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1 School of Biomedical Sciences, Worsley Building, University of Leeds, Leeds LS2 9NQ, UK
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Abstract |
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(Received 5 August 2004;
accepted after revision 28 October 2004;
first published online 4 November 2004)
Corresponding author J. Deuchars: School of Biomedical Sciences, Worsley Building, University of Leeds, Leeds LS2 9NQ, UK. Email: j.deuchars{at}leeds.ac.uk
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Introduction |
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While the precise cellular organization of the hippocampus and cerebellar cortex facilitates identification of the ion channel complement and distribution within specific neuronal classes (e.g. Lorincz et al. 2002), this is more difficult in less-ordered CNS regions. One such area is the nucleus of the solitary tract (NTS) in the dorsomedial brainstem. The NTS plays a pivotal role in maintaining many homeostatic mechanisms as it integrates information from numerous other brain areas, as well as peripheral structures via sensory nerves (Barraco et al. 1992). Many studies have examined the neurochemistry of the NTS (e.g. Maley, 1996; Lawrence & Jarrott, 1996), and others have revealed that the NTS contains neurones with diverse firing properties (e.g. Haddad & Getting, 1989; Fortin & Champagnat, 1993; Kawai & Senba, 1996, 1999; Deuchars et al. 2000). However, the molecular identity of ion channels that underlie the firing properties of NTS neurones has rarely been determined.
Here we report Kv3.1b subunit immunoreactivity throughout the medulla oblongata in cell somata and presynaptic terminals. Electrophysiological recordings from NTS neurones in slices provide evidence for functional roles consistent with the localization of Kv3.1b. Some of these data have been published in abstract form (Atkinson & Deuchars, 2000; Dallas et al. 2002, 2004).
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Immunohistochemistry
Wistar rats (150–200 g, n = 10) were anaesthetized intraperitoneally with Sagatal (60 mg kg–1) and transcardially perfused with 4% paraformaldehdye (PFA) and between 0.05 and 0.2% glutaraldehyde (in 0.1 M phosphate buffer, PB; pH 7.4). For GABA and glutamate immunohistochemistry, Wistar rats (150–200 g, n = 6) were anaesthetized with intraperitoneal Sagatal (60 mg kg–1) and transcardially perfused with 4% PFA/4% carbocyamide/0.05% glutaraldehdye in 0.1 M PB. The brains and/or nodose ganglia were removed and post fixed in the same solution for 2 h at 4°C. Coronal (50 µm) sections of the relevant tissues were then cut on a vibrating microtome (Leica, Milton Keynes, UK) and collected into phosphate buffered saline (PBS; pH 7.2).
Immunohistochemistry was performed by incubating sections in primary antibody raised against the end portion of the intracellular C-terminus (residues 567–585) of the rat Kv3.1b channel subunit (Alomone Laboratories, Jerusalem, Israel; 1:1000 in PBS/0.1% Triton X-100, raised in rabbit) or rat Kv3.2 (1:1000 in PBS; Alomone Laboratories) for 12–18 h at 4°C. For fluorescence, these antibodies were visualized using Cy3-conjugated antirabbit antibodies (1:1000 in PBS; Stratech Scientific, Luton, UK) for 
4 h at room temperature. Sections were then air-dried onto slides and mounted under a coverslip using VectaMount mounting medium (Vector Laboratories, Burlingame, CA, USA).
Dual labelling.
To determine the percentage of neurones that contained Kv3.1b immunoreactivity (Kv3.1b-IR), sections containing Cy3-labelled Kv3.1b-IR were incubated in a neuronal nuclear marker (NeuN) primary antibody (1:1000 in PBS; Chemicon, Hertfordshire, UK; raised in mouse) overnight at 4°C. Following washes in PBS, the sections were incubated in a biotinylated mouse secondary antibody (1:500; Stratech Scientific) for 4 h, washed again in PBS and then incubated in streptavidin-conjugated Alexa488 (1:1000; Molecular Probes) for 2 h. The number of NeuN-IR and Kv3.1b-IR neurones were counted in representative sections of levels of the medulla relative to the area postrema. For counting purposes the NTS was subdivided into medial (classically defined medial and commissural aspect of the tractus solitarius, TS) and lateral (encompassing the TS and surrounding subdivisions: interstitial, dorsolateral, ventrolateral, ventral, intermediate and dorsal). Dual-labelled cells were expressed as a percentage of the total number of cells. To determine the neurochemistry of Kv3.1b-IR neurones, some labelled sections were incubated in mouse anti-GABA and mouse antiglutamate (both at 1:1000 in PBS/0.1% Triton X-100; Sigma, Dorset, UK), visualized and detected as above.
Some sections were also processed for light and electron microscopy as described in detail previously (Atkinson & Deuchars, 2000). In brief, sections were incubated in anti-Kv3.1b (1:1000 in PBS) which was localized using biotinylated antirabbit secondary antibody (1:200 in PBS, Stratech Scientific) prior to detection with ExtrAvidin peroxidase (1:1500 in PBS; Sigma) and visualization using diaminobenzidine (DAB) as the chromogen. Sections were then postfixed in 0.5% osmium tetroxide (in 0.1 M PB) and dehydrated before embedding in Durcupan ACM resin (Fluka, Switzerland). Kv3.1b-IR NTS cells were drawn under the x20 objective using a camera lucida drawing tube. Areas with suitable staining were prepared for electron microscopy, and viewed on a Phillips CM10 transmission electron microscope (EM). Negatives were digitized using an Umax 2200 scanner, and adjusted in Corel Draw 10 (Corel, Maidenhead, Berkshire, UK) until the desired brightness, contrast and gamma levels were reached.
Control sections were incubated for 12–24 h as above, either in PBS in place of the primary antibody or in primary antibody which had been preabsorbed with peptide antigen (1 µg per 1 µg of primary antibody; Alomone Laboratories) for 1 h at room temperature. These sections were then incubated in biotinylated secondary antibody, followed by ExtrAvidin peroxidase, and reacted using DAB as described previously. All control sections were free from immunoreactivity.
Vagal efferent cell and afferent fibre labelling combined with Kv3.1b immunohistochemistry
To retrogradely label vagal efferent cells, four male Wistar rats (150–250 g) were injected intraperitoneally with 100 µl of Fluorogold (2% in distilled water; Fluorochrome Inc., Denver, CO, USA) 7 days prior to perfusion. Vagal afferent fibres were labelled by injection of 5–10 µl of 10% biotinylated dextran amine (BDA, molecular mass 10 000 kDa; Molecular Probes) into the right nodose ganglion of seven Wistar rats (150–250 g) under halothane anaesthesia (5% in O2) 7–10 days prior to perfusion and sectioning as described previously. BDA was visualized using ExtrAvidin peroxidase and a DAB reaction, while anti-Kv3.1b antibodies were visualized using pre-embedding gold procedures (Atkinson et al. 2000).
Kv3.1b-IR and GAD65/VGLUT2 in situ hybridization in the NTS
Wistar rats (150–200 g, n = 6) were anaesthetized with intraperitoneal sagatal (60 mg kg–1), perfused transcardially with 4% PFA, and coronal (30 µm) sections of the medulla were cut as described previously. In situ hybridization was conducted using previously published protocols (Stornetta et al. 2002). To visualize GABAergic neurones single-stranded digoxigenin (DIG)-UTP-labelled (Roche Molecular Biochemicals, Switzerland) sense (control) and antisense riboprobes were transcribed from a 2.3 kb GAD65 DNA template plasmid kindly supplied and previously characterized by A. Tobin (Wuenschell et al. 1986; Esclapez et al. 1993). For glutamatergic neurones, sense and antisense VGLUT2 riboprobes were transcribed from a 3.3 kb DNA template kindly supplied and previously characterized by R. Stornetta (Stornetta et al. 2002). Hybridized mRNA was visualized with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) diluted in Tris-HCl/MgCl2, pH 9.5. The sections were then incubated in Kv3.1b primary antibody (1:500) for 12–18 h at 4°C, washed for 3 x 10 min in PBS, and visualized by incubation in a Cy3-conjugated secondary antibody as described previously.
Image capture
Slides were viewed on a Nikon E600 microscope equipped with epifluorescence. Images were captured directly from the slide using an Aquis Image Capture system (Synoptics, Cambridge, UK), and adjusted in CorelDraw 10 until the desired gamma, brightness and contrast levels were reached.
RT-PCR
Wistar rats (150–200 g, n = 5) were anaesthetized with Sagatal (60 mg kg–1), and transcardially perfused with artificial cerebrospinal fluid (aCSF). The medulla and nodose ganglia were removed, and RNA was isolated from these tissues using TRI reagent (according to the manufacturer's instructions). RT-PCR was then performed using the Kv3.1 primers (sense: 5'-CAAGAGATTGGCGCTCAGTGAC-3', antisense: 5'-CCCAGRGCCAGRAAGATGATMAGCA-3') which detects a 584 bp sequence of the Kv3.1 channel corresponding to the amino acid sequence 153–354. The PCR cycling conditions were: 94°C for 5 min, 94°C for 45 s, 57°C for 30 s, 72°C for 45 s for 35 cycles. The RT-PCR product was then run on a 2% agarose gel and viewed under a Hybaid Illuminator.
Electrophysiological experiments
Slice preparation.
Wistar rats (18–21 days) were terminally anaesthetized with intraperitoneal sodium pentobarbitone (120 mg kg–1). The brain was removed and placed in ice-cold sucrose aCSF containing (mM): sucrose (217); NaHCO3 (26); KCl (3); MgSO4 (2); NaH2PO4 (2.5); CaCl2 (2); and glucose (10); that received a continual supply of mixed gas (95% O2, 5% CO2). Slices (300 µm) of the medulla and cerebellum were cut using a Vibroslice (Campden Instruments, Sileby, UK) and transferred to a holding chamber containing aCSF (mM): NaCl (124); NaHCO3 (26); KCl (3); MgSO4 (2); NaH2PO4 (2.5); CaCl2 (2); glucose (10); equilibrated with 95% O2, 5% CO2. Visualized patch-clamp recordings were carried out at room temperature using an upright microscope with DIC optics (BX50WI; Olympus Optical, Tokyo, Japan). Patch electrodes were filled with intracellular solution consisting of (mM): potassium gluconate (110); EGTA (11); MgCl2 (2); CaCl2 (0.1); Hepes (10); Na2ATP (2); NaGTP (0.3); and had a resistance of 6 M
. In some experiments, Lucifer yellow (0.1%) and/or the primary antibody raised against Kv3.1b (1:1000; Alomone Laboratories), or a primary antibody raised against the hyperpolarization-activated cyclic nucleotide-gated K+ channel 1 subunit (HCN1, 1:1000; Alomone Laboratories) were added to the intracellular solution.
Current-clamp mode recordings
Whole-cell patch-clamp recordings were made in current-clamp mode using an Axopatch ID (Axon Instruments) from neurones ventral, and immediately adjacent to, the TS at the level of the area postrema, where the majority of Kv3.1b-IR neurones are situated. In particular, larger neurones with proximal dendrites running horizontally were selected (Fig. 2E) as their morphological characteristics corresponded to those of Kv3.1b-IR neurones detected by immunohistochemical experiments. Neurones were characterized as previously been described (Deuchars et al. 2001), following which depolarizing current pulses (+ 10 to +300 pA) were applied to examine the firing properties of the neurones. Tetraethylammonium chloride (TEA; 0.5 mM) and 4-aminopyridine (4-AP; 30 µM) (both from Sigma) were applied to the bathing medium at the rate of 3–5 ml min–1. While TEA may act at other channels at this concentration, overlapping sensitivity to both drugs strongly suggests an action on Kv3.1 subunits (Coetzee et al. 1999; Deuchars et al. 2001). To investigate the presynaptic actions of 4-AP and TEA, a bipolar stimulating electrode (voltage 9–18 V, duration 100 µs; model DS2A; Digitimer, Hertfordshire, UK) was placed in the TS to evoke postsynaptic potentials within NTS neurones. Recordings were carried out in current-clamp mode since we were particularly interested in how the drugs affected the firing characteristics of the neurones. Consequently, measurements of the electrophysiological characteristics were made as has previously been described (Deuchars et al. 2001). In addition, firing patterns were analysed in response to 1 s depolarizing current pulses, and early accommodation was calculated using the following equation: (instantaneous frequency – frequency at 500 ms)/instantaneous frequency. Late accommodation was calculated as follows: (frequency at 500 ms–frequency at 1000 ms)/instantaneous frequency.
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Voltage-clamp recordings
To isolate outward voltage-dependent K+ currents, tetrodotoxin (TTX; 1 µM; Sigma) was bath applied, and the Ca2+ was replaced with magnesium (4 mM) in the aCSF. Preliminary experiments revealed a low-threshold outward delayed rectifier K+ current characteristic of that carried by Kv1 channels, so in addition alpha-dendrotoxin (
-DTX; 100 nM; Alomone) was bath applied. Outside-out somatic patches were obtained from NTS neurones from the same location as those used for current-clamp recordings. Series resistance in the patches (18.2–20.8 M, mean 19.4 ± 0.92 M) was monitored periodically, and compensated (60–80%) throughout. Where the series resistance increased to a value greater than 30 M, or was unstable, recordings were discontinued. Recordings were corrected for a liquid junction potential of –14.4 mV, as calculated using Clampex 8.1 software. Currents were leak subtracted online using a P/4 subtraction protocol from a holding potential of –110 mV. Signals were low-pass filtered at 5 kHz (four-pole Bessel), and sampled at 50 kHz. K+ currents recorded from the outside-out patches are an average of five trials recorded at each voltage step. K+ conductance (Gv)–voltage curves were fitted using Kaleidograph software with a single, first-order Boltzmann function of the form: Gv
=
Gmax/{1 + exp[–(V – V
)/k]}, where V is the membrane voltage, V is the half-activation voltage, and k is the slope factor. Offline analysis was carried out using the Clampfit 8.1 analysis package.
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Results |
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At the levels studied (1.3 mm rostral to 0.4 mm caudal to the obex), Kv3.1b subunit immunoreactivity (Kv3.1b-IR) was observed in a large number of medullary areas (Fig. 1A), where it was present in the somatic membrane and proximal dendrites of neurones, with limited labelling of dendritic arborizations, similar to that described in the cortex (Sekirnjak et al. 1997). Dual immunohistochemistry for the Kv3.1b subunit and the neuronal nuclear antibody NeuN revealed that Kv3.1b-IR was present in 100% of neurones in the cuneate (408/408) and gracile (530/530) nuclei (Fig. 1B and C). A low number of Kv3.1b-IR cells were observed in the vicinity of the nucleus ambiguus; however, these Kv3.1b-IR cells were not vagal efferent neurones, as they were not labelled following intraperitoneal injection of Fluorogold (Fig. 1E). Kv3.1b-IR was observed in 100% of the densely packed neurones in the paratrigeminal (240/240) and spinal trigeminal nuclei (Fig. 1F, 1240/1240). Approximately 98% of neurones in the raphe obscurus nucleus were also Kv3.1b-IR (63/63). Extremely rare Kv3.1b-IR somata were present in the hypoglossal nucleus, but these never contained Fluorogold and were therefore unlikely to be motoneurones (not shown). Somatic Kv3.1b-IR was not observed in the dorsal vagal motor nucleus (DVN; Fig. 6C), the inferior olive, raphe pallidus or the area postrema. In these areas, punctate Kv3.1b-IR was observed suggestive of terminal labelling, and such punctate labelling was also observed in the hypoglossal nucleus (Fig. 1G).
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Kv3.1b-IR was present in approximately 13% (111/825) of NeuN-labelled NTS neurones. The soma of Kv3.1b-IR neurones were predominantly round or elongated, and ranged from 25 to 50 µm in length. Kv3.1b-IR neurones also had visible proximal dendrites that ran predominantly in the mediolateral axis. Camera lucida mapping revealed that Kv3.1b-IR NTS neurones were primarily located in the vicinity of the TS. Caudally (at the level of the central canal) a few Kv3.1b-IR neurones were observed in the commissural and medial subdivisions of the nucleus and within the TS (Fig. 2A). More rostrally, a greater number of Kv3.1b-IR cells were present in the medial, dorsolateral, interstitial and ventrolateral subdivisions and within the TS (Fig. 2B and C). Kv3.1b-IR neurones were approximately 36% of NeuN-IR cells in these areas (111/309 NeuN cells, 36%). However, since it was sometimes difficult to differentiate whether NTS neurones themselves contained Kv3.1b-IR or were closely apposed by Kv3.1b-IR structures, this analysis of colocalization is likely to be an underestimate.
At the light microscope level, Kv3.1b-IR NTS cells contained reaction product which was predominantly located close to the cell membrane of the soma and proximal dendrites, similar to that observed in the other areas of the medulla. Ultrastructural examination of such Kv3.1b-IR neurones revealed that Kv3.1b reaction product was indeed located close to the somatic and proximal dendritic membranes indicating that the channel is membrane bound (Fig. 2D). Kv3.1b reaction product observed in the cytoplasm of labelled NTS neurones may represent channels being transported to or from the plasma membranes, as it was often detected near to cellular structures such as the Golgi apparatus (Fig. 2D).
Both GABA-IR and Glut-IR cells in the proximity of the tractus contained Kv3.1b-IR (Fig. 3A–D). In addition, VGLUT2 or GAD65 mRNA was detected in Kv3.1b-IR cells (Fig. 3E–H). Since immunohistochemistry for GABA and glutamate in the NTS may provide either false-negative or false-positive results (Fremeau et al. 2002; Kaneko & Fujiyama, 2002), and in situ markedly reduced the number of visible Kv3.1b-IR neurones, we did not quantify the degree of colocalization in either case.
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Whole-cell patch-clamp recordings were obtained from 102 neurones in the ventral aspect of the TS at the level of the area postrema. This area was targetted due to the relatively high density of Kv3.1b-IR neurones in the NTS. In addition, large neurones with proximal dendrites running in the mediolateral plane were preferentially selected (Fig. 2E) as this corresponds to the morphology of Kv3.1b-IR neurones. A total of 81/102 neurones displayed electrophysiological characteristics which may be supported by the presence of the Kv3.1b subunit (Table 1): rapid action potentials, high firing frequencies and sensitivity to TEA and 4-AP represented by increases in action potential duration (TEA, 4.6 ± 0.5 to 10.4 ± 1.5 ms; 4-AP, 4.2 ± 0.8 to 10.9 ± 1.2 ms; P < 0.05, n = 81; Fig. 4A, B and F). This effect was due to the repolarization phase being prolonged, and these pharmacological agents also reduced the AHP amplitude (TEA, 16.4 ± 1.3 to 11.9 ± 3 mV; 4-AP, 15.6 ± 1.1 to 10.9 ± 0.8 mV; P < 0.05; Fig. 4A, B and F). As a negative control, 14 neurones located in the DVN, which do not contain Kv3.1b immunoreactivity (Figs 1 and 6C), did not respond to the same concentrations of TEA (action potential duration: pre-TEA, 5.4 ± 0.9 ms; post-TEA, 5.4 ± 1.2 ms; AHP amplitude: pre-TEA, 17.9 ± 6.7 mV; post-TEA 17.7 ± 5.6 mV; n = 14, P = n.s.; Fig. 4C) or 4-AP (action potential duration: pre-4-AP, 5.3 ± 1.3 ms; post-4-AP 5.3 ± 1.1 ms; AHP amplitude: pre-4-AP, 18.6 ± 7.5 mV; post-4-AP 18.5 ± 5.3 mV; P = n.s.; Fig. 4D).
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Whilst the majority of 4-AP- and TEA-responsive neurones were located close to the solitary tract in ventral and ventrolateral regions, those that were unresponsive were predominantly localized more medially towards the commissural NTS (Table 1). The 4-AP- and TEA-sensitive neurones displayed shorter action potential half-widths, larger AHP amplitude, higher instantaneous firing frequency and less late accommodation than those insensitive to 4-AP and TEA (Table 1).
The Kv3.1b subunit contributes to action potential repolarization and firing frequency of NTS neurones
Our electrophysiological studies with 4-AP and TEA are consistent with a Kv3.1-subunit-containing channel contributing to action potential repolarization in NTS neurones, supporting our immunohistochemical data that the Kv3.1b subunit was present in somata in the NTS. Previous studies have used Kv-subunit-specific antibodies to identify the subunit composition of ion channels, and there is growing evidence that these can have functional effects (Trimmer & Rhodes, 2004), which in expression systems commence within 8–10 min, and display time-dependent blockade (Murakoshi & Trimmer, 1999; Conforti et al. 2000), whilst first effects are observed with a longer time course in native cells (Archer et al. 1998; Lu et al. 2002; Sanchez et al. 2002). Since there is a lack of pharmacological tools that are selective for Kv3.1 isoforms, we sought to confirm the involvement of the Kv3.1b subunit in these cells by introducing the antibody to the inside of the cell via the patch pipette. Since the antibody is directed specifically against the intracellular C-terminus of the Kv3.1b subunit, our reasoning was that it would disrupt only channels containing the Kv3.1b subunit.
In NTS neurones, intracellular application of anti-Kv3.1b antibody resulted in progressive prolongation of the action potential duration (from 4.6 ± 0.7 to 7.7 ± 0.8 ms at 30 min; P < 0.05, n
= 7; Fig. 5A). After 30 min, 4-AP could still produce a further significant increase in the action potential duration (7.7 ± 0.8 to 10.1 ± 1.1 ms; P < 0.05, n
= 7), which was reversed upon washout of the 4-AP (Fig. 5Ba). However, repeated applications of 4-AP revealed a progressive blockade of 4-AP-sensitive channels due to the antibody, since at 65 min, 4-AP had no further effect on the action potential duration (11.2 ± 0.8 to 11.4 ± 0.7 ms; P
= n.s., n
= 7; Fig. 5Bb). In addition, decreases were observed in instantaneous (t
= 0 min, 61.5 ± 2.8 Hz; t
= 30 min, 57 ± 2.5 Hz; t
= 60 min, 54.8 ± 3 Hz; P < 0.05, n
= 7) and steady-state (t
= 0 min, 50.2 ± 3.2 Hz; t
= 30 min, 45.5 ± 2.3 Hz; t
= 60 min, 41.4 ± 2 Hz; P < 0.05, n
= 7) firing frequencies as well as an increase in late accommodation (t
= 0 min, 1.7 ± 0.3%; t
= 30 min, 5 ± 0.6%; t
= 60 min, 7.8 ± 0.7%; P < 0.05, n
= 5). There was no significant change in the input resistance (t
= 0 min, 796 ± 41 M; t
= 30 min, 801 ± 39 M; n
= 7) or resting membrane potential (t
= 0 min, 65.8 ± 5.1 mV; t
= 30 min, 67.4 ± 6.3 mV; n
= 7) during the time taken for the antibody to have an effect. There was no significant change in the duration of the action potential over the 60 min recording period when the antibody was not present (t
= 0 min, 4.1 ± 0.2 ms; t
= 30 min, 4.2 ± 0.4 ms; t
= 60 min, 4.1 ± 0.4 ms;
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Additionally, to test whether the effects of intracellular application of the Kv3.1b antibody to NTS neurones were not merely due to intracellular application of an antibody, we conducted the same experiments using an antibody raised against HCN1 which is rarely present in the NTS (Brooke et al. 2004a). Intracellular application of the HCN1 antibody at the same concentration as the Kv3.1b antibody, which is also the effective concentration for immunohistochemistry (Brooke et al. 2004a), did not significantly alter the action potential characteristics in NTS neurones (action potential duration: control, 4.0 ± 0.2 ms; HCN1 Ab, 4.2 ± 0.1 ms; AHP amplitude: control, 24.2 ± 1.3 mV; HCN1 Ab, 24.9 ± 1.3 mV; P = n.s., n = 6; Fig. 5C) or DVN neurones (data not shown). The neurones located within the NTS were, however, still responsive to TEA following intracellular dialysis of the HCN1 antibody. As before, TEA led to a significant increase in the action potential duration and a decrease in the AHP, and was fully reversible on wash out (action potential duration: HCN1 Ab, 4.1 ± 0.1 ms; HCN1 Ab + TEA, 7.6 ± 0.2; P < 0.05; AHP amplitude: HCN1 Ab, 24.9 ± 1.4 mV; HCN1 Ab + TEA, 19.9 ± 0.9 mV; P < 0.05, n = 6; Fig. 5C). This indicates that introduction of the antibody intracellularly does not interfere with nonspecific targets.
As further verification that the Kv3.1b antibody was acting on Kv3.1b-containing channels, we recorded from neurones in the dorsal column nuclei (DCN), which all contained Kv3.1b-IR (Fig. 1B and C). In control recordings without antibody in the pipette DCN, neurones responded to TEA (2/2) and/or 4-AP (4/4) with an increase in action potential duration (4-AP, 2.1 ± 0.3 to 3 ± 0.1 ms; P < 0.05). When recordings were made with the antibody present in the pipette during the experiments the action potential duration progressively increased (AP duration: t = 0 min, 2 ± 0.2 ms; t = 50 min, 2.9 ± 0.3 ms; P < 0.05, n = 4; Fig. 5Da). During initial stages of recordings, 4-AP prolonged action potential duration similar to control cells (AP duration: t = 15 min, 2.3 ± 0.2 to 2.8 ± 0.1 ms; P < 0.05, n = 4; Fig. 5Da). However, after 50 min, 4-AP had no further effect (AP duration: 2.91 ± 0.2 to 2.93 ± 0.1 ms; P = n.s., n = 4, Fig. 5Db).
We further tested whether the Kv3.1b antibody could be acting on other Kv3 subunits by intracellular application to cerebellar Purkinje neurones which contain Kv3.2 (Fig. 6E), Kv3.3 and Kv3.4 subunits (Weiser et al. 1994; Goldman-Wohl et al. 1994; Martina et al. 2003) but not Kv3.1b subunits (Fig. 6D; Weiser et al. 1995; Sekirnjak et al. 1997). The presence of the Kv3.1b antibody had no effect on the duration of the action potential (Fig. 6B), measured from first action potential at t = 0 min, 1.2 ± 0.04 ms; t = 30 min, 1.2 ± 0.03 ms; t = 60 min, 1.2 ± 0.04 ms; P = n.s., n = 6) or amplitude of the AHP (t = 0 min, 21.2 ± 3.6 mV; t = 30 min, 20.8 ± 2.2 mV, t = 60 min 19.8 ± 2.3 mV; n = 6). TEA (100 µM) applied to three of these six cells increased the action potential duration (applied at t = 45 min, the duration increased from 1.2 ± 0.1 to 1.8 ± 0.2 ms), and this effect was reversed upon removal and wash of TEA (recovery upon 15 min washout = 1.3 ± 0.1 ms; Fig. 6B). This TEA-induced increase in action potential duration observed in the Purkinje neurones was consistent with previous studies (Sacco & Tempia, 2002). These data indicate that the Kv3.1b antibody did not interfere with the Kv3 channel subunits present in cerebellar Purkinje cells.
Voltage-clamp studies reveals a TEA-sensitive K+ current in NTS neurones
To investigate the basic properties of, and provide further evidence for, the Kv3 current within NTS neurones, we conducted voltage-clamp experiments using outside-out patches in the presence of 100 nM
-DTX to block Kv1-subunit-containing channels (Coetzee et al. 1999). Somatic outside-out patches were excised within 30 s of establishing the whole-cell configuration, and were held at a holding potential of –110 mV. Depolarizing voltage steps to +30 mV (10 mV increments, 100 ms duration) revealed a voltage-dependent outward current (Fig. 7A, upper trace). All patches yielded a significant outward current of at least 200 pA when stepped to +30 mV. The steady-state outward current had a mean amplitude of 719 ± 114 pA (n
= 8) for a voltage step to +30 mV. The mean maximum conductance was 8.1 ± 0.7 nS (n
= 8). Initial trials indicated that we were unable to maintain high-resistance seals and satisfactory voltage control for sufficient durations when the antibody was present in the pipette and, therefore, we used pharmacological manipulations to isolate Kv3 currents, as previously reported (e.g. Coetzee et al. 1999; Southan & Robertson, 2000).
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-DTX, TEA was applied to patches at a concentration of 100 µM, which is known to block Kv3-subunit-containing channels (Coetzee et al. 1999). All patches were sensitive to TEA at this concentration (n
= 8), and a reduction in the total outward K+ current was observed (48.4 ± 6.5%, n
= 6/6
-DTX-sensitive patches). Recovery (>60%) was observed following washout of TEA in all patches. Offline digital subtraction revealed the TEA-sensitive component (mean steady amplitude 414 ± 132 pA; n
= 6), which activated in a voltage-dependent manner from a threshold of around –30 mV (Fig. 7A, lower trace). K+ conductance–voltage curves of the TEA-sensitive current were fitted with a single, first-order Boltzmann function, yielding a mean V of 0.1 ± 5.3 mV, and slope factor of 10.1 ± 2.2 mV (n
= 5; Fig. 7B). The kinetics of this -DTX-insensitive, TEA-sensitive current correlate with those previously published for Kv3-containing channels in both expression systems and native neurones (see Rudy et al. 1999; Lien & Jonas, 2003; Baranauskas et al. 2003; McCrossan et al. 2003). Kv3.1b immunoreactivity is present in presynaptic terminals in the NTS, some of which are vagal afferent in origin
Ultrastructural examination revealed Kv3.1b-IR in numerous axons running in the TS (Fig. 8B) and presynaptic terminals (n = 35) in the NTS. These labelled terminals appeared to be restricted to the levels more rostral to the area postrema in the medial area of the NTS, and they formed synapses of both the symmetric and asymmetric type with Kv3.1b-labelled and unlabelled dendrites within this region (Fig. 8A, C and D). In the majority of Kv3.1b-IR terminals detected with DAB, the reaction product was often restricted to discrete zones within the terminals, predominantly observed close to the membrane, yet often distant to the active zone of the synapse (Fig. 8A). In addition, vagal afferent fibres detected by DAB formed synaptic contacts with Kv3.1b-gold-containing structures (Fig. 8D; n = 9).
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Although the cell bodies giving rise to vagal afferent fibres in the NTS reside in the nodose ganglion, Kv3.1b-IR was present in the cytoplasm and not their somatic membrane (Fig. 8E; n = 10 nodose ganglia). Nevertheless, RT-PCR on nodose ganglion RNA revealed Kv3.1 transcripts (Fig. 8F). This represents an unusual organization of the Kv3.1b subunit since it appears to be targeted to the terminals of the afferent neurones, yet absent from their somata.
Pathway-specific enhancement of neurotransmitter release by 4-AP and TEA correlates with the presence of Kv3.1b.
Since we detected Kv3.1b in presynaptic terminals, we examined the effects of 4-AP and TEA on synaptic potentials evoked in NTS neurones. Electrical stimulation in the region of the TS, which contains vagal afferent axons and Kv3.1b-IR cells, elicited monosynaptic excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) in recorded NTS neurones (Fig. 9A and B). We considered these to be monosynaptic since they exhibited constant latency, all or nothing responses and a low failure rate in response to high-frequency stimulation (Neff et al. 1998; Doyle & Andresen, 2001). To determine if the effects of 4-AP and TEA were presynaptic, we examined the paired-pulse ratio (PPR), which is the ratio of the amplitude of the second EPSP compared to that of the first EPSP when two EPSPs are evoked in quick succession. If an effect is mediated postsynaptically, the ratio should remain unchanged, whereas a significant change in the PPR would be indicative of a presynaptic effect. Both 4-AP and TEA evoked increases in the amplitude of EPSPs elicited by both first (4-AP, 12.3 ± 2.4%, n = 28; TEA, 28.3 ± 3.1%, n = 23) and second (4-AP, 7.2 ± 1.0%, n = 28; TEA, 10.8 ± 1.8%, n = 23) stimuli (Fig. 9A). This was likely to be due to a presynaptic action since the PPR significantly decreased in the presence of 4-AP from 0.82 ± 0.01 to 0.73 ± 0.01, and with TEA from 0.78 ± 0.01 to 0.66 ± 0.01 (Fig. 9A; P < 0.05). IPSPs were also elicited by electrical stimulation of the TS and isolated by blockade of excitatory amino acid receptors using kynurenic acid (1 mM), as well as depolarizing the neurone to a holding potential of 0 mV. The amplitude of both IPSPs increased (Fig. 9B) with 4-AP (first, 23 ± 2.8%; second, 11.3 ± 1.4%; n = 22, P < 0.05) or TEA (first, 29.6 ± 4.02%; second, 9.8 ± 2.3%; n = 20, P < 0.05), and the PPR decreased with both compounds (4-AP, 0.7 ± 0.01 to 0.61 ± 0.02; TEA, 0.88 ± 0.02 to 0.65 ± 0.01). Postsynaptic membrane potential and input resistance did not change during these presynaptic experiments.
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Subunit selective disruption reveals that Kv3.1b subunits contribute to the shape of action potentials in NTS neurones
NTS cells that were sensitive to low concentrations of 4-AP and TEA had faster action potential kinetics and firing frequencies than insensitive neurones. Whilst overlapping sensitivity to both TEA and 4-AP is consistent with the presence of Kv3.1 subunits (Coetzee et al. 1999; Deuchars et al. 2001), the action potential duration and firing frequencies were lower than observed for other cells containing Kv3.1b subunits (e.g. Wang et al. 1998; Erisir et al. 1999). Nevertheless, our data provide direct evidence that Kv3.1b contributes to the action potential waveform, since intracellular application of anti-Kv3.1b antibody elicited effects likely to be specific to Kv3.1b subunits as: (1) the actions of the antibody mimicked and occluded the effects of TEA (100 µM) and 4-AP (30 µM), suggesting that they are acting on the same target; (2) action potentials of neurones in areas that do not contain Kv3.1b immunoreactivity (dorsal vagal nucleus and cerebellar Purkinje neurones) were unaffected, while it was effective in all cells in the dorsal column nuclei, which all contained Kv3.1b-IR – thus the presence of Kv3.1b correlates with the antibody action; (3) the antibody did not act on other Kv3 subunits since the action potential characteristics of cells containing Kv3.4 (dorsal vagal neurones, Brooke et al. 2003, 2004b) or Kv3.2, 3.3 and 3.4 subunits (cerebellar Purkinje cells, Martina et al. 2003) were not affected by the antibody – these experiments also show that the antibody does not affect the wide variety of channels (e.g. Na+ and other K+ channels) determining action potential waveform in cells that do not contain Kv3.1b; (4) the effects of anti-Kv3.1b antibody are not due to a nonspecific interaction of the antibody with cellular machinery since inclusion of an antibody directed against HCN1, another ion channel absent from these cells, had no effect; (5) finally, the regions in the NTS where we performed our electrophysiological recordings correlate with those that contained Kv3.1b-immunoreactive somata. The mechanism of action of the antibody could not be determined from these experiments, but possibilities include disruption of trafficking, changes of conformation or physical blockade of the pore. Nevertheless, the evidence accumulated here indicates that Kv3.1b subunits are highly likely to contribute to the firing patterns of these NTS cells. Since they do not fire as fast as other Kv3.1b-containing neurones, these slower firing rates may be a consequence of phosphorylation (Macica et al. 2003) and/or association with accessory subunits such as MiRP1/2 or MinK, which can modulate the rate of channel activation and deactivation without affecting 4-AP sensitivity (Lewis et al. 2003; McCrossan et al. 2003).
NTS neurones display a Kv3-like current
The currents recorded in voltage clamp also display properties consistent with those carried by Kv3-containing channels. For instance, a large proportion of the current was sensitive to low concentrations of TEA (in the presence of -DTX); the activation curve for the TEA-sensitive current had a threshold of around –30 mV, and a half-activation voltage of around 0 mV. These features have all been observed in mammalian cell lines expressing Kv3-subunit-containing channels (Rudy et al. 1999) as well as neurones containing Kv3 channels in the CNS (Wang et al. 1998; Erisir et al. 1999; Hernandez-Pineda et al. 1999; Baranauskas et al. 2003; Martina et al. 2003). At this stage we are unable to rule out the possibility that association with accessory subunits may alter the biophysical properties of the Kv3 channels expressed in NTS neurones (Lewis et al. 2003; McCrossan et al. 2003).
Potential role of presynaptic Kv3.1 subunits in neurotransmitter release in the NTS
While Kv3.1 subunits have previously been identified in presynaptic terminals (Sekirnjak et al. 1997; Wang et al. 1998; Parameshwaran et al. 2001; Ozaita et al. 2002; Ishikawa et al. 2003), their physiological role in this location has been rarely addressed. It is known that terminals can display currents typical of Kv3 channels since direct recordings from presynaptic ‘pinceau’ terminals in the cerebellum revealed a dendrotoxin-resistant, TEA-sensitive current (Southan & Robertson, 2000); however, these terminals contain only Kv3.4 (Laube et al. 1996) and possibly Kv3.3 (Taylor & Perney, 2001; McMahon et al. 2004) channel subunits. Kv3.1b subunits have been detected in terminals of the calyx of Held, where neurotransmitter release is regulated by a Kv3 channel (Ishikawa et al. 2003). The terminals in the NTS are too small for direct patch-clamp recordings, and we therefore used the paired-pulse protocol as an indirect measure of terminal function and neurotransmitter release (Clements, 1990). Consistent with blockade of a K+ channel, we found that low concentrations of TEA and 4-AP (likely to act on Kv3.1 subunits, Coetzee et al. 1999; Deuchars et al. 2001) increased release of neurotransmitter, as reflected in a decrease in PPR. However, this effect was observed only in a pathway where Kv3.1b-IR was detected (TS), and not in a control pathway lacking Kv3.1b-IR (reticular formation). Although the electrophysiological data correlate well with immunohistochemistry, we were limited to an indirect measurement, and there remains the possibility that even the low concentrations of TEA and 4-AP used may act on other channels that affect transmitter release. In particular, we cannot exclude a contribution from Kv3.1a subunits, since these appear to be trafficked preferentially to terminals of neurones in other CNS regions (Ozaita et al. 2002). The two splice variants exhibit similar currents and sensitivity to TEA and 4-AP (Coetzee et al. 1999), thus, the effects observed could be due to blockade of a heteromeric complex of these two isoforms. In addition, it is possible that in these terminals, Kv3.1 subunits participate in heteromeric complexes with other Kv3 subunits such as Kv3.2 (Hernandez-Pineda et al. 1999; Tansey et al. 2002), Kv3.3 (Rudy et al. 1999) and Kv3.4 (Baranauskas et al. 2003) in other CNS regions. Neither studies of expression (Weiser et al. 1994) nor protein (L. Atkinson, unpublished results) detect Kv3.2 in the medulla oblongata, but Kv3.3 (J. Deuchars, unpublished results) and Kv3.4 (Brooke et al. 2004b) are present. Therefore, we can conclude that our data are consistent with the proposed role of Kv3 in regulation of transmitter release from the calyx of Held (Ishikawa et al. 2003).
Functional role of Kv3.1b in the NTS
In vivo, few functionally identified cell types in the NTS exhibit the fast-firing phenotype typical of Kv3.1b-containing neurones. Possible candidates include ‘pump’ neurones, which are driven by afferents from the pulmonary slowly adapting receptors, and may be part of the Hering–Breuer reflex (Berger, 1977). During prolonged lung inflation, these cells maintain high-frequency discharge with little or no adaptation (Kalia & Richter, 1985; Miyazaki et al. 1998). Consistent with this, Kv3.1b-IR NTS cells correspond in location, size and approximate orientation to pump neurones in the rat (Ezure et al. 2002). In addition, Kv3.1b cells contain markers for GAD or VGLUT2, and some pump cells can synaptically inhibit cells (Ezure & Tanaka, 2000), while others have been proposed to be excitatory, since respiratory neurones are excited by lung inflations (Hayashi et al. 1996). The Kv3.1b-IR vagal afferent terminals may therefore represent slowly adapting receptors from the lung since these also fire at high frequencies with little or no adaptation (Miyazaki et al. 1998). In support of this possibility, we observed Kv3.1b-IR vagal afferent terminals in synaptic contact with Kv3.1b-IR NTS neurones. Electrophysiologically, we also observed 4-AP-sensitive vagal afferent inputs to neurones in which the repolarization of the action potential was mediated by Kv3.1. The role of the Kv3.1b subunit in the NTS, similar to that in the auditory brainstem (Brew & Forsythe, 1995; Wang et al. 1998), may therefore be to facilitate high-frequency discharge and phase-lock cell activity to enable faithful distribution of the lung inflation signal throughout the appropriate circuitry.
Functional role of Kv3.1b in other regions of the medulla oblongata
Throughout the CNS, the Kv3.1b subunit imparts fast firing properties to neurones which are critical for the pattern of activity in neuronal circuits (Rudy & McBain, 2001). Here we have shown that the Kv3.1b subunit is present in many neurones of the medulla oblongata that we speculate may be interneurones. For example, in the nucleus ambiguus, the origin of vagal and glossopharyngeal projections to supra-diaphragmatic structures (Bieger & Hopkins, 1987), Kv3.1b-IR was absent from the efferent neurones themselves, but present in neurones that surrounded this nucleus. Similarly, in the hypoglossal nucleus, the few Kv3.1b-IR neurones were not motoneurones. This is analogous to the situation in the spinal cord where sympathetic preganglionic neurones lack Kv3.1b-IR, yet presympathetic interneurones express the Kv3.1b subunit (Deuchars et al. 2001; Brooke et al. 2002). Since fast firing neurones, such as those containing Kv3.1b, may govern the activity of output neurones in other brain regions (Cobb et al. 1995), it will be of interest to determine the role of Kv3.1b neurones throughout the medulla in shaping the activity of neuronal circuits such as those regulating autonomic output.
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Footnotes |
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References |
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