Functional role of A-type potassium currents in rat presympathetic PVN neurones

Authors


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@psychiatry.uc.edu

Abstract

Despite the fact that paraventricular nucleus (PVN) neurones innervating the rostral ventrolateral medulla (RVLM) play important roles in the control of sympathetic function both in physiological and pathological conditions, the precise mechanisms controlling their activity are still incompletely understood. In the present study, we evaluated whether the transient outward potassium current IA is expressed in PVN-RVLM neurones, characterized its biophysical and pharmacological properties, and determined its role in shaping action potentials and firing discharge in these neurones. Patch-clamp recordings obtained from retrogradely labelled, PVN-RVLM neurones indicate that a 4-AP sensitive, TEA insensitive current, with biophysical properties consistent with IA, is present in these neurones. Pharmacological blockade of IA depolarized resting Vm and prolonged Na+ action potential duration, by increasing its width and by slowing down its decay time course. Interestingly, blockade of IA either increased or decreased the firing activity of PVN-RVLM neurones, supporting the presence of subsets of PVN-RVLM neurones differentially modulated by IA. In all cases, the effects of IA on firing activity were prevented by a broad spectrum Ca2+ channel blocker. Immunohistochemical studies suggest that IA in PVN-RVLM neurons is mediated by Kv1.4 and/or Kv4.3 channel subunits. Overall, our results demonstrate the presence of IA in PVN-RVLM neurones, which actively modulates their action potential waveform and firing activity. These studies support IA as an important intrinsic mechanism controlling neuronal excitability in this central presympathetic neuronal population.

The hypothalamic paraventricular nucleus (PVN), a key centre for the integration of autonomic, endocrine and neuroendocrine functions (Swanson & Sawchenko, 1983), comprises a variety of functionally discrete neurones, including magnocellular neuroendocrine, parvocellular neuroendocrine and preautonomic neurones (Swanson & Sawchenko, 1980).

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.

Methods

Male Wistar rats (n= 51, 200–300 g) were purchased from Harlan Laboratories (Indianapolis, IN, USA), and housed in a 12 h: 12 h light–dark cycle with free access to food and water. All procedures were carried out in agreement with both the University of Cincinnati and Wright State University Institutional Animal Care and Use Committees' guidelines.

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.

Figure 1.

PVN-RVLM projecting neurones are identified following microinjections of a fluorescent retrograde tracer in the RVLM
A, representative example of a retrograde tracer injection site in the RVLM at three rostrocaudal brainstem levels (A1: Bregma – 11.30, A2: Bregma – 11.96; A3: Bregma – 14.08). Sections in the right panels show the rhodamine beads injection site (arrowheads). Bright light and fluorescence images were superimposed to better depict the injection site. Images on the left panels were obtained from similar rostrocaudal levels, and were counterstained to better depict the anatomy of the region. Arrows in A1, A2 and A3 point to the facial nucleus, the nucleus ambiguous and the area postrema, respectively. CC, central canal. Ba, a representative example of an intracellularly labelled PVN neurone located in the posterior subnucleus. b, the same neurone shown at an expanded scale, along with an ABC-DAB staining. C, PVN-RVLM neurones displayed low threshold spikes (LTS) (arrows) of varying shapes and magnitudes.

Hypothalamic slices

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 MΩ) 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 MΩ, 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 (pH∼7.3) 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 (τ) 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 (τ) 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 τ 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 τ 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 (Δ10 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 (τ) 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 MΩ; 4-AP, 829.4 ± 244.3 MΩ; 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 MΩ; 4-AP, 1167 ± 147.5 MΩ; 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.

Figure 6.

Effects of 4-AP on evoked Na+ action potential waveforms
A, representative examples of evoked (500 pA, 5 ms pulse) single action potentials before and after 5 mm 4-AP, at holding potentials of −80 (left) or −50 mV (right). B, summary data showing the effects of 4-AP on action potential width and decay time at these two holding potentials. Note that 4-AP significantly prolonged spike width and decay times, effects that were significantly larger when neurones were held at −80 mV ***P < 0.0001 versus control within same holding potential group. #P < 0.05, ##P < 0.01 and ###P < 0.0005 compared to same treatment at −80 mV.

Immunohistochemistry

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); 63× oil immersion, zoomed × 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 × 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).

Results

Whole-cell patch clamp recordings were obtained from 119 retrogradely labelled PVN-RVLM neurones, located in the three main PVN subnuclei known to contain long-descending preautonomic neurones: the ventromedial, dorsal cap and posterior subnuclei (Swanson & Kuypers, 1980; Swanson & Sawchenko, 1983; Stern, 2001). In many instances, neurones were efficiently loaded intracellularly with biocytin, and identified following an ABC-DAB procedure (see Fig. 1B). Overall, 40, 16 and 24 neurones were found to be located within the ventromedial, dorsal cap and posterior subnuclei, respectively. Mean input resistance and cell capacitance of recorded neurones were 992.7 ± 49.3 MΩ 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.

Figure 2.

Isolation and voltage-dependent activation of IA in PVN-RVLM neurones
A, outward currents were activated in the presence of 30 mm TEA using depolarizing steps (from −70 to +25 mV in 5 mV increments, 400 ms) following a conditioning step to −90 mV (340 ms). Responses included a transient (IA, arrow) and a TEA-insensitive sustained component (IKDR, arrowheads) (a). Using a conditioning step to −40 mV, the same depolarizing steps resulted only in the activation of IKDR (b). IA was then electronically isolated by digitally substracting traces in a and b (c). B, representative example of the rapid rate of activation of IA following a command potential to −10 mV. C, plot of the normalized chord conductance versus the command potential was created, and a Boltzmann function was fitted to the I–V plot. The mean half-activation potential was −24.5 ± 1.6 mV (n= 23), with a slope factor of 11.7 ± 0.6 mV.

Activation properties of IA in PVN-RVLM neurones

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).

Figure 3.

Voltage dependence and kinetics of inactivation of IA
A, time dependence of inactivation of IA. A command pulse to −10 mV (300 ms) was applied from a conditioning step of −45 mV of successively longer durations (10–200 ms, 10 ms increments). Note that the amplitude of the evoked IA rapidly decreased as a function of the duration of the conditioning step. The inset shows a plot of the evoked IA peak amplitude as a function of the conditioning step duration. The plot was fitted with a monoexponential function, and a time constant (τ) of 27.0 ms was obtained. B, representative example of IA currents evoked by voltage steps to −10 mV from conditioning step to between −120 and −35 mV in 5 mV increments (50 ms). C, the mean normalized current amplitude was plotted against the conditioning potential and a Boltzmann function was fitted to the I–V plot. The mean half-inactivation potential was −87.4 ± 3.1 mV (n= 21), with a slope factor of 13.1 ± 0.5 mV. Inset, a single exponential function (thick line) was fitted to the IA decay phase. The mean time constant of inactivation (τ) was 33.9 ± 3.0 ms at a command potential of −10 mV. D, when the mean normalized IA amplitude obtained with the voltage-dependent activation (s) and inactivation (▪) 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 ∼−50 mV.

The voltage dependence of inactivation was then studied. To remove variable amounts of IA inactivation, neurones were depolarized using a command pulse to −10 mV, from a range of conditioning steps (−120 to −35 mV, 5 mV increments, 50 ms duration), preceded by a fixed pulse to −40 mV (65 ms) (Fig. 3B). I–V plots were generated and fitted with a Boltzmann function, and the voltage-dependent inactivation properties were then calculated (see Methods) (Fig. 3C). The mean IA half-inactivation potential was −87.4 ± 3.1 mV, with a slope factor of 13.1 ± 0.5 mV. Similar values were obtained when conditioning steps were prolonged in duration from 50 ms to 115, 120 and 150 ms (results not shown).

The mean inactivation τ 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 (ΔVm: 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 (τ) of 65.7 ± 4.5 ms.

Figure 4.

Time course of recovery from inactivation of IA
A, representative example of IA currents evoked with command steps to −10 mV (200 ms), from conditioning steps to −100 mV of increasing duration (10 ms increments). Note that longer conditioning steps removed increasing amounts of inactivation of IA, allowing for larger IA amplitudes at the command step. B, the mean normalized current amplitude evoked at the command test was plotted against the conditioning step duration. A single exponential function was fitted to the plot, and the mean time course of recovery from inactivation (τ) of IA in PVN-RVLM neurones was calculated to be 65.7 ± 4.5 ms.

4-AP Inhibits IA in PVN-RVLM neurones

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).

Figure 5.

Inhibition of IA by 4-AP is both concentration and voltage dependent
A, representative trace of isolated IA before and after 5 mm 4-AP, at a command potential of −10 mV. B, summary data showing partial block of IA with 1 and 5 mm 4-AP. Note the larger inhibition induced by the larger 4-AP concentration. C, the mean current amplitude of IA in control ACSF and in the presence of 5 mm 4-AP was plotted versus the command potential. #P < 0.01 and *P < 0.0001 vs. control ACSF.

In a subset of neurones (n= 3), the effect of 5 mm 4-AP was tested at a wide range of command steps (−70 mV to +25 mV) (Fig. 5C). As shown in the mean I–V plot generated from these recordings, IA sensitivity to 4-AP was found to be voltage dependent (F= 63.3; n= 3; P < 0.0001, 2-way ANOVA), with larger inhibition observed at more depolarized membrane potentials.

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 MΩversus 896.8 ± 157.4 MΩ 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.

Table 1.  The effects of 4-AP upon action potential waveform parameters
 4-AP stimulated group4-AP inhibited group
ACSF5 mm 4-APACSF5 mm 4-AP
  1. P < 0.05 within same group; †P < 0.05 between different groups (same drug treatment). IA restrains firing activity in a subset of PVN-RVLM neurones.

Peak amplitude (mV)64.6 ± 1.4  70.3 ± 1.3*  61.1 ± 2.7   67.8 ± 3.1*  
Half-width (ms)3.0 ± 0.095.1 ± 0.3 *4.1 ± 0.2† 7.5 ± 0.6*†
90–10% decay time (ms)1.5 ± 0.062.9 ± 0.2* 2.0 ± 0.08†5.2 ± 0.6*†
Threshold (mV)−31.2 ± 0.8    − 32.8 ± 0.8*   −31.0 ± 0.9    −31.8 ± 1.7    

For purposes of simplicity, further results obtained from these two differently responsive PVN-RVLM neurones are presented below in separate sections.

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).

Figure 7.

4-AP increases firing discharge in a subset of PVN-RVLM neurones
A, representative examples of firing discharge in a continuously (upper trace) and bursting (lower trace) firing neurones before (left), during (middle) and after (right) bath application of 5 mm 4-AP. As summarized in the bar graphs, the firing frequency was significantly increased by 4-AP. B, representative examples of averaged spontaneous action potential waveform before (thin line) and after (thick line) application of 5 mm 4-AP, obtained from a neurone whose firing activity was increased by 4-AP. Inset, the HAP peaks were normalized and overlapped in order to more clearly depict the effects of 4-AP on the HAP slope. The mean effects of 4-AP on HAP properties are summarized in the bar graphs. *P < 0.05 versus control. C, another example of a 4-AP-induced increment in firing discharge in a PVN-RVLM neurone. In this case, negative current was injected in the presence of 4-AP, in order to diminish firing rate back to control levels. A plot of firing frequency as a function of time (10 s binning) is shown in the middle panel. In the right panel, a plot of action potential half-width as a function of time (10 s binning) is shown. Note that in the presence of 4-AP, the width of the action potentials was still prolonged, even when firing rate was similar to control levels.

To determine whether IA also modulates action potential waveform during repetitive firing activity, action potentials recorded in periods before and during 4-AP application were analysed (Methods). Similarly to the effects observed on single evoked spikes (see above), bath application of 4-AP prolonged action potential duration (∼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).

Figure 8.

4-AP dimished firing discharge in a subset of PVN-RVLM neurones
A, representative examples of firing discharge in a continuously (upper trace) and a bursting (lower trace) firing neurone before (left) and during (right) bath application of 5 mm 4-AP. As summarized in the bar graphs, the firing frequency was significantly diminished by 4-AP. B, representative examples of averaged spontaneous action potential waveforms before (thin line) and after (thick line) application of 5 mm 4-AP, obtained from a neurone whose firing discharge was diminished by 4-AP. Inset, the HAP peaks were normalized and overlapped in order to more clearly depict the effects of 4-AP on the HAP slope. The mean effects of 4-AP on HAP properties are summarized in the bar graphs. *P < 0.05 and **P < 0.01 versus control.

Similarly to the other subset of PVN-RVLM neurones, bath application of 4-AP in the subset of PVN-RVLM neurones inhibited by 4-AP, increased the amplitude of the action potential (∼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.

Figure 9.

Effects of TEA on outward K+ currents and action potential waveform in PVN-RVLM neurones
A, representative traces of outward currents evoked by a depolarizing step to −10 mV, from a conditioning step to −100 mV (500 ms), before and during bath application of 30 mm TEA. Note that TEA completely abolished the sustained outward component, while having very little effect on the transient A-type K+ component. B, representative examples of spontaneous action potentials before and during bath application of 30 mm TEA. Note that TEA induced a long lasting plateau potential on the spontaneous spike. C, summary data showing a significant decrease in the firing frequency of PVN-RVLM neurons in the presence of 30 mm TEA. *P < 0.05 versus control.

The effects of 4-AP on action potential waveform and firing activity in PVN-RVLM neurones are Ca2+-dependent

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).

Figure 10.

4-AP effects on PVN-RVLM firing discharge are Ca2+-dependent
A, the afterhyperpolarizing potential (AHP), a Ca2+-dependent process, is blocked by bath application of 200 μm Cd2+. B, in the presence of 200 μm Cd2+, 4-AP failed to affect the firing discharge of PVN-RVLM neurones. Representative traces and the summary data are shown in the upper and lower panels, respectively.

In the presence of Cd2+, 4-AP was still able to prolong the duration of the action potential. However, the magnitude of this effect was smaller (though the difference did not reach statistical significance) as compared to control conditions (4-AP-ACSF, 77.2 ± 8.7%; 4-AP-Cd2+, 55.5 ± 14.1%; P= 0.09). These results indicate that 4-AP-induced spike broadening is likely to result from both the slower action potential decaying phase and the increased Ca2+ influx during the depolarizing phase of the action potential, as previously shown in magnocellular neurosecretory neurones (Bourque et al. 1985). In fact, the 90–10% decay time course of the action potential was still significantly slowed down by 4-AP (P < 0.05) in the presence of Cd2+, to a similar extent as that observed in control ACSF (4-AP-ACSF = 115.5 ± 15.2%, 4-AP-Cd2+= 97.7 ± 26.7%, P= 0.3).

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).

Figure 11.

Kv1.2, 4.2 and 4.3 immunoreactivity in retrogradely labelled PVN-RVLM neurones
A, low magnification confocal images of the PVN (10×) showing Kv1.4 (A1), Kv4.2 (A2) and Kv4.3 (A3) immunoreactivities. The inset in A2 shows Kv4.2 immunoreactivity in the median eminence. B, high magnification confocal images of retrogradely labelled PVN-RVLM neurones (B1) and Kv 1.4 immunoreactivity (B2). Both images were superimposed in B3 to better depict colocalization of the two signals. Arrowheads in B3 point to representative Kv1.4 immunoreactive clusters located at the surface of the retrogradely labelled neurone. A magnified image is shown in the inset. C, high magnification confocal images of retrogradely labelled PVN-RVLM neurones (C1) and Kv4.2 immunoreactivity (C2). Both images were superimposed in C3. Note the lack of Kv4.2 immunoreactivity in the neurones displayed. D, high magnification confocal images of retrogradely labelled PVN-RVLM neurones (D1) and Kv 4.3 immunoreactivity (D2). Both images were superimposed in C3 to better depict colocalization of the two signals. Arrows in B–D point to the location of PVN-RVLM retrogradely labelled neurones. Scale in A1 = 100 mm (Applies to A1-A3). Scale in B1 = 10 mm (Applies to B1-D3).

Immunoreactivities for these Kv subunits were sampled in a total of 68 retrogradely labelled PVN-RVLM neurones at higher magnification. Strong immunoreactivities for Kv1.4 and Kv4.3 subunits were clearly present in PVN-RVLM neurones (32/33 and 15/16, respectively), as well as in nearby, unlabelled neurones. Representative examples are shown in Fig. 11B1–3 (for Kv1.4) and Fig. 11D1–3 (for Kv4.3). Immunoreactivities for these subunits were highly punctate in nature, with immunoreactive clusters located within the cytoplasm as well as near the surface membrane, both in somatic and dendritic compartments. In the case of Kv4.3 subunits, nuclear immunoreactivity was also evident. On the other hand, weak or undetectable Kv4.2 immunoreactivity was observed in PVN-RVLM, or other PVN neurones (Fig. 11C1–3).

Discussion

The PVN is a highly heterogeneous region containing both magnocellular neurosecretory (type I) and parvocellular (types II–III) neurons, the latter including both neurosecretory and preautonomic neurons (Swanson & Sawchenko, 1980, 1983). Previous studies indicate that these two major PVN populations can be distinguished based on their electrophysiological properties. Thus, while magnocellular neurosecretory neurons are characterized by the expression of a robust transient outward rectification (TOR) (Bourque & Renaud, 1991; Stern & Armstrong, 1995; Luther & Tasker, 2000), parvocellular neurons, including the preautonomic ones, are characterized by their ability to generate a Ca2+-dependent low-threshold spike (LTS) (Luther & Tasker, 2000; Stern, 2001). Elegant work by Tasker and colleagues showed that while both IA and IT are in fact present in both types of PVN neurons, differences in their relative expression and voltage-dependent properties determine their relative contribution to the general membrane properties characteristic of these two neuronal populations (i.e. TOR and LTS, respectively). In agreement with these previous studies, results from our present work support that in addition to IT (Stern, 2001), preautonomic PVN neurons also express a functionally relevant IA, which shapes the Na+ action potential waveform and modulates their firing activity. As discussed below, the Ca2+ dependency of IA effects on PVN-RVLM firing activity suggests that K+ and Ca2+ conductances actively interact to modulate neuronal excitability in this PVN neuronal population.

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 MΩ, compared to neuronal input resistance of ∼1000 MΩ), (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 blockade of IA, and/or its voltage-dependent inactivation, affected both action potential waveform and firing discharge properties of PVN-RVLM neurones. Importantly, the firing activity of these neurones was differentially affected by IA blockade: while the majority of the neurons (60%) responded to 4-AP with an increased firing rate, opposite effects were observed in the remainder, suggesting a differential role of IA within PVN-RVLM neurones. The precise mechanisms underlying such disparate roles are at present unknown. However, some insights could be drawn from the present results. Worth noting is the fact that some basic intrinsic properties differed between the two subpopulations of 4-AP sensitive PVN-RVLM neurones (e.g. resting Vm, degree of spontaneous activity, action potential duration), suggesting the presence of distinct subsets of PVN-RVLM neurones. This is in agreement with previous observations indicating a high degree of functional and neurochemical heterogeneity among preautonomic PVN neurones, even within those innervating a common target (Swanson & Sawchenko, 1980, 1983; Stern, 2001). Alternatively, differences in 4-AP sensitivity could represent variability within a single population of PVN-RVLM neurones. Future studies comparing for instance the neurochemical identity of these subsets of PVN-RVLM neurones will provide more insights into this important phenomenon. Nonetheless, the overall lack of significant correlations between 4-AP-induced changes in firing activity and any of the parameters that differed between the two subpopulation of PVN-RVLM neurones indicates that the opposing effects of 4-AP on firing discharge were not due per se to differences in these parameters. Similarly, both 4-AP sensitive subgroups of PVN-RVLM neurones displayed either continuous or bursting firing patterns, indicating that initial firing patterns were not factors influencing the type of response induced by IA blockade.

The general properties of individual action potential waveforms in the two subsets of PVN-RVLM neurones were similarly affected by 4-AP (i.e. spikes were increased in amplitude and prolonged in duration, the latter likely to be due to increased spike width and slower decay times). These results indicate that IA shapes action potential waveform in PVN-RVLM neurones, as previously described in other PVN and SON neurones as well (Hlubek & Cobbett, 1997; Luther & Tasker, 2000), and also suggest that changes in these action potential parameters per se do not underlie the differential effect of 4-AP on the firing discharge of these neurones.

Conversely, other neuronal properties were differentially affected by 4-AP in these two subsets of PVN-RVLM neurones. As previously shown in SON and PVN magnocellular neurosecretory neurones (Andrew & Dudek, 1984; Bourque et al. 1985), the repolarizing phase of the action potential in PVN-RVLM neurones was followed by a prominent HAP. In those neurones in which an increase in firing rate was observed, the HAP peak reached a more hyperpolarized membrane potential in 4-AP, an effect expected to result in a more efficient removal of Na+ channel inactivation following each action potential. This in turn may contribute to the more hyperpolarized Na+ spike threshold observed in this subset of PVN-RVLM neurones. In fact, previous studies have shown that modulation of the degree of Na+ channel inactivation is an effective mechanism by which IA modulates firing properties (Colbert et al. 1997; Hoffman et al. 1997). Moreover, a steeper HAP decay slope, with concomitant shorter interspike intervals, was also observed in this group in the presence of 4-AP. Thus, these combined actions of 4-AP (i.e. hyperplolarized HAP peak, faster HAP decay time course, shorter interspike intervals, and hyperpolarized Na+ spike threshold) are likely to contribute to the enhanced firing discharge found in this subset of PVN-RVLM neurones.

On the other hand, in those neurones in which 4-AP decreased their firing discharge, these parameters were either unchanged, or affected in opposite ways by 4-AP. For example, a slower HAP decay time course, leading in turn to an overall enhanced HAP area, was induced by 4-AP, effects that are likely to have contributed to the decreased interspike interval found in this subgroup of PVN-RVLM neurones.

In summary, our results indicate that IA differentially modulates firing discharge in PVN-RVLM neurones, actions that seem to be in part mediated by differential and/or opposing modulatory effects on HAP parameters. These results raise the intriguing question as to how such disparate actions can be mediated by IA.

The effects of IA on PVN-RVLM firing activity are Ca2+-dependent

An important finding of the present work is that 4-AP effects on the firing activity of PVN-RVLM neurones were blocked by the broad spectrum Ca2+ channel blocker Cd2+. Several mechanisms could be proposed to underlie this Ca2+-dependent effect. Firstly, it could be argued that these effects are due to Ca2+-dependent inhibition of IA, as previously shown in magnocellular supraoptic neurons (Bourque, 1988). However, the fact that a large IA component was always present in a 0 Ca2+/EGTA/Cd2+-containing ACSF (see Figs 2–5), along with lack of evidence for a diminished IA amplitude before and after addition of Cd2+ to control ACSF (not shown), argues against this possibility. Secondly, blockade of IA could result in amplification and/or prolongation of the Ca2+-dependent LTS typically found in preautonomic PVN neurons (Fig. 1; Stern, 2001), as previously shown in other neuronal populations (Cavelier et al. 2003; Pinato & Midtgaard, 2005). Whether the magnitude of the LTS in PVN-RVLM neurones is dependent on IA availability remains to be determined.

Finally, 4-AP effects on PVN-RVLM firing discharge could be dependent on downstream Ca2+-dependent mechanisms activated by enhanced Ca2+ entry per spike, as a consequence of prolonged action potentials following blockade of IA. In various neuronal types, including magnocellular neurosecretory neurons, action potential-mediated Ca2+ influx leads to activation of a variety of Ca2+-dependent K+ channels (e.g. BK and SK channels), which reduce in turn neuronal excitability by inducing HAPs and spike frequency adaptation (Bourque et al. 1985). Conversely, activity-dependent Ca2+ influx my lead to increased excitability, by means of Ca2+-dependent decrease in resting K+ currents (Li & Hatton, 1997), or through Ca2+-activated non-selective cation currents (Partridge & Swandulla, 1988; Ghamari-Langroudi & Bourque, 2002). These latter conductances have been proposed to underlie depolarizing afterpotentials (DAPs) in vasopressin neurosecretory neurones, a property that effectively competes with HAPs to influence the firing discharge in these neurones. Thus, depending on the complement of Ca2+ and Ca2+-dependent conductances available within particular neuronal types, accumulation of intracellular Ca2+ during spiking activity may result in either increased or decreased membrane excitability. Thus, it is tempting to speculate that the differential effects of 4-AP on the firing discharge of PVN-RVLM neurones could be due to the expression of different complements of Ca2+ and Ca2+-dependent K+ currents in subsets of PVN-RVLM neurones. Future studies are warranted to characterize the expression and functional properties of such conductances in these preautonomic PVN neurons, and their interaction with IA.

In summary, these studies provide the first characterization of the biophysical and pharmacological properties of IA in identified sympathetic-related PVN neurones, and support an active role of IA in the regulation of PVN-RVLM action potential waveform and firing activity. It will be important to determine in future studies whether changes in IA properties may contribute to altered PVN-RVLM neuronal excitability during pathological conditions, as previously shown in NTS neurones such as hypertensive disorders (Belugin & Mifflin, 2005).

Appendix

Acknowledgements

This grant was supported by National Heart, Lung and Blood Institute Grant ROI-HL-68725 (J. E. Stern).

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