in the regulation of subthreshold variations of membrane potential in PVN-RVLM neurones.
By regulating action potential waveform and interspike intervals (Rogawski, 1985; Rudy, 1988; Magee et al. 1998), IA has been shown to play important roles in shaping temporal firing patterns of various neuronal types. Our present studies indicate this to be the case in PVN presympathetic neurones as well: pharmacological
) protocols are plotted together as a function of the command potential, a region of overlap between –50 mV and –35 mV is observed (grey area). Note that the plots were expanded to better depict the overlapping region. Inset, summary data showing that blockade of IA with 5 mM 4-AP induced a significant membrane depolarization (*P < 0.005, n
= 4). Neurones were current clamped at 
