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Seizures and Reduced Life Span in Mice Lacking the Potassium Channel Subunit Kv1.2, but Hypoexcitability and Enlarged Kv1 Currents in Auditory Neurons

¹Virginia Merrill Bloedel Hearing Research Center, ²Department of Otolaryngology–Head and Neck Surgery, ³Graduate Program in Neurobiology and Behavior, and ⁴Department of Pharmacology, University of Washington, Seattle, Washington; and ⁵Department of Physiology and ⁶Waisman Center, University of Wisconsin–Madison, Madison, Wisconsin

Submitted 19 June 2006; accepted in final form 14 July 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Genes Kcna1 and Kcna2 code for the voltage-dependent potassium channel subunits Kv1.1 and Kv1.2, which are coexpressed in large axons and commonly present within the same tetramers. Both contribute to the low-voltage–activated potassium current I_Kv1, which powerfully limits excitability and facilitates temporally precise transmission of information, e.g., in auditory neurons of the medial nucleus of the trapezoid body (MNTB). Kcna1-null mice lacking Kv1.1 exhibited seizure susceptibility and hyperexcitability in axons and MNTB neurons, which also had reduced I_Kv1. To explore whether a lack of Kv1.2 would cause a similar phenotype, we created and characterized Kcna2-null mice (–/–). The –/– mice exhibited increased seizure susceptibility compared with their +/+ and +/– littermates, as early as P14. The mRNA for Kv1.1 and Kv1.2 increased strongly in +/+ brain stems between P7 and P14, suggesting the increasing importance of these subunits for limiting excitability. Surprisingly, MNTB neurons in brain stem slices from –/– and +/– mice were hypoexcitable despite their Kcna2 deficit, and voltage-clamped –/– MNTB neurons had enlarged I_Kv1. This contrasts strikingly with the Kcna1-null MNTB phenotype. Toxin block experiments on MNTB neurons suggested Kv1.2 was present in every +/+ Kv1 channel, about 60% of +/– Kv1 channels, and no –/– Kv1 channels. Kv1 channels lacking Kv1.2 activated at abnormally negative potentials, which may explain why MNTB neurons with larger proportions of such channels had larger I_Kv1. If channel voltage dependence is determined by how many Kv1.2 subunits each contains, neurons might be able to fine-tune their excitability by adjusting the Kv1.1:Kv1.2 balance rather than altering Kv1 channel density.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Neuronal information processing is shaped by voltage-dependent potassium (Kv) channel tetramers, whose properties depend partly on which four subunits they contain. The subfamily of mouse genes Kcna1–Kcna8 codes eight Kv1 subunit types, Kv1.1–Kv1.8, potentially leading to high functional diversity in Kv1 channels (Lock et al. 1994). Alternatively, if there is functional redundancy, it may occur between subunit types Kv1.1 and Kv1.2 because they are coded by the two most closely related Kcna genes, are present at many of the same CNS locations, and either type forms channels with rapid activation and slow inactivation when expressed in oocytes (Hopkins et al. 1994; Lock et al. 1994; Stuhmer et al. 1989; Wang et al. 1993, 1994). Kv1.1 and Kv1.2 are also the two most abundant Kv1 subunit types and are commonly in the same tetramers; Kv1.1:Kv1.2 and Kv1.1:Kv1.4 are common combinations detected by coimmunoprecipitation from mammalian brains along with several different combinations of Kv1.1, Kv1.2, Kv1.3, Kv1.4, and Kv1.6 (Coleman et al. 1999; Rhodes et al. 1997; Scott et al. 1994; Shamotienko et al. 1997; Wang et al. 1993, 1999). This makes it hard to discern whether Kv1.1 and Kv1.2 possess distinct functional roles.

One approach to dissecting out separate functions for individual Kv1 subunit types is genetic deletion. For example, evidence for an axonal role of Kv1.1 was provided by Kcna1-null mice in which several axonal and axon terminal sites exhibited hyperexcitability, thought to underlie their seizure susceptibility, cold-swim–induced myoclonus, and hyperalgesia (Clark and Tempel 1998; Smart et al. 1998; Zhang et al. 1999; Zhou et al. 1998). Such evidence is difficult to obtain in direct recordings from the sites where Kv1.1 and Kv1.2 are likely most strongly expressed, at the juxtaparanodes of large-diameter axons, because they are beneath myelin, which also impedes toxin access and makes their physiological role controversial (David et al. 1995; Wang et al. 1993). In the present study we created Kcna2-null (–/–) mice lacking Kv1.2 and compared them with control +/– and +/+ littermates, as well as previous results from Kcna1-nulls. The –/– mice had reduced life spans and exhibited spontaneous generalized seizures. To explore developmental processes pertaining to the postnatal day 15 (P15) onset of –/– seizures, we performed quantitative polymerase chain reaction assays (qPCR) on +/+ brain stems that showed Kcna2 mRNA was strongly upregulated from P7 on.

Kv1 currents have been characterized at some neuronal somata using direct recordings and toxins that block channels containing at least one sensitive subunit type. For example, channels containing at least one Kv1.2 subunit are blocked by tityustoxin-K (TsTx), a component of Tityus serrulatus scorpion venom (Hopkins 1998; Werkman et al. 1993), and dendrotoxin-I (DTX) from black mamba snake venom (Strydom 1976) blocks channels containing at least one of the DTX-binding subunits Kv1.1, Kv1.2, and Kv1.6 (Robertson et al. 1996). This probably means DTX blocks all Kv1 channels native to neurons, especially in auditory neurons of the medial nucleus of the trapezoid body (MNTB) where Kv1.1, Kv1.2, and Kv1.6 are the predominant Kv1 subunit types (Dodson et al. 2002; Fonseca et al. 1998). Applying DTX to murine MNTB neurons and other auditory neurons strongly expressing Kv1.1 and Kv1.2 has shown that Kv1 channels underlie a rapidly activating low-voltage–activated potassium current I_kl (Adamson et al. 2002; Bal and Oertel 2001; Brew et al. 2003; Grigg et al. 2000; Wang et al. 1994). Proposed functions of auditory I_kl are to powerfully repolarize large synchronized excitatory postsynaptic potentials, reduce membrane time constants, minimize temporal summation, facilitate temporally precise transmission across synapses, and preserve phase locking to sound peaks (Brew and Forsythe 1995; Manis and Marx 1991; Oertel 1983; Trussell 1999).

The homogeneity of the MNTB (90% principal neurons) makes it a prime location for combining the above-cited approaches: direct recordings, toxin applications, and genetic deletion. For example, MNTB neurons in brain stem slices from Kcna1-null mice had 30% reduced I_kl amplitudes and were hyperexcitable [they fired more action potentials (APs) and had smaller threshold currents than those of controls; Brew et al. 2003]. In analogous recordings described below, Kcna2-null MNTB neurons were hypoexcitable and possessed enlarged I_kl, exactly opposite to the changes in Kcna1-null MNTB. To distinguish subcomponents of I_kl due to Kv1 channels with or without Kv1.2 subunits, TsTx and DTX were applied. One unexpected finding was that all Kv1 channels in +/+ mouse MNTB neurons contained Kv1.2, in marked contrast to rat MNTB neurons in which only a subset contained Kv1.2 (Dodson et al. 2002). An analysis of voltage dependence suggests Kv1.2-free channels produce larger currents because they activate at more negative potentials than Kv1.2-containing channels, whose voltage dependence may also depend on the number of Kv1.2 subunits they contain. Thus neurons may adjust their balance between Kv1.2 and Kv1.1 expression to fine-tune their excitability for specialized information-processing tasks. Some MNTB results were previously described in abstracts (Brew et al. 2000, 2001).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Generation of Kcna2-null mice

A genomic phage clone containing the Kcna2 gene was isolated from a 129/Sv mouse liver library (Stratagene, La Jolla, CA). For the targeting construct, the neomycin resistance cassette (provided by Dr. R. Behringer, The University of Texas M. D. Anderson Cancer Center, Smithville, TX) was inserted between a Kcna2 5' Xba I–Eag I and 3' Nco I–Xba I genomic fragment (see Fig. 1A). The thymidine kinase cassette (provided by Dr. R. Behringer) was cloned into the 3' portion of the targeting construct. The targeting vector was linearized and electroporated into AB-1 embryonic stem (ES) cells (gift of Dr. A. Bradley, Wellcome Trust Sanger Institute, Cambridge, UK). The ES cells were then subjected to positive–negative selection for 8 days in 300 µM G418 and 200 nM fialuridine. Doubly resistant clones were expanded and analyzed by Southern blot analysis.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Generation and verification of Kcna2-null mice. A: diagram of the targeting strategy used to remove the Kcna2 open reading frame (ORF). Homologous recombination of the Kcna2 chromosomal locus (top) with the targeting vector construct (middle) generated a targeted locus (bottom) in which the neomycin resistance cassette (Neo) has replaced the Kcna2 ORF. B: Southern blot analysis of genomic DNA from offspring of a heterozygote intercross. An Eco RV digest probed with the 5' (Xba I–Xba I) fragment detected an 8.5-kilobase (kb) wildtype allele and an 11.3-kb targeted allele. In this litter there were 2 wildtype (+/+) mice, 3 heterozygous (+/–) mice, and one Kcna2-null (–/–) mouse. C: Western blot analysis of Kv1.2 in protein isolated from whole brains. Kv1.2 protein was detected in +/+, reduced in +/–, and not detected in –/– brains. The broad band of Kv1.2 staining probably reflects variable levels of glycosylation. Blots were reprobed with anti--actin to control for loading. D: quantitative polymerase chain reaction (qPCR) expression of mRNA for Kcna2, Kcna1, and Kcna6 in whole brains from mice aged postnatal day 14 (P14). For each qPCR experimental run, the expression of the 3 Kcna genes was measured and normalized relative to the geometric mean of the expression levels for 3 reference genes: -actin; -actin; and succinate dehydrogenase complex, subunit A (Table S1 and METHODS). Mean relative expression levels from 1 to 4 qPCR runs were calculated for each mouse and each gene (only 2 mice had only a single qPCR run per gene) before averaging across mice, and plotting the mean and SE. Kcna1 and Kcna6 mRNA expression were similar across genotypes. Kcna2 mRNA expression was approximately halved in +/– brain compared with +/+ controls, and Kcna2 mRNA was absent from –/– brain (the calculated –/– expression level and error bar were both smaller than the thickness of the x-axis).

Breeding and genotyping

The mutation was transferred into C3HeB/FeJ mice (backcross generations N5–20) to create Kcna2-null mice (referred to here as –/– mice) and their wildtype (+/+) and heterozygous (+/–) littermates. A chimeric founder was also crossed to C57BL/6J mice, establishing a hybrid line and providing mutant mice referred to as –/– (B6/129) in this text. Breeding of the +/– mice took place in AAALAC-approved specific-pathogen–free facilities. Offspring were genotyped using tail clips taken from mice aged P6 or older and ear-punched for later identification on the day of experiments. When necessary, mice were killed by CO₂ exposure, followed by decapitation. All animal protocols were reviewed and approved by the University of Washington IACUC or the University of Wisconsin IACUC.

DNA was isolated from the tail and then PCR-amplified using two primer sets (supplemental Table S1)¹ : one to match the ORF of Kcna2, indicating the presence of an intact Kcna2 gene; the other to match the neomycin resistance cassette, indicating a Kcna2-null chromosome. The size of the DNA fragments generated told us which mice in a litter were wildtypes, heterozygotes, and Kcna2-nulls. (For further details, see http://depts.washington.edu/tempelab/Protocols/KCNA2.html.)

Western blot analysis of Kv1.2 protein expression

Total protein was isolated from whole brains and enriched for membrane-associated proteins. Brains were homogenized in 320 mM sucrose with protease inhibitors and cellular debris was pelleted by centrifugation at 1,000 x g. The supernatant was centrifuged at 120K x g for 1 h and the resulting pellet resuspended in 10 mM Tris (pH 7.4), 150 mM NaCl, and 1% Triton X-100 with protease inhibitors. Unsolubilized membrane proteins were removed by centrifugation at 120K x g for 1 h and the supernatant was saved. All steps were done on ice or at 4°C. Protein concentrations were determined using the BCA Protein Assay (Pierce, Rockford, IL).

Protein samples from each animal (20 µg) were denatured at 50°C for 10 min in loading buffer containing 100 mM dithiothreitol and separated by 8% SDS–PAGE. Protein was then electroblotted to a nitrocellulose membrane (BioRad, Hercules, CA). Membranes were blocked in phosphate-buffered saline Tween-20 with 10% nonfat milk and probed with an anti-Kv1.2 monoclonal antibody (1:5,000; Upstate, Waltham, MA). Anti-mouse horseradish peroxidase–conjugated secondary antibodies (1:1,000) were used for detection with enhanced chemiluminescent reagents (Amersham Biosciences, Piscataway, NJ). Blots were exposed to film for the amount of time necessary to obtain a clear image. Blots were then reprobed with anti--actin monoclonal antibody (1:1,000; Abcam, Cambridge, MA) and reprocessed for chemiluminescence detection.

Quantitative real-time PCR

qPCR techniques used here were similar to recently published procedures (Duncan et al. 2006; Silverstein and Tempel 2006), including the normalization of Kcna expression levels to the geometric mean of the three most stable internal reference genes from a panel of ten candidate reference genes, as described in Vandesompele et al. (2002). To measure Kcna gene expression versus genotype, +/+, +/–, and –/– mice were killed at age P14, and the whole brain was homogenized in 6 ml of Trizol (Invitrogen, Carlsbad, CA) and purified. To measure changes in brain stem Kcna expression during development C3HeB/FeJ control mice were killed at various ages (P1–P29). The brain was placed in RNAlater (Qiagen), after which the brain stem was dissected away and frozen at –80°C, for subsequent purification. Total RNA yield was measured spectrophotometrically and RNA integrity was verified by gel electrophoresis.

cDNA was reverse transcribed from 1 µg of total RNA using 50 pM of Random Hexamers (Amersham Biosciences) and 1 µl Powerscript Reverse Transcriptase (Clontech/BD Biosciences, Franklin Lakes, NJ) as detailed in Duncan et al. (2006). For each transcript of interest, qPCR primer pairs were designed using Primer3 software (Rozen and Skaletsky 2000) and following design criteria of: amplicon size 50–150 bp, primer T_m of 60–64°C, primer %GC of 35–65%, and complementarity between and within primers minimized (see supplemental Table S1 for sequences).

qPCR using about 5 ng starting total RNA was measured using SybR Green Supermix (BioRad). Cycling parameters were as follows: 95°C x 3 m (activate enzyme), 40 repeats of 95°C x 30 s, 60°C x 30 s (amplification), 95°C x 1 m, 55°C x 1 m (premelt curve), 90 repeats of 10 s each starting at 55°C and incrementing 0.5°C per step (melt curve). Real-time fluorescence was measured using the iCycler I/Q Module (BioRad). In each experiment, all samples were divided into two replicate reactions (same biological sample and reaction mixture), and the average threshold cycle number for the two replicates was used as a single data point. For each primer set melt curves were inspected and efficiencies determined as described in Duncan et al. (2006).

Kcna expression levels were normalized to the geometric mean of multiple internal reference genes, as previously described (Vandesompele et al. 2002). For all of the samples of a given data set, the qPCR threshold cycles were determined for a panel of ten candidate reference genes; the three most stable reference genes were used, as ranked by geNORM software (http://medgen31.ugent.be/jvdesomp/genorm/). These were -actin, -actin, and succinate dehydrogenase complex, subunit A for the cross-genotype study (Fig. 1D); and -actin, -actin, and hydroxymethylbilane synthase for the developmental study (Fig. 3). The primer sequences used for the three Kcna genes and these four reference genes are shown in supplemental Table S1.

View larger version (14K): [in this window] [in a new window]   FIG. 3. Developmental expression in brain stem of mRNA for Kcna2, Kcna1, and Kcna6. A: expression of Kcna2 mRNA in +/+ brain stems from 14 mice killed at ages P1–P29. Expression is given relative to the geometric mean of the 3 control genes -actin, -actin, and hydroxymethylbilane synthase (supplemental Table S1 and METHODS). At each age, the expression level is plotted for 2 mice, as the mean of 3–5 qPCR measurements (SE also plotted for each mouse using either narrow or wide error bars). Line is a Boltzmann fit to the 14 mean values plotted. B: relative mRNA expression as in A, but shown as the average of both mice at each age, for Kcna2 (triangles), Kcna1 (inverted triangles), and Kcna6 (diamonds). At each age, Kcna1 and Kcna6 expression are shown as the mean and SE for 4 qPCR measurements, 2 from each mouse. Kcna2 expression was the mean and SE from 6–9 qPCR measurements. Each line is a Boltzmann fit to the 7 mean values for that gene.

Motor tests and seizure testing

We tested gross motor behavior and susceptibility to flurothyl-evoked seizures in 28 mice aged P14 (three litters). Mice were weighed before the motor tests, which included 1) lowering the mice toward a cage top, to see whether they could grasp the rungs with their forelimbs, and testing the grip strength by pulling gently upward on their tail, 2) letting the mice climb vertically upward on cage rungs, and 3) letting them balance on a 1-cm-diameter aluminum rod held horizontally about 10 cm above the cage floor.

Later on the same day, these mice were tested for seizure susceptibility. After placing a mouse into a Plexiglas chamber of volume 10.7 liters, we applied the volatile convulsant flurothyl (Sigma–Aldrich, St Louis, MO) by dripping it in liquid form onto a filter paper at 20 µl/min. The chamber was cleaned and aerated between subjects. Two human observers noted the latencies from the first drip of flurothyl to the onset of various seizure-related behaviors, which included "flagpole" tail dorsiflexion (Straub tail), unilateral myoclonic jerks, tremors, and bilateral forelimb clonus. Also noted was the latency to a fully generalized seizure, which consisted of 2 to 5 s of running-bouncing seizure (RBS) followed by full tonic extension. At this point, the mouse was rapidly removed into fresh air and given abdominal massage to restore breathing, before being placed in a cage for observation.

Because Kcna1-null mice display a dramatic tremor when forced to swim in cold water (Zhou et al. 1998) we performed similar swim tests on Kcna2-null mice [in this case the –/– (B6/129) mice]. A tank, 18 x 29 cm (width x length), was filled with water to a depth of 7 cm. Mice were placed in the middle of the tank to swim. The water temperature was 17°C. The swim time was 2 min. After swimming, the mice were placed on a dry platform at room temperature for observation of abnormal motor behavior.

MNTB electrophysiology

BRAIN STEM SLICE PREPARATION AND RECORDING TECHNIQUES. The preparation of brain stem slices containing MNTB neurons was carried out using previously published techniques (e.g., Brew et al. 2003). In brief, mice aged P9–P16 were killed by brief CO₂ exposure, followed by decapitation and brain dissection. A tail clip was collected for confirmation of prior genotyping (see previous text). Dissection and slicing took place in ice-cold sucrose-based solution containing (in mM): 250 sucrose; 2.5 KCl; 26 NaHCO₃; 1.25 NaH₂PO₄; 2 CaCl₂; 1 MgCl₂; 10 glucose; 2 Na pyruvate; 0.5 Na ascorbate; and 3 myo-inositol (pH was maintained at 7.4 by gassing with 95% O₂-5% CO₂ mixture). After separation of the forebrain by a transverse cut through the colliculi, the cut rostral surface of the brain stem was glued to the chamber of a vibrating slicer (Vibratome Series 1000; Technical Products International, St. Louis, MO). Slices of 150 µm each were cut, up to five of which contained MNTB. Slices were kept for 1 h at 37°C in an incubation chamber filled with artificial cerebrospinal fluid (ACSF) and gassed with 95% O₂-5% CO₂ mixture. The ACSF was identical to the sucrose-based solution described earlier except that it contained 125 mM sodium chloride instead of the 250 mM sucrose. The incubation chamber was allowed to cool to room temperature and slices were maintained there for up to 8 h before use. Slices were then placed in a recording chamber, also perfused with gassed ACSF at room temperature (22–25°C), sited on a stable recording platform built around a microscope (on an X–Y stage) fitted with differential interference contrast optics (Axioskop or Axioskop FS2, Zeiss, www.zeiss.com).

Recording pipettes were borosilicate glass (Vertical Pipette Puller 700B; David Kopf Instruments, Tujunga, CA) and filled with a solution containing (in mM): K gluconate 97.5, KCl 32.5, EGTA 5, HEPES 10, MgCl₂ 1 (adjusted to pH 7.2 using 14 mM KOH). For voltage-clamp recordings, pipettes were coated with Sylgard (Dow-Corning, Midland, MI) to reduce their capacitance. Pipettes were connected to the patch-clamp amplifier (Axopatch 200; Axon Instruments, Foster City, CA) by the amplifier headstage mounted on a micromanipulator [Narishige (Tokyo, Japan) or EXFO (Burleigh, Victor, NY)] bolted to the recording platform. Pipette resistance was 3–6 M before gigaohm seal formation, measured from the responses to –5-mV voltage-clamp steps. Although recording locations were not measured, attempts were made to record from a variety of locations throughout the MNTB, to try to fully represent the properties of all MNTB neurons, and also to minimize bias from any variations in Kv1-based potassium current amplitudes across the tonotopic axis (e.g., as reported in rat MNTB; Brew and Forsythe 2005). The access resistance was monitored frequently throughout recordings and typically rose to about 10 M on achieving the whole cell recording configuration (range 5–18 M). Recordings were discontinued if the pipette access resistance went to >20 M. There was a –7-mV liquid junction potential that is included in all the subsequently given membrane potential values.

Soon after the start of voltage-clamp recordings, the slice was perfused with ACSFV (a low-calcium version of the ACSF) containing 0.5 mM CaCl₂, 2.5 mM MgCl₂, and 0.5–1 µM tetrodotoxin to minimize sodium currents, calcium currents, and synaptic activity. The pipette access resistance was compensated using the series resistance compensation circuitry of the patch-clamp amplifier ("correction" dial at 85% and "prediction" dial at 85%).

Chemicals were from one source (Sigma–Aldrich) except sucrose and NaH₂PO₄ (J.T. Baker, Phillipsburg, NJ). Tetrodotoxin (TTX), dendrotoxin-I (DTX), and tityustoxin-K (TsTx) were from another source (Alomone Labs, Jerusalem, Israel). TsTx (100 nM) or DTX (100 nM) plus TsTx (100 nM) was added to the ACSFV and applied by perfusion. Because DTX block is almost irreversible in slices, for recordings following previous toxin applications, we used a fresh slice, replaced the perfusion tubing and reservoirs, and either replaced the recording chamber or washed it with dilute HCl for 60 min.

STIMULUS GENERATION AND DATA ACQUISITION. The software for stimulus generation and data acquisition [either Synapse (Synergy, Bethesda, MD) or Axograph (Axon Instruments)] ran on a Macintosh computer (7100/80AV or G3) and sent command sequences to the patch-clamp amplifier by an AD/DA interface (ITC-16; Instrutech, Port Washington, NY). This interface also digitized signals on two channels from the patch-clamp amplifier so that they could be digitally recorded by the software. During current-clamp experiments, the two channels recorded were the current-clamp pulse amplitude and the resulting pipette potentials, each recorded at a digitization rate of 10 kHz (amplifier filtering 5 kHz). During voltage-clamp experiments, the two channels recorded were the voltage-clamp pulse amplitude and resulting pipette currents, each recorded at a digitization rate of 5 kHz (amplifier filtering 2 kHz).

Current-clamp pulses were applied at 1-s intervals, were of 180-ms duration, and increased in 10-pA increments from 0 to 200 pA, or from –100 to 200 pA. This is the same protocol as that previously used for the study of MNTB neurons in Kcna1-null mice, to allow straightforward comparisons between the data sets (e.g., supplemental Table S2 and Table 1 of Brew et al. 2003).

To measure current–voltage (I–V) relations in voltage-clamped MNTB neurons, a standard I–V protocol was used (also used previously for the Kcna1-null MNTB study; Brew et al. 2003). Voltage-clamp command test pulses were applied at approximately 1.3-s intervals, were of 180-ms duration, and increased in 10-mV increments from –40 to +90 mV. These test pulses were applied over a continual background potential of –60 mV, set using the amplifier, and the –7-mV liquid junction potential. This –67-mV holding potential was necessary to inactivate the large A-currents present in murine MNTB neurons (see Fig. 7A and supplemental METHODS). The resulting patch pipette potentials were –107 to +23 mV. Each pulse sequence included an initial 150-ms pulse to –107 mV (intended to remove any potassium channel inactivation that had occurred during previous test pulses), an 870-ms prepotential of –67 mV (to inactivate A-type potassium currents), the 180-ms test pulse, a 50-ms postpotential at –37 mV, and a subsequent step back to –67 mV for 50 ms. The 245-ms section recorded included the final 15 ms of the prepotential, the 180-ms test pulse, and the 50-ms postpotential.

View larger version (21K): [in this window] [in a new window]   FIG. 7. Outward K+ currents in +/+, +/–, and –/– MNTB neurons. A: qualitatively similar currents recorded from 2 MNTB neurons, each typical of their genotypes (+/+, left; –/–, right), in response to voltage-clamp test pulses –107 through –37 mV, from within the standard I–V protocol (test pulses were applied in 10-mV increments between –107 and +23 mV, at 1.3-s intervals, each preceded by a –67-mV prepotential and followed by a –37-mV postpotential; see METHODS). Slices were perfused with ACSFV (a low-calcium version of the ACSF; see METHODS). A large transient A-type potassium current was visible during the –37-mV postpotential if the test pulse was –67 mV or more negative, but its inactivation by the –67-mV prepotential meant it was absent or very small during test pulses. Bars show the times of measurement of sustained currents for B and C. B: sustained current amplitudes from 11 +/+ neurons (8 mice) and 10 –/– neurons (6 mice) at test potentials –107 through +23 mV. For each run of the standard I–V protocol, the sustained current amplitudes were measured toward the end of each test pulse (during time marked by bars in A) and leak currents were subtracted (see METHODS). Then, for each neuron, the sustained current amplitudes were averaged across 2–3 runs of the I–V protocol, before averaging across the neurons within each genotype. Asterisks indicate significant differences between +/+ and –/– neurons (P < 0.05, Mann–Whitney U test). C: boxed region from B, plotted on an expanded scale to highlight the potential range where there were significant differences between genotypes (asterisks as in B). Also shown are the sustained currents from 8 +/– neurons (7 mice). There were no significant differences between the +/+ and +/– currents. Circumflexes (hats) indicate significant differences between +/– and –/– current amplitudes (P < 0.05, Mann–Whitney U tests).

View larger version (29K): [in this window] [in a new window]   FIG. 12. Voltage-dependence curves for +/+, +/–, and –/– Gkl and GKv1 conductances. A–C: symbols show G/Gmax values from individual MNTB neurons plotted against test potentials –87 through –37 mV (see METHODS for conductance calculations and fitting methods). Conductances were calculated from the current responses obtained using the standard I–V protocol (or the high resolution I–V protocol if available) after leak subtraction and averaging across 2–3 runs. Fits of Boltzmann functions to these data are shown as solid lines, or dashed lines for slightly "less good" fits (see METHODS). Gray bars are visual aids showing the range –57 to –48 mV. A: symbols show Gkl/Gmax values plotted against test potential for 9 +/+ neurons (top), 8 +/– neurons (middle), and 8 –/– neurons (bottom, same neurons as in Fig. 7C but excluding three neurons asterisked in Fig. S2A, and one –/– neuron with a poor fit). Half-activation voltage (Vhalf) values for the +/+ neurons and –/– neurons fell in distinct ranges, whereas the +/– neurons had intermediate Vhalf values (approximately within the gray bar). B: GKv1/Gmax values from individual MNTB neurons plotted against test potentials for 17 MNTB neurons (same neurons as in Figs. 9, 10, but excluding 1 +/+ neuron and 2 –/– neurons yielding poor fits, and one +/– neuron for which the DTX-sensitive component was too small to be well fitted for C). Mean Vhalf values and their ranges and the genotypic differences between them were generally similar to those from A, although the mean Vhalf values were a little more negative than those in A. C: fits as in A and B, but to the distinct components of +/– GKv1 either TsTx-sensitive or TsTx-insensitive but DTX-sensitive. GKv1/Gmax values of TsTx-sensitive components became half-activated at less-negative potentials than those of the TsTx-insensitive components, but the Vhalf values for these 2 components did not fall neatly into the +/+ and –/– ranges, respectively.

View larger version (25K): [in this window] [in a new window]   FIG. 9. Effects of tityustoxin-K (TsTx) and DTX in +/+, +/–, and –/– MNTB neurons. A: sustained current amplitudes in response to –47-mV test pulses for an example MNTB neuron of each genotype (+/+, left; +/–, middle; –/–, right). Currents were measured first in control ACSFV, then while the perfusion solution was switched to ACSFV containing 100 nM TsTx, and finally to ACSFV containing both 100 nM TsTx and 100 nM DTX-I (times of toxin applications shown by black bars). In the +/+ neuron, TsTx led to substantial current block but the additional presence of DTX caused no further block (left). In the +/– neuron, TsTx led to substantial current block and the addition of DTX also led to substantial current block (middle). In the –/– neuron, TsTx had no effect but DTX led to substantial current block. Here leak current was not subtracted because most data points came from the toxin-monitoring protocol and only 9–12 data points came from the I–V protocols that allow leak current subtraction (see METHODS). For each neuron in each solution, 3 runs of the standard I–V protocol described in Fig. 7 were applied at times when the currents appeared approximately stable (e.g., shown by the letters c, t, and d in each panel, which also denote when the data in B were collected). For some neurons the "high-resolution" I–V protocol was also applied for 3 runs per solution (see METHODS). B: current responses during –47-mV pulses of the same example neurons as in A (directly above) at the approximate times shown by letters c, t, or d in A, i.e., in ACSFV, TsTx, or (TsTx + DTX). Each trace is the average response from 2–3 runs of the standard I–V protocol. In the +/+ neuron, the current block appeared identical in the TsTx solution and the (TsTx + DTX) solution (left). In the +/– neuron, the current amplitude in TsTx fell about halfway between the control amplitude and that in the solution containing both toxins (middle). In the –/– neuron, the current appeared identical in the control ACSFV or in the TsTx solution, and only the (TsTx + DTX) solution led to substantial current block (right). These characteristic effects of toxins on the current amplitudes of neurons of each genotype were as evident at the beginning of test pulses as at the end, when sustained current amplitudes were measured (see bars in Fig. 7A). C: voltage dependence of the mean sustained current amplitudes in +/+, +/–, and –/– neurons, in control ACSFV (c, solid lines), ACSFV plus TsTx (t, dashed lines), and ACSFV plus TsTx and DTX (d, dotted lines). For each neuron, the currents from each run of the standard I–V protocol were subjected to leak subtraction (see METHODS) before averaging the amplitudes from 2–3 runs per solution. Inserts show mean current amplitudes for all the test pulses –107 through +23 mV, and the larger panels show the same data plotted on an expanded scale to highlight effects of the toxins in the range of potentials relevant to Kv1 channels. In the +/+ neurons, the current amplitudes at each potential were similar in TsTx or (TsTx + DTX). In the +/– neurons, TsTx led to substantial current block and (TsTx + DTX) caused even greater block. In the –/– neurons, TsTx had no effect at any potential and DTX led to substantial current block.

Current-clamp data for each neuron were measured from a single run of the current-pulse protocol before any averaging across runs or between neurons was performed. All membrane potentials presented include the addition of the –7-mV junction potential and a voltage correction for current flow across the pipette tip access resistance, both carried out off-line. The sustained potential response to a particular current pulse amplitude was the average potential during a 10-ms time window toward the end of the pulse (100 data points, 160 to 170 ms after current-pulse initiation). The resting potential was taken to be the sustained potential response to the 0-pA current pulse. Sustained potential responses were also used for calculations of input resistance (in supplemental Table S2).

In voltage-clamped neurons, the sustained outward current amplitudes (I) were measured from each test potential of the standard I–V protocol or the "high-voltage–resolution" I–V protocol as follows. First the absolute current amplitude was measured at the end of each 180-ms test pulse, as the average of the 50 data points between 160 and 170 ms after test pulse initiation. The current amplitudes at membrane potentials between –97 and –77 mV represented background "leak current" and were used to calculate the linearly extrapolated current at other test potentials, which was then subtracted from the absolute currents to yield the sustained outward current amplitudes, I. For each neuron and in each perfusion solution, these values of I were also averaged across two to three runs of the respective I–V protocol. To obtain a measure of that neuron's current through Kv1 channels, the sustained I_Kv1, these average I values in the presence of both TsTx and DTX, were subtracted from the I values measured in ACSFV alone.

The membrane potentials referred to in the subsequent voltage-clamp results and figures are the pipette potentials with a –7-mV correction for the liquid junction potential, but do not include any voltage correction for current flow across the uncompensated 15% of the pipette tip access resistance (range 1–3 M). This omission facilitated the averaging of data collected at the same pipette test potentials and is justified because the estimated corrections were all small in the potential range relevant to Kv1 channels (e.g., at –37 mV, the mean amplitude of the sustained current was +760 pA before leak subtraction, resulting in an average correction of –1.5 mV).

To fit the apparent activation time course of an MNTB neuron's I_Kv1, the current records were averaged from two to three runs of the standard I–V protocol in ACSFV and two to three runs in the presence of both TsTx and DTX; the latter were subtracted from the former (which removed capacitative current transients). The resulting currents were well fitted by curves increasing with a single-exponential time course at test potentials –47 and –57 mV. Fits were performed on a time period of 20 ms (i.e., 100 data points) beginning soon after the start of the test pulse (1.2–21.1 ms). Because data were available from a larger number of neurons in ACSFV alone, the apparent activation time course was also fitted from each neuron's averaged I–V responses in ACSFV, which required a nonstandard method of subtracting the leak and capacitative transient currents, as follows. First, the record containing the –77-mV pulse was subtracted from that containing the –87-mV pulse, which yielded the estimated passive "leak plus capacitance" current response to a single –10-mV pulse (the –67-mV pulse could not be used because some sustained outward current including I_Kv1 was usually present). Next, this passive "leak plus capacitance" estimate was multiplied by the relevant n before adding it to the current responses to test pulses differing by n increments of 10 mV from the –67-mV holding potential. Finally the current fits were performed (as described earlier for I_Kv1).

DATA SETS AND DATA EXCLUSION CRITERIA. The current-clamp data are from a total of 68 MNTB neurons and 27 mice (26 from 15 –/– mice, 23 from 12 +/+ mice, and 19 from 10 +/– mice). The voltage-clamp data are from another 29 MNTB neurons and 21 mice (11 from 8 +/+ mice, 8 from 7 +/– mice, and 10 from 6 –/– mice). The recordings from neurons of each genotype had similar mean pipette access resistances and capacitances, as read off the amplifier dials, and were from mice of similar ages (see supplemental Table S2).

Current-clamped neurons were excluded if the resting potential was smaller than –57 mV (including the –7-mV liquid junction potential). Voltage-clamped neurons were excluded if a holding current larger than –100 pA was required to clamp at the background holding potential of –67 mV. We made exceptions to this criterion for two voltage-clamped neurons to which we subsequently successfully applied both toxins (one +/+ neuron and one –/– neuron requiring –160 and –190 pA, respectively). On average, the holding current was near zero for the remaining +/+ and –/– voltage-clamped neurons (+/+, –2 ± 24 pA, n = 10; –/–, –5 ± 18 pA, n = 9) and slightly larger for +/– neurons (–50 ± 11 pA, n = 8).

FITTING OF CONDUCTANCE DATA. The conductance (G) and its toxin-sensitive component (G_Kv1) were calculated at each test potential (V) by dividing sustained current amplitudes (I or I_Kv1) by the driving force (V minus –80 mV, the approximate reversal potential for DTX-sensitive channels; Brew and Forsythe 1995; Stansfeld and Feltz 1988). The calculated G values were plotted versus V (–107 through –37 mV) and fitted with a single Boltzmann function G = G_max/{1 + exp[(V – V_half)/–k]} with variable parameters G_max, the maximum conductance, V_half, the half-activation voltage, and k, the slope factor. The fitting used a least-squares curve-fitting method (the Pearson's R general curve fit within Kaleidagraph software, which finds the minimum value of ², the sum of the squared residuals, using partial derivatives according to the Levenburg–-Marquardt algorithm, described in Press et al. 1992). In Fig. 12, 56 fits and data are shown normalized to their fitted G_max values, to facilitate visual comparisons, but the subsequent description of goodness-of-fit is based on the fits before normalization.

The 56 fits shown in Fig. 12 appeared good to the naked eye, had small mean errors returned with each fitted parameter (G_max, 0.9 nS; V_half, 1.6 mV; and k, 0.8 mV), and values of R² > 0.93 (53 fits had R² > 0.98). Of these, 46 fits were judged "very good" because the returned errors were <2 units for all three parameters (shown by solid lines in Fig. 12). Fits were judged too poor to be included if one or more parameters were returned with estimated errors >10 units, but in most cases the same neuron's current responses from an alternative protocol returned a good fit. Thus only one neuron was excluded from a plot because of a poor fit (a –/– neuron excluded from Fig. 12A). If both protocols returned very good fits, those from the high-voltage–resolution protocol were preferred for inclusion in Fig. 12 and statistical comparisons. Also shown are 10 "less-good" fits, for which at least one parameter was returned with an error estimate >2 units but <10 units (dashed lines in Fig. 12). The majority of these "less-good" fits came from +/+ neurons (7 of 10). This was probably partly because the conductance was not near its maximum value at –37 mV in +/+ neurons, whereas at that potential the +/– and –/– conductances had reached or closely approached their maxima (Fig. 12A; also see RESULTS). The results and discussion therefore focus on the +/– and –/– results, and only tentative conclusions are made from the +/+ data.

Statistical evaluation

Tests were unpaired two-tailed Student's t-tests assuming equal variance in each sample, except as noted for paired comparisons or comparisons involving appreciably nonnormal distributions, for which we used Mann–Whitney U tests. All tests were performed using StatView (SAS Institute, Cary, NC) or Kaleidagraph. All averaged values are expressed in text and figures as mean ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Verification of Kcna2 knockout and Kcna expression in +/+, +/–, and –/– brains

Our targeting strategy successfully knocked out the entire open reading frame of the Kcna2 gene (Fig. 1, A and B). The lack of Kv1.2 protein in the brains of Kcna2-null (–/–) mice was confirmed by Western blot (Fig. 1C). By qPCR there was also no Kcna2 mRNA present in brains from –/– mice aged P14, and Kcna2 mRNA expression was approximately halved in +/– compared with +/+ littermates' brains (Fig. 1D). The Western blot is suggestive of a reduced amount of Kv1.2 protein in +/– brain relative to +/+ brain (Fig. 1C). These results verify that both Kcna2 mRNA and Kv1.2 protein are absent in the –/– mouse brain.

To test whether other Kcna genes had altered expression as a result of the absence of Kcna2 in –/– mice, the qPCR experiments also measured Kcna1 and Kcna6 mRNA expression, which were found to be very similar between +/+, +/–, and –/– brains (Fig. 1D). These two genes code for Kv1.1 and Kv1.6 subunits, which are known to be expressed along with Kv1.2 in both rat and mouse MNTB neurons (Brew et al. 2003; Dodson et al. 2002; Fonseca et al. 1998). Because the expression of each Kcna gene was normalized to the same three reference genes, these data suggest that Kcna1 mRNA may be expressed at a higher level than either Kcna2 or Kcna6 in +/+ brain. The brains of P14 –/– mice did not exhibit any signs of compensatory changes or greater variability in mRNA expression for Kcna1 or Kcna6 because both their mean expression and variability were similar to those of +/+ and +/– brains (Fig. 1D).

Life span and gross motor behavior in Kcna2-null mice

The –/– mutation did not cause embryonic lethality, judging by the Mendelian proportions of each genotype in 14 litters consisting of 88 mice, none of which was subjected to any experimental testing (+/+, n = 21, 23.8%; +/–, n = 45, 51%; and –/–, n = 22, 25%). However, the –/– mice had a severely reduced life span, averaging 17 ± 0.2 postnatal days (Fig. 2 A, range P16–P19). Their littermate +/+ and +/– mice had normal life spans. In litters removed from the specific-pathogen–free facility for experimental testing, some –/– mice had even shorter life spans (range P14–P19). The mean life span was also 17 ± 2 postnatal days in eight –/– (B6/129) mice, although the range was much larger [one –/– (B6/129) mouse died at P6, the rest at P14–P25] perhaps because the mutation was in a genetic background with seizure-resistant (B6) and relatively seizure sensitive (129) alleles sorting in the mixed background (Frankel et al. 2001). Nonetheless, the data suggest the null mutation was penetrant in more than one genetic background. Adult +/– mice were good breeders, suggesting that they flourish despite the substantially reduced expression of Kcna2 mRNA and Kv1.2 protein demonstrated at P14 (Fig. 1, C and D).

View larger version (16K): [in this window] [in a new window]   FIG. 2. Survival curves and seizure susceptibility in +/+, +/–, and –/– mice. A: percentage of mice surviving through each postnatal age (e.g., if they died at age P16, they survived through P15). All of the 22 –/– mice died at age P19 or younger, whereas none of the 21 +/+ mice or the 45 +/– mice died during the first 30 days. In total, 88 mice were from 14 litters, with roughly Mendelian proportions of each genotype and balanced gender proportions (45 males and 43 females). Line through the points was drawn by eye. B: mean latencies to the first seizure-related behavior (left bars) and running-bouncing seizure (RBS, middle bars) for 6 +/+ mice, 15 +/– mice, and 7 –/– mice, following the first drip of flurothyl onto the filter paper in the exposure chamber. Mean time between the first seizure-related behavior and the RBS is also shown (right bars). Significant differences between genotypes are denoted by single or double asterisks (P < 0.05 or P < 0.005, respectively; Mann–Whitney U tests). –/– mice differed significantly from +/+ and +/– mice on all 3 measures.

"Spontaneous" seizures in Kcna2-null mice

Many –/– mice were observed undergoing apparently spontaneous episodes that began with a sudden explosive onset of wild running and jumping, followed after 5–10 s by full tonic extension (TE). This sequence is highly reminiscent of (RBS), leading to TE, which is a typical feature of a fully generalized seizure in rats and mice (Gale 1992). When –/– mice were observed recovering from TE, they often exhibited myoclonic jerks and tremors, followed by 5–20 min of relative immobility before recovery of normal motor behavior, reminiscent of a postictal phase. The fatality rate of observed RBS/TE events was high, about 50%, and was probably due to the cessation of movement and breathing that accompanied TE. It is likely that all the –/– mice eventually experienced fatal RBS/TE because all those that died unobserved were found with their limbs and bodies fully extended. Thus the RBS/TE episodes in –/– mice are probably seizures with generalized onset, and the proximal cause of the reduced –/– life spans was probably fatal apnea occurring during TE.

The exact frequencies, fatality rates, and ages of occurrence of all RBS/TE events could not be recorded, although some conclusions can be drawn from the following quantitative estimates, as remembered by two human observers (L.R. and H.B.) who were with litters including –/– mice for 60 min per day of routine mouse care or experimental preparations. At least 50 RBS/TE episodes were observed in about 100 –/– mice aged P15–P19. This is more than would be expected if observed and unobserved RBS/TE events occurred at the same rate and had the same fatality rate (50%), in which case a typical –/– mouse would undergo only one or two RBS/TE events in its lifetime, and the expected number of events in 100 –/– mice would be 200, only eight of which should coincide with the daily hour of observation. Both observers reported that many RBS/TE events began when the observer moved the cage slightly, perhaps suggesting they were evoked by sounds or accelerations or stress. The fatality rate did not appear to increase with age because RBS/TE was fatal in the two youngest –/– mice ever observed undergoing RBS/TE, aged P14, and nonfatal RBS/TE events were observed in several –/– mice at ages P17 and P18, and in a –/– (B6/129) mouse aged P17, which survived but died later the same night. RBS/TE events were very rare and about 50% fatal during several days of quiet extended observation of five –/– mice in a cage with a foster mother, and were initiated from sleep or normal awake behavior without any provoking stimuli apparent to the human observer. Thus RBS/TE events in –/– mice were probably infrequent (less than one per day between P15 and P19), 50% fatal, and some observed RBS/TE events may have been evoked by unknown stimuli.

Susceptibility to seizure induction of Kcna2-null mice

To test whether the –/– CNS was abnormally susceptible to evoked seizures, mice aged P14 were tested for their latencies to flurothyl-induced seizures (see METHODS; same 7 –/– mice, 15 +/– mice, and 6 +/+ mice as in the gross motor testing described earlier). At this age, it was likely that none of the –/– mice had yet experienced an RBS/TE generalized seizure event. All mice behaved normally for a few minutes after the first drip of flurothyl into the exposure chamber, then exhibited seizure-related behaviors (see METHODS), eventually culminating in RBS followed by TE. The mean latency to occurrence of the first seizure-related behavior was shortest in –/– mice, intermediate in +/– mice, and longest in +/+ mice (Fig. 2B, left). The mean RBS latency for –/– mice was 40% shorter than that in +/+ or +/– mice (Fig. 2B, middle). The –/– mice progressed very rapidly from their first seizure-related behavior to RBS, taking an average of 23 s, whereas their +/+ and +/– littermates displayed ongoing seizure-related behaviors for about 2 min before progressing to RBS (Fig. 2B, right). There was no overlap in the range of latencies to flurothyl-induced seizures aged P14 in the seven –/– mice (182–251 s) and their +/– and +/+ littermates (311–465 and 292–455 s, respectively). Abdominal massage restored breathing for 18 of these 28 mice, including three –/– mice. The shorter latencies to RBS in –/– mice suggest there was network hyperexcitability present in the –/– CNS at P14.

Although the seizure profile of –/– mice (described earlier) is distinct from that of Kcna1-null mice (partial seizures from age P21 onward) the Kcna1-null mice did also have reduced seizure latencies in response to flurothyl at ages as young as P10 (Rho et al. 1999). Because Kcna1-null mice showed abnormal tremor after a cold swim, we performed similar cold-swim tests on five –/– (B6/129) mice (see METHODS). However, unlike the Kcna1-null mice, the Kcna2-null mice did not exhibit any signs of hyperexcitability (body tremors) after a cold swim.

Could sound be one of the stimuli able to induce RBS/TE in –/– mice? This question arises because RBS/TE can be induced audiogenically in certain strains of rats and mice that are either genetically susceptible or have been made susceptible by early partial deafening (see DISCUSSION). Typically, loud sounds of 100–130 dB lead after 2–20 s to RBS, closely followed by clonic seizures and/or TE (Ross and Coleman 2000). In preliminary tests, 70 of 71 applications of an octave band stimulus 8–16 kHz (112 dB SPL, 20-s duration) failed to induce RBS or TE in six –/– mice tested at ages P14–P18 (data not shown).

Developmental expression of Kcna mRNA in +/+ brain stem

To explore whether the second and third postnatal weeks might be an especially important period in the development of CNS Kv1 channels, the qPCR technique was used to measure the developmental time course of Kcna2 gene mRNA expression in the brain stems of 14 +/+ mice, two mice at each of seven ages tested (Fig. 3A). The expression level increased 10-fold between P1 and P29 for Kcna2 mRNA (Fig. 3A). This increase in +/+ Kcna2 expression occurs at approximately the same age as the onset of seizures in –/– mice (see DISCUSSION).

The developmental time course of Kcna1 and Kcna6 gene mRNA expression was also measured using the same brain stem tissue samples (Fig. 3B). The mRNA expression increased approximately thirtyfold for Kcna1 but only twofold for Kcna6 (Fig. 3B). Kcna1 mRNA and Kcna2 mRNA reached their half-maximal expression at P12 and P11, respectively (values from fits to Boltzmann functions; Fig. 3B). A comparison of the relative expression of these three Kcna genes is also of interest. The data suggest that neonate brain stems had higher expression of Kcna6 mRNA than Kcna1 and Kcna2 mRNA, whereas the opposite was true for juvenile brain stems (Fig. 3B). Also, Kcna1 and Kcna2 mRNA were present at similar copy numbers in neonatal mice, whereas in juvenile mice Kcna1 mRNA contributed more than twice as many copies as Kcna2 mRNA (Fig. 3B). If this reflects the ratios of Kv1 subunit proteins produced, it suggests that the order of expression strength in neonatal brain stem may be Kv1.6 >> Kv1.2 > Kv1.1, whereas this order is reversed during maturation, becoming Kv1.1 > Kv1.2 >> Kv1.6.

Kcna2-null MNTB neurons were hypoexcitable

One obvious way in which a lack of Kv1.2 might cause network hyperexcitability in the –/– CNS would be by reduced potassium currents causing hyperexcitability in individual neurons. Although it is probably unlikely that MNTB neurons within the brain stem auditory system play a role in seizure susceptibility in either Kcna1-null or Kcna2-null mice, MNTB neurons are a useful model for study because even small reductions in their I_Kv1 have large effects on excitability (Brew and Forsythe 1995; Brew et al. 2003).

To find out whether –/– MNTB neurons had abnormal excitability, we recorded and analyzed the responses to current pulses (180-ms duration, –100 to 200 or 0 to 200 pA, in 10-pA increments) of 63 MNTB neurons in brain stem slices from mice aged P9–P16 (9 +/+ mice, 14 +/– mice, and 15 –/– mice). The responses of three example MNTB neurons, each typical of their genotype, are shown in Fig. 4A. The –/– neuron fired the smallest numbers of APs, e.g., only a single initial AP at the start of a 200-pA pulse contrasting with three APs generated by the +/+ neuron (Fig. 4A, top traces). The –/– neuron also had the highest threshold current amplitude (defined as the smallest current pulse amplitude that generated at least one AP) of 160 pA, contrasting with 80 pA for the +/+ neuron and 130 pA in the +/– neuron. Overall, the threshold current amplitudes were significantly smaller in the 21 +/+ MNTB neurons than those in the 25 –/– neurons (P < 0.0001) or the 19 +/– neurons (P < 0.005, Fig. 4B). The +/– neurons’ threshold current amplitudes were distinctly intermediate between +/+ and –/– neurons because they were also significantly smaller than those of the –/– neurons (P < 0.05).

View larger version (23K): [in this window] [in a new window]   FIG. 4. –/– Medial nucleus of the trapezoid body (MNTB) neurons are less excitable by current pulses than +/+ neurons. Threshold currents and action potential (AP) numbers were measured from +/+, +/–, and –/– MNTB neurons subjected to 180-ms-duration current pulses –100 to 200 pA, applied at 1-s intervals, incrementing by 10 pA. A: example responses of typical +/+, +/–, and –/– MNTB neurons to the current-pulse protocol, shown here at 50-pA intervals. Linked arrows on the top traces mark the time during which sustained membrane potential was measured (used for Fig. 5 and for calculating the input resistances and resting potentials as described in RESULTS and supplemental Table S2). B–D: summary data on threshold currents and AP numbers from 21 +/+ neurons (filled circles), 17 +/– neurons (triangles), and 25 –/– neurons (open circles). For each neuron, data came from a single run of the current-clamp protocol. Significant differences between the genotypes are shown by single, double, or triple symbols (P < 0.05, P < 0.01, and P < 0.005, respectively). B: threshold current amplitudes required to generate at least one AP in the MNTB neurons of each genotype. Threshold current amplitudes were larger in –/– neurons and +/– neurons than in +/+ neurons. C: mean number of APs generated at each current-pulse amplitude. Error bars show the SE. –/– and +/– neurons fired significantly fewer APs than the +/+ neurons. Significant differences between +/+ and –/– values are shown by small asterisks, between +/+ and +/– values by large asterisks, and between +/– and –/– values by circumflexes. D: mean numbers of APs during the 5 largest current steps tested (160–200 pA). This value was termed the APN(160–200) and it was largest in +/+ neurons, smallest in –/– neurons, and intermediate in +/– neurons.

To more accurately represent the excitability of each MNTB neuron, and compare the distribution of excitability within each genotype, the AP numbers were averaged across the five largest current pulses tested, 160–200 pA, and this average was termed the APN(160–200). The APN(160–200) values were significantly larger in +/+ neurons than those in –/– neurons or +/– neurons (Fig. 4D).

In Fig. 4 and the statistical comparisons related earlier, five of an original total of 68 recordings were excluded because they fired so many APs that they were statistical outliers (supplemental Fig. S1A, arrows) and their tonic firing also disallowed their inclusion in Fig. 5 and supplemental Table S2 (see following text). These recordings were probably not from MNTB principal neurons and their exclusion made very little difference to any of the statistically significant differences between the genotypes (see legend to Fig. S1).

View larger version (16K): [in this window] [in a new window]   FIG. 5. Sustained potentials were more depolarized in +/+ than in –/– or +/– MNTB neurons. A: mean sustained potential responses to current pulses, measured in artificial cerebrospinal fluid (ASCF) for 21 +/+ MNTB neurons and 13 –/– neurons. Depolarizing current pulses led to larger depolarizations in the +/+ neurons than in the –/– neurons. (Same current-pulse protocol as in Fig. 4; same +/+ and –/– neurons as in Fig. 4C and Table S2, except excluding data from 12 –/– neurons to which only depolarizing pulses were applied. Mean responses of these 12 neurons were identical to within 1 mV of the 13 –/– neurons shown.) Sustained potential was defined as the mean of 600 data points toward the end of the current pulse, during the 30-ms period marked by linked arrows in Fig. 4A. Sustained potentials were corrected for current flow across the pipette access resistance. SE is shown by error bars, sometimes obscured by the size of the symbols. Significant differences between genotypes are denoted by single and double asterisks (P < 0.05 and P < 0.005, respectively). B: as in A, but comparing 17 +/– neurons with the 21 +/+ MNTB neurons. Depolarizing current pulses led to larger depolarizations in the +/+ neurons than in the +/– neurons.

These data show that –/– MNTB neurons and +/– MNTB neurons were hypoexcitable compared with their +/+ counterparts. This is especially surprising because it was previously shown that Kcna1-null MNTB neurons were hyperexcitable and had the reduced I_kl amplitudes expected because of their Kv1.1 deficit (Brew et al. 2003). In marked contrast, the deficits of Kv1.2 subunits in –/– and +/– MNTB neurons were associated with hypoexcitability, suggesting their I_kl amplitudes were enlarged, not reduced.

Altered resting membrane properties and I–V relations in –/– MNTB neurons

An enlarged I_kl would be expected to cause slightly less depolarized steady-state I–V relations in the range –70 to –40 mV, as well as enlarged conductance (reduced membrane resistance). Analysis showed that –/– MNTB neurons did have less depolarized sustained membrane potentials than those of +/+ MNTB neurons, for all depolarizing current-pulse amplitudes tested, as well as shallower I–V relations around rest (Fig. 5A; same neurons and pulse protocol as in Fig. 4; linked arrows in Fig. 4A show the time window of sustained potential measurement). The +/– neurons' sustained potentials were similar to those of +/+ neurons, although there were small significant differences in potential at a few current amplitudes (Fig. 5B). The abnormal sustained I–V relations in –/– MNTB neurons support the idea that their sustained I_kl amplitude is enlarged compared with +/+ neurons.

Although the mean resting potentials were slightly larger in the –/– and +/– neurons (–66 and –67 mV) than those in the +/+ neurons (–65 mV) the differences were not significant (supplemental Table S2, resting potential was defined as the sustained membrane potential during the 0-pA current pulse). Table S2 compares the MNTB neurons of each genotype across a range of excitability-related membrane parameters. The mean resting input resistance was about 90 M in –/– neurons, significantly lower than the 130–140 M in +/+ neurons (Table S2; see legend for values and details of both methods used to quantify input resistance). The mean resting input resistance of +/– neurons was similar to that of +/+ neurons (Table S2). These data do not support the idea that there had been variable levels of compensation for the Kcna2 deficit in +/– and –/– neurons because the SDs for each parameter were generally similar in the MNTB neurons of each genotype (Table S2). As expected for mouse strains repeatedly backcrossed into the same inbred C3HeB/FeJ background, the distributions of AP numbers, threshold currents, and other membrane properties for the +/+ MNTB neurons reported here were very similar to those for MNTB neurons from the control littermates of Kcna1-nulls (compare Figs. 4, 5, and supplemental S1 and Table S2 in the present study with Figs. 1 and 5 and Table 1 of Brew et al. 2003).

Action potentials and other properties were similar in +/+ and –/– MNTB neurons

Aside from the above-described differences that could be ascribed to potassium currents, MNTB neurons had similar properties irrespective of genotype. For example, the –/– and +/– sodium channels were probably functioning normally because MNTB neurons of all three genotypes had a similar initial AP during a 200-pA pulse, with an approximate mean latency of 3 ms, mean half-width of 0.9 ms, and a rapid rising phase beginning at –45 mV (supplemental Table S2). MNTB neurons of each genotype were probably of similar size because they had similar capacitances (Table S2). Also, the size of MNTB and its principal neuron somata appeared similar in brain stem slices of each genotype viewed in our recording chamber (data not shown). The pipette access resistances and ages were similar for MNTB neurons of each genotype (Table S2). Thus the genotypic differences in threshold currents, AP numbers, and I–V relations described earlier could be produced solely by differences in potassium currents.

Dendrotoxin-I had greater effects on –/– MNTB neurons and abolished genotypic differences

Next, we measured the effects of dendrotoxin (DTX; see INTRODUCTION) on threshold current amplitudes, AP numbers, and sustained potentials in five +/+ and four –/– MNTB neurons, to test whether –/– hypoexcitability could be caused solely by an enlarged I_Kv1 (the subcomponent of I_kl clearly attributable to Kv1-type channels because of its DTX sensitivity). If so, DTX should have greater effects on the excitability of –/– neurons than +/+ neurons and there should be no differences in excitability between +/+ and –/– neurons when DTX is present. The typical effect of DTX on MNTB neuron firing is a reduction in the threshold current for an AP and a conversion from phasic to tonic firing (e.g., in rat MNTB; Brew and Forsythe 1995). This phasic firing during a prolonged current pulse was shown earlier for two typical example +/+ and –/– MNTB neurons in control ACSF (Fig. 4A) and both converted to tonic firing throughout current