Characterization of Myenteric Sensory Neurons in the Mouse Small Intestine

doi: 10.1152/jn.00204.200696:998-1010, 2006. ;J Neurophysiol 

Yukang Mao, Bingxian Wang and Wolfgang KunzeMouse Small IntestineCharacterization of Myenteric Sensory Neurons in the

You might find this additional info useful...

 80 articles, 40 of which you can access for free at: This article citeshttp://jn.physiology.org/content/96/3/998.full#ref-list-1

 11 other HighWire-hosted articles: This article has been cited by http://jn.physiology.org/content/96/3/998#cited-by

including high resolution figures, can be found at: Updated information and serviceshttp://jn.physiology.org/content/96/3/998.full

can be found at: Journal of Neurophysiology about Additional material and informationhttp://www.the-aps.org/publications/jn

This information is current as of June 3, 2013.

at http://www.the-aps.org/. Copyright © 2006 by the American Physiological Society. ISSN: 0022-3077, ESSN: 1522-1598. Visit our websitetimes a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991.

publishes original articles on the function of the nervous system. It is published 12Journal of Neurophysiology

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

Characterization of Myenteric Sensory Neurons in the Mouse Small Intestine

Yukang Mao,1,* Bingxian Wang,3,* and Wolfgang Kunze1,2

1Brain-Body Institute, 2Department of Psychiatry and Behavioral Neurosciences, and 3Department of Medicine, McMaster University,Hamilton, Ontario, Canada

Submitted 25 February 2006; accepted in final form 11 April 2006

Mao, Yukang, Bingxian Wang, and Wolfgang Kunze. Character-ization of myenteric sensory neurons in the mouse small intestine.J Neurophysiol 96: 998–1010, 2006; doi:10.1152/jn.00204.2006. Werecorded from myenteric AH/Dogiel type II cells, demonstrated mech-anosensitive responses, and characterized their basic properties. Re-cordings were obtained using the mouse longitudinal muscle myen-teric plexus preparation with patch-clamp and sharp intracellularelectrodes. The neurons had an action potential hump and a slowafterhyperpolarization (AHP) current. The slow AHP was carried byintermediate conductance Ca2�-dependent K�-channel currents sen-sitive to charybdotoxin and clotrimazole. All possessed a hyperpolar-ization-activated current that was blocked by extracellular cesium.They also expressed a TTX-resistant Na� current with an onset nearthe resting potential. Pressing on the ganglion containing the patchedneuron evoked depolarizing potentials in 17/18 cells. The potentialspersisted after synaptic transmission was blocked. Volleys of presyn-aptic electrical stimuli evoked slow excitatory postsynaptic potentials(EPSPs) in 9/11 sensory neurons, but 0/29 cells received fast EPSPinput. The slow EPSP was generated by removal of a voltage-insensitive K� current. Patch-clamp recording with a KMeSO4-containing, but not a conventional KCl-rich, intracellular solutionreproduced the single-spike slow AHPs and low input resistances seenwith sharp intracellular recording. Cell-attached recording of interme-diate conductance potassium channels supported the conclusion thatthe single-spike slow AHP is an intrinsic property of intestinalAH/sensory neurons. Unitary current recordings also suggested thatthe slow AHP current probably does not contribute significantly to thehigh resting background conductance seen in these cells. The charac-terization of mouse myenteric sensory neurons opens the way for thestudy of their roles in normal and pathological physiology.

I N T R O D U C T I O N

The ability of the intestine to function independently of theCNS has been attributed to the presence of the enteric nervoussystem (ENS) (Costa et al. 1998; Furness and Costa 1987).Consistent with this, the ENS of the guinea pig contains thecomponents of an independent, integrative nervous system,including sensory, inter- and motor neurons (Kunze and Fur-ness 1999). In the guinea pig, enteric sensory neurons (AHcells) have large oval somas with multiple long processes(Dogiel type II morphology) and they make up about 20–30%of enteric neurons. Because of their sensory role and largenumbers in the gut wall, Dogiel type II neurons have becomean object of considerable experimental interest (see Brookes2001; Furness et al. 1998; Holzer 2001; Holzer et al. 2001;Kunze and Furness 1999). It is generally assumed that Dogieltype II neurons in species other than guinea pig are also

sensory, although intrinsic sensory neurons have been directlyidentified only in the guinea pig where responses to chemicalor mechanical stimulation were recorded under conditions ofsynaptic blockade (Bertrand et al. 1997; Kunze et al. 1995,2000).

For historical reasons the great majority of electrophysio-logical recordings from enteric neurons have been made in theguinea pig small intestine. With the advent of knockout andtransgenic technology, however, the mouse is being increas-ingly used in the study of physiology (Picciotto and Wickman1998) including the activity of the intestine (see Bullard andWeaver 2002; Der et al. 2000; Gershon 1999; Spencer 2001).Despite this there have been few published reports (Bian et al.2003; Furukawa et al. 1986; Nurgali et al. 2004; Ren et al.2003) of nerve cell recording from the intact mouse entericnervous system and these were done only in current-clampmode. Nevertheless, neurons with clear AH cell electrophysi-ology have been recorded in mouse small intestine (Bian et al.2003; Ren et al. 2003). The voltage-clamp device (Cole 1982)has been the conventional method for studying currents inenterocytes, intestinal myocytes, or interstitial cells of Cajal.Yet, reports of voltage-clamp recordings from myenteric neu-rons are scarce in species other than guinea pig. Three studiesused cultured rat neurons (Franklin and Willard 1993; Haschkeet al. 2002; Hirning et al. 1990) and one used cultured mouseneurons (Liu et al. 2002); all used the patch-clamp technique.Liu et al. (2002) reported that cultured mouse small intestinalmyenteric neurons constitute an electrophysiologically homo-geneous population that discharges phasically in response toprolonged depolarization.

The aim of the present experiments was to provide an initialdescription of mouse enteric AH cell electroresponsiveness andmajor somatic currents. Among guinea pig myenteric neurons,a slow afterhyperpolarization (AHP) current, a tetrodotoxin(TTX)-resistant persisting Na� current and a hyperpolariza-tion-activated cationic current are predominantly expressed inAH cells (Furness et al. 2004a; Kunze and Furness 1999).These currents profoundly influence AH cell electrorespon-siveness and they were thus the ones we chose to initiallyinvestigate. We made recordings from intact ganglia becausedissociation and isolation of enteric neurons erase currents thatare expressed in situ (Rugiero et al. 2002, 2003) and disruptnatural synaptic connections. In preliminary experiments, wefound that the technique of patch-clamp recording from my-enteric neurons in the longitudinal muscle myenteric plexuspreparation (LMMP) works as well for the mouse (Kunze et al.2002) as it does for the guinea pig (Kunze et al. 2000). This* Y. Mao and B. Wang contributed equally to this work.

Address for reprint requests and other correspondence: W. Kunze, St.Joseph’s Healthcare, Hamilton, North Tower, Room T3306, 50 CharltonAvenue East, Hamilton, Ontario, Canada L8N 4A6 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

J Neurophysiol 96: 998–1010, 2006;doi:10.1152/jn.00204.2006.

998 0022-3077/06 $8.00 Copyright © 2006 The American Physiological Society www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

allowed us to make voltage- and current-clamp recordingsfrom mouse myenteric neurons that could be directly comparedwith previous guinea pig data (Kunze et al. 2000; Rugiero et al.2002) using the identical technique.

M E T H O D S

Preparation

We used inbred C57BL/6 female mice (20–25 g) obtained fromCharles River laboratories (http://www.criver.com). All procedureswere in line with University of Tubingen and McMaster guidelines forthe use and care of animals. A 2-cm segment of ileum was removedfrom deeply anesthetized [Na pentobarbitone, 70 mg kg�1, adminis-tered intraperitoneally (IP)] mice, after which the animals were killedby exsanguination. The tissue was placed in a 2-ml recording dishlined with silastic and filled with oxygenated extracellular Krebssaline of the following composition (in mM): NaCl 118.1, KCl 4.8,NaHCO3 25, NaH2PO4 1.0, MgSO4 1.2, glucose 11.1, and CaCl2 2.5.Nicardipine (2–3 �M) was routinely added to the saline to preventspontaneous muscle contraction. The segment was opened along a lineparallel to the mesenteric attachment and pinned flat, under moderatetension, mucosa uppermost. The myenteric plexus was exposed bydissecting away the mucosa, submucosa, and circular muscle. Therecording dish was then mounted on an inverted microscope and thetissue continuously superfused (4 ml min�1) with physiological sa-line, gassed with 95% O2-5% CO2, and warmed to 35–37°C. A singleganglion was prepared for patch clamping as described in Kunze et al.(2000); briefly, the ganglion was exposed for 10–15 min to 3 ml of0.01–0.02% protease type XIV (Sigma, http://www.sigmapaldrich.com), then the upper surfaces of myenteric neurons were revealed bycleaning part of the ganglion with a fine hair until individual neuronsoma became just visible. As described previously (Kunze et al. 2000;Rugiero et al. 2002) there was no evidence of cell swelling after thisgentle treatment.

Other salines that were substituted for the standard extracellularKrebs saline were those in which CaCl2 was reduced to 0.25 mM andMgCl2 increased to 10 mM (saline for synaptic blockade), and anotherfor which 90% of NaCl was replaced by N-methyl-D-glucamine-Cl(NMDG-Cl) (saline to remove Na� currents). CdCl2 (0.5 mM) or 2mM CsCl was also added to some saline solutions, when, to preventdivalent cation precipitation, 10 mM HEPES buffer (pH � 7.4) wasadded, bicarbonate and phosphate salts omitted, and NaCl adjusted tomaintain osmolarity. Aliquots of stock solutions of clotrimazole,charybdotoxin, and apamin (Sigma) were kept at �4°C and added towarm, oxygenated, extracellular saline 10 min before use.

Electrophysiology

Signals were measured in voltage- or current-clamp modes using anAxon Instruments Multiclamp 700A computer amplifier (Axon In-struments, http://www.axon.com) and a Digidata 1322A (Axon In-struments) digitizer was used for A/D conversion.

A bipolar stimulation electrode, constructed from two twisted75-�m insulated stainless steel wires, was placed over one of theconnecting internodal strands lying circumferentially to the ganglionbeing recorded from. Nerve fibers were electrically stimulated using0.1-ms, 0.1- to 1-mA constant-current pulses delivered from anISO-flex stimulus isolation unit (AMPI, http://www.ampi.co.il/).

Patch pipettes were pulled on a Flaming-Brown P97 (Sutter Instru-ments, http://www.sutter.com) electrode puller to produce micropi-pettes with resistances 4–6 M�. The error arising from uncompen-sated series resistance for a 130-mV voltage command was 2–4 M�for typical values of cell input and access resistances obtained inwhole cell mode. Signals were low-pass, four-point Bessel filtered at2 or 5 kHz, and then digitized at 5 or 20 kHz. Conventional sharp

electrodes were made from thin-wall borosilicate glass and filled with1 M KCl and 0.5% Neurobiotin.

Data were stored on computer and analyzed off-line. Voltage orcurrent commands were delivered to the amplifier under computercontrol using Clampex 8 (Axon Instruments) software. To allow directcomparison with earlier work (Rugiero et al. 2002) using in situpatch-clamp recording from guinea pig myenteric neurons, patchpipettes were filled with a standard KCl-rich intracellular saline of thefollowing composition (in mM): KCl 140–146, NaCl 10, CaCl2 1,MgCl2 2, HEPES 10, Na3GTP 0.2, and EGTA 2, to which 0.2%(wt/vol) Neurobiotin had been added; pH was titrated to 7.3 using 0.1mM KOH. This solution had a predicted (Maxchelator: http://www.stanford.edu/�cpatton/maxc.html) free [Ca2�] of 0.09 �M at 37°C(Bers et al. 1994). This value is close to the resting free intracellular[Ca2�] as estimated using Ca2�-sensitive dyes in guinea pig Dogieltype II neurons (Hillsley et al. 2000; Tatsumi et al. 1988).

A solution favoring preservation of the slow AHP similar to thatrecommended by Velumian and Carlen (1999) was used for somecells. Its composition (in mM) was: KMeSO4 110–115, NaCl 9,CaCl2 0.09, MgCl2 1.0, HEPES 10, Na3GTP 0.2, and BAPTA.K4 0.2with 0.2% Neurobiotin and 14 mM KOH to bring the pH to 7.3. Thesame saline was used to perfuse the cytoplasmic face of inside-outpatches, except that total Ca2� was altered to produce free [Ca2�] of0.1 or 0.5 �M. Total and free [Ca2�] were calculated using Max-Chelator.

About �50 hPa pressure was applied to the pipette before its tipentered the extracellular saline; the pressure was maintained until thetip was in close apposition to a neuron membrane. Only recordingswith seal resistances �4 G� were used and about half of these formedspontaneously (cf. Kunze et al. 2000) when pipette pressure wasreleased; the rest were formed by applying mild (�10 hPa) suction.Whole cell recording mode was entered by further suction, then theamplifier was switched to current-clamp mode and brief current pulsesdesigned to evoke a single action potential (AP) were injected by thepatch pipette. Thus resting membrane potential, AP shape, and theexistence of a slow AHP were all noted within seconds of rupturingthe cell membrane. Access resistance and cell membrane resistance,capacitance, and time constants were periodically monitored by soft-ware programmed switching to the Pclamp membrane test protocol,which injects square-wave pulses oscillating about the holding poten-tial (Vhold). Quasi-steady-state current–voltage (I–V) plots were madeusing voltage-clamp mode and by slowly depolarizing the membrane(ramp speed � 25 mV s�1) from an initial hyperpolarizing step.

Responses to local deformation in and around the ganglion-con-taining patched neurons were sought as previously described (Kunzeet al. 2000). Briefly, after obtaining a stable whole cell recording,surfaces of the ganglion and surrounding muscle were gently andsystematically prodded to depths of 25 and 50 �m from “touch” witha calibrated Von Frey hair (Kunze et al. 2000).

Descriptive statistics are given as means � SD. When a statisticaltest was performed, the P value given is the probability of the teststatistic being at least as extreme as the one observed if the nullhypothesis of no difference is admitted.

Histochemistry

At the end of each recording, neurons were ionophoretically loadedwith Neurobiotin by passing forty 500-ms duration, �0.1 nA currentpulses by the patch pipette. The tissue was fixed in Zamboni’s fixative(2% vol/vol picric acid, 4% paraformaldehyde in 0.1 M Na2HPO4/NaH2PO4 buffer, pH � 7.0) overnight at 4°C, and then cleared usingthree 10-min washes of DMSO followed by three 10-min washes withphosphate-buffered saline (PBS). The tissue was then exposed tostreptavidin–Texas Red (Vector, http://www.vectorlabs.com), diluted1:50, to reveal Neurobiotin. After a final rinsing, the tissue wasmounted in PBS containing 80% glycerol and 0.1% NaN3 and viewedunder fluorescence epi-illumination on a Leitz DM RBE microscope

999MOUSE MYENTERIC SENSORY NEURONS

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

as part of a Quantimet 600 high-resolution image analysis system(Leica, http://www.leica.com). Texas Red (596 and 620 nm excitationand emission peaks) fluorescence was viewed using a N2.1 Leicafilter. Images were recorded and digitized with a black and white CCDcamera (Cohu, San Diego, CA) connected to the Quantimet system.

R E S U L T S

Resting potentials and input resistances

In agreement with previous LMMP patch-clamp studies thatused the standard KCl-rich intracellular solution of Rugiero etal. (2002, 2003), we use the term AH cell (Hirst et al. 1974) todescribe those neurons whose spikes had humps (Clerc et al.1998; Kunze et al. 2000; Schutte et al. 1995) on the repolar-ization phase and expressed a slow AHP current as revealed bytwo successive voltage-ramp commands (see following text).

Results using the standard KCl-rich intracellular saline weretaken from 32 myenteric AH cells in 31 ganglia from 30animals. All neurons included for electrophysiological analysishad APs with a hump on the recovery phase. Cells that lackedAP humps were also recorded (n � 31) and none of these hada slow AHP current as tested for by the double voltage-rampprotocol; these S cells are not included in the present analysis.There were no cells recorded that had humps but lacked a slowAHP current or had the current but lacked the hump.

Twenty-two of the 32 AH cells and 17/31 non-AH cellsrecorded with the standard intracellular solution were injectedwith Neurobiotin and later recovered for morphological iden-tification. The correlation between morphology and electro-physiology was unambiguous. All 22 AH cells had Dogiel typeII morphology with smooth oval somas and multiaxonal orpseudounipolar projections, having from two to four longprocesses that projected circumferentially. All non-AH cellswere uniaxonal with Dogiel type I or filamentous soma shapes.

AH cells had a resting membrane potential (Vrest) of(mean � SD) �55 � 7 mV (n � 32). Vrest did not changeduring recording periods of �20 min. Input resistances (Rin)were calculated from instantaneous voltage deflections elicitedby the injection of 500-ms-duration hyperpolarizing currentpulses (Fig. 1). The slope (Fig. 1B) of the voltage–current(V–I) relation was extrapolated to Vrest to give a resting inputimpedance (Rrest) of 500 � 52 M� (n � 30). The membranetime constant (�) was 28 � 8 ms (n � 32).

All 32 AH cells showed a time-dependent sag in the voltagetrace during hyperpolarizing current injection (Fig. 1A). Posi-tive to �90 mV peak voltage response was linearly related tocurrent intensity, but negative to �90 mV the onset of theinward rectifier (Baidan et al. 1992; Hanani et al. 2000;Rugiero et al. 2002) was signaled by a conductance increase.Consequently, AH cells had a high resistance state near Vrest,but when hyperpolarized input impedance decreased conspic-uously. This trend was quantified by comparing slope conduc-tances (Gs) at V � 0 and �90 mV taken from quasi-steady-state I–V curves. These curves had an inflection close to Vrestwhen Gs approached 0 (Fig. 1C) or sometimes became nega-tive (e.g., trace 1, Fig. 7), matching the N-shaped I–V relationdescribed for guinea pig AH cells (Rugiero et al. 2002). For 29AH cells, Gs was 2.3 � 0.7 nS at Vrest compared with 4.5 � 1.9nS at V � �90 mV.

The action potential and slow AH current

Action potentials of the 32 AH cells recorded from, usingthe standard KCl-rich patch pipette saline, had the character-istic shape of Dogiel type II neuron spikes; they were broadwith a hump on the repolarization phase. The hump was alwaysconfirmed by the presence of an inflection in the time deriva-tive of the voltage trace (Fig. 2A) (Clerc et al. 1998). Spikeshad a peak amplitude and width at half-amplitude (half-width)of 102 � 14 mV and 2.7 � 0.8 ms. These parameters are thesame as those reported for the guinea pig (Rugiero et al. 2002).An effective method for evoking slow AHP currents andmeasuring their voltage dependency has been to inject twosuccessive, slow (25 mV s�1) depolarizing (�110 to 0 mV)voltage ramps (Rugiero et al. 2002). The interval between theend of the first ramp and the beginning of the second ramp was50 ms. The first ramp activates the current, the second de-scribes the voltage relation during activation, and the differ-ence between them gives the current (IKCa) that was evoked(Rugiero et al. 2002). When this experiment was performed onmouse myenteric neurons with the standard KCl intracellular

FIG. 1. Input resistance and voltage–current (V–I) curve. A, top: whole cellvoltage responses to hyperpolarizing current pulses (bottom traces) injected bypatch pipette. Responses displayed a time-dependent relaxation. Instantaneousand time-dependent voltages were measured at positions of square and triangle,respectively. B: plots of instantaneous and steady-state V–I relationships.Slopes of straight lines indicate maximal input resistance (640 M�) near Vrest

and reduced resistance (440 M�) at hyperpolarized levels. C: quasi-steady-state I–V curve generated by slowly (25 mV s�1) depolarizing voltagecommand. Low-resistance state (closed arrowhead) with slope conductance of2.7 nS at V � �90 mV. Conductance decreased to 1.7 nS near Vrest, causingnegative inflection at open arrowhead.

1000 Y. MAO, B. WANG, AND W. KUNZE

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

solution, all (32/32) AH cells tested exhibited a distinct out-ward difference current. With voltage adjusted for a 9 mVjunction potential (Gola and Niel 1993) the difference currentreversed at the Nernst equilibrium potential for K� (EK)between �91 and �87 mV (88 � 2 mV, n � 12). IKCa waswell fitted by the Goldman–Hodgkin–Katz (GHK) equation forK� current

I �

PKVF2

RT�K�i � K�oe

�VF/RT�

1 � e�VF/RT

where R, T, and F have their usual meanings. [K�]o was set at4.8 mM, but [K�]i and K� current permeability (PK) were freeto vary during the fitting process. For the current shown in Fig.2B, [K�]i � 140 mM and PK � 0.32 � 10�9 cm3 s�1.Although this current was present in all AH cells tested, PKvaried considerably between neurons (Rugiero et al. 2002),ranging from 0.0054 to 0.32 � 10�9 cm3 s�1. For all 32 AHcells, [K�]i � 144 � 5 mM and PK � 0.13 � 0.03 � 10�9 cm3

s�1. This current was Ca2� dependent because addition of theCa2� channel blocker CdCl2 (0.5 mM) to the extracellularsaline (Fig. 2C) completely abolished it.

Only five of the 32 cells had a discernable slow AHP lasting�2 s (duration: 3.6 � 1.1 s; amplitude: �2.3 � 1.4 mV) aftera single action potential. The proportion of single spike slowAHPs recorded with our standard solution patch pipettes (5/32)is less (P � 0.003, Fisher’s exact test) than that (12/12)reported for sharp electrode recording by Ren et al. (2003),who also found a lower Rin value of 136 � 121 M� than the497 � 52 M� given above (P � 0.001, t-test, two-tailed). Weaddressed this discrepancy by making recordings from a fur-ther 18 myenteric AH cells using various pipette configura-tions; all 18 neurons had Dogiel type II shape as revealed afterinjecting Neurobiotin. Sharp intracellular pipettes filled with 1M KCl were used to record from nine neurons and whole cellpatch-clamp recordings were made from another nine usingpipettes filled with a slow AHP-favoring solution containingMeSO4 (METHODS). All 18 AH cells recorded with eithermethod had single-spike slow AHPs (Fig. 3), which is a greaterproportion (P � 0.001, Fisher’s exact test, two-tailed) than thatfor standard pipette solution recording (5/32) (Table 1). For

four of four AH cells, the slow AHP was unmitigated byexposure to 100 nM extracellular apamin for 20 min (Fig. 3B)(Kunze et al. 1994). After 20- to 30-min washout with normalKrebs solution, the same neurons were then exposed to 100 nMcharybdotoxin for a further 20 min, which blocked the slowAHP. The block was irreversible for 30 min final washout(Kunze et al. 1994). Extracellular clotrimazole (20 �M) re-versibly abolished the slow AHP for four of four AH cells (Fig.3C) and after 5 to 10 min application, washout occurred within3 min. Consistent with the current-clamp slow AHP data, AHPcurrent availability as measured by K� permeability (PK) wasdoubled to 0.27 � 0.02 � 10�9 cm3 s�1 when KMeSO4 pipettesolution was substituted for the KCl-rich one (Table 1). Vrestwas comparable between recording modes, but sharp orKMeSO4 pipette recording was associated with a greater thanthreefold increase in background conductance (Table 1). Inaddition, Rin values for sharp and KMeSO4 pipette recordingswere not discernibly different. Sharp electrode recordingsyielded smaller-amplitude APs than those of patch-clamp re-cordings, irrespective of the filling solution, yet AP half-widthswere unaffected by recording modality (Table 1). Attenuationof AP amplitudes by sharp electrodes is a well-known phe-nomenon and can be ascribed to the poor high-frequencyresponse of the electrode, and not damage to the neuron (Li etal. 2004).

Neurons recorded using the KMeSO4 solution or sharpelectrodes were less excitable than those recorded with thestandard patch-clamp solution. Action potential thresholds(rheobase) measured as the minimal intensity for 500-mscurrent pulses required to evoke single APs 50% of the time,were about four times lower for standard solution recordings(Table 1). Action potential firing accommodation was alwaystested with a 500-ms-duration positive current pulse at twofoldrheobase intensity. For neurons recorded using the standardsolution, 18/19 discharged with a tonic firing pattern (Fig. 3A),i.e., throughout the test pulse. Those recorded with sharpelectrodes or KMeSO4 solution (17/18) discharged phasically(Fig. 3, B and C), i.e., accommodation occurred within the first250 ms of the 500-ms stimulus pulse.

To minimize perturbation of the intracellular milieu whilerecording the slow AHP current, we also attempted to record

FIG. 2. Action potential (AP) shape and slow af-terhyperpolarization (AHP) potential and current. A,top: AP with hump evoked by passing brief supra-threshold current pulse by patch pipette into Dogieltype II neuron soma. Bottom: hump was made morenoticeable as a transient slowing (arrow) of repolar-ization in the smoothed time derivative of AP traceabove. B: gK,Ca revealed by depolarizing a Dogieltype II neuron using 2 successive voltage ramps (25mV s�1). Top: first ramp (trace 1) activated thecurrent and then the second ramp was executed.Bottom: difference current (2 � 1) between those intop panel was fitted with the Goldman–Hodgkin–Katz (GHK) equation for K� currents, giving thepermeability and internal [K�] indicated. C, top: first(1) and second (2) voltage ramps from different AH/Dogiel type II neurons showing activation of com-pound AHP current (HEPES extracellular saline).Bottom: Ca2� dependency of compound AHP currentwas revealed when 0.5 mM CdCl2 was added to theextracellular saline; the I–V curve for the first voltageramp (1) did not substantially differ from that for thesecond (2).

1001MOUSE MYENTERIC SENSORY NEURONS

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

the slow AHP ion channel (Fig. 4) from the nine neurons thatwere patched with the KMeSO4 pipettes. After G� seals wereformed, APs were evoked on passing 20 ms-duration inwardcurrent pulses by stepping the voltage clamp from 0 to �80mV. Unitary currents caused by AHP channel opening (Kunzeand Mueller 2002; Vogalis et al. 2002a) were detected after theAP in four of the nine cells (Fig. 4). Before the AP, the channelhad a low open probability (P0 � 0.05 � 0.02, supposing threechannels in the patch), but this increased to a maximum of0.24 � 0.12 postspike. All-points histograms made from thepostspike openings (Fig. 4C) were fitted with multiple Gauss-ians to reveal unitary currents of 1.6 � 0.3 pA, which,

assuming Vrest � �60 � 8 mV (Table 1), yielded a unitaryconductance of 27 � 6 pS. Ensemble averages of �10 indi-vidual traces gave postspike currents that activated rapidly butdecayed to 0 pA over 4–10 s (Fig. 4B)—a time courseanalogous to that for the whole cell slow AHP current (Vogaliset al. 2002a). After recording in cell-attached mode, inside-outpatches were pulled from each of the active patches. Each cellwas repatched with a new electrode for whole cell recording.The cytoplasmic face of the patch was exposed to a gravity-fedstream of the KMeSO4 pipette solution whose [Ca2�] waseither 0.1 or 0.5 �M (see METHODS). Channel openings wereextremely rare with 0.1 �M cytoplasmic Ca2� but when this

FIG. 3. Effect of recording method on slow AHP. A, top: whole cell recording using patch pipette filled with KCl-rich solution. AP discharge on injectionof 500-ms-duration current pulses (middle traces) at 1 � and 2 � threshold intensities. Near threshold the neuron fired between 0 and 2 APs, discharge was tonicat supraliminal stimulation. Bottom: this neuron had a modest (1.6-mV amplitude, 2-s-duration slow AHP). B, top: intracellular recording using sharp electrodesfilled with 1 M KCl. Responses to threshold and 2 � threshold current injection (middle) were phasic. Bottom: this neuron had a 10-s-duration, slow AHP aftera single AP that was blocked after adding 50 nM charybdotoxin to the extracellular saline. C, top: whole cell recording using KMeSO4-rich pipette solution.Phasic responses to threshold and 2 � threshold stimulation. Bottom: from same neuron as top panel, 10-s-duration, single spike, slow AHP abolished by 20�M extracellular clotrimazole.

TABLE 1. Effect of recording method on slow AHP current and action potential parameters

Slow AHP

Vrest, mV Rin, M� Rheobase, pA

Action Potential

PK, 10�9 cm3

s�1No. ofcells Amplitude

Amplitude,mV

Half-Width,ms

Whole cell KCl 5/32a �2.3 � 1.4 �55 � 7 497b � 52 (30) 43c � 19 (24) 102 � 14 (24) 2.4 � 0.3 (24) 0.13d � 0.03Sharp 9/9 �4.1 � 1.2 �57 � 5 134 � 46 188 � 110 76e � 13 2.1 � 0.5 —Whole cell MeSO4 9/9 �3.8 � 3.7 �60 � 8 161 � 38 169 � 73 105 � 17 2.3 � 0.4 0.27 � 0.02

Cell numbers in parentheses where they are less than the total recorded for that category. a Differs from that for sharp or KMeSO4 pipettes (P � 0.001, Fisher’sexact test for proportions). b Differs from that for sharp or KMeSO4 pipettes (P � 0.05, Bonferroni–Holm test for pairwise comparisons). c Differs from that forsharp and KMeSO4 pipettes (P � 0.05, Bonferroni–Holm test for pairwise comparisons). d Differs from that for KMeSO4 pipettes (P � 0.0001, Mann–Whitneytest, two-tailed). e Differs from that for KCl or KMeSO4 pipettes (P � 0.05, Bonferroni–Holm test for pairwise comparisons).

1002 Y. MAO, B. WANG, AND W. KUNZE

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

was increased to 0.5 �M the average number of open channels[NP0, calculated from averaged unitary currents as in Kunze etal. (2000)] noticeably increased (Fig. 4D) and did not decreaseeven when the low Ca2� solution was again applied (cf.Vogalis et al. 2002b). For Fig. 4D the number of activechannels in the patch was at least three, giving an upper limitfor P0 of 0.4. Current amplitudes changed with transpatchvoltage (Fig. 4, Ea and F), although NP0 was poorly voltagedependent. Single-channel conductances were calculated usingthe slope of the dashed line connecting single-channel open-state currents in all-points current histograms that were fittedwith multiple Gaussians (Fig. 4Eb). The channel conductancewas 26 � 5 pS (n � 4), which is in the intermediate conduc-tance class for KCa channels recorded with 140 mM symmet-rical K�. The calcium sensitivity, conductance, and the poorvoltage sensitivity together suggest that the slow AHP channelis related to the IKCa (IK1, KCa3.1 type) channel (IUPHAR2002).

Inward currents active near the resting potential

HYPERPOLARIZATION-ACTIVATED CURRENT. Two inward cur-rents that are active near Vrest have been described in guineapig AH cells: 1) a hyperpolarizing-activated cationic current(Ih), which produces the sag in membrane voltage duringapplication of sustained negative current (Galligan et al. 1990;Rugiero et al. 2002); and 2) a TTX-resistant, persisting Na�

current (INa,P), which results in a negative-going inflection inthe steady-state I–V graph (Rugiero et al. 2002). Therefore wetested whether similar currents are present in mouse myentericAH neurons.

We examined the properties of the Ih by injecting 1 to 2s-duration hyperpolarizing voltage command pulses from aholding potential of �50 mV. All 32 AH cells exhibited atime-dependent inward current, which was further analyzedwith voltage-step protocols for 24/32 cells. The Ih amplitudewas determined from the difference between the steady-state(Iss) and instantaneous (Ii) currents (Fig. 5A), where Ii wasmeasured from a single exponential fit to the current traceextrapolated to the beginning of the step command (Fig. 5B).Maximal Ih (Imax) was determined by plotting Ih amplitudeagainst voltage, followed by fitting with a single-factor Boltz-mann equation

I �Imax

1 � e�V�V1/2)k

to yield Imax for each neuron (Fig. 5C). Fractional activation(open probability for the gating variable: P0 � ti/ti,max) wasdetermined from the amplitude of instantaneous tail currents(ti) using voltage steps as in Fig. 5A. To avoid contaminationby capacitative transients and a transient outward rectifier, timeasurements were determined by fitting a single exponentialto the tail current and extrapolating back to the time of offsetof the step voltage command (e.g., Fig. 5F). Using this method,

FIG. 4. Ion channels that determine slow AHP in Dogiel type II neuron. A: digital image of Neurobiotin-filled, multiaxonal neuron from which theserecordings were made. B, top: cell attached unitary current recording; dashed lines identify closed states. AP evoked by passing a brief positive current pulse(S) by pipette evokes ion channel opening. Bottom: ensemble average from 11 single traces of pre- and poststimulus channel activity gives postspike current withsimilar duration (about 5 s) and relaxation as the whole cell slow AHP. C: all-points–amplitude histogram of current taken from postspike activity of all 11 tracesfitted with multiple Gaussians gives unitary current amplitude of 1.5 pA (whole cell RMP � �55 mV). D: inside-out patch pulled from cell-attached one in B.Cytoplasmic membrane side initially exposed to nominally Ca2�-free intracellular KMeSO4-rich saline with NP0 �0.01. Exposure of 30 s to 0.5 �M Ca2�

increased channel opening to NP0 � 1.3 (open circles). Three active channels in the patch would give P0 � 0.4 (line graph) after exposure to 0.5 �M Ca2�.Ea: unitary currents from same inside-out patch as for D at transpatch potentials indicated after exposure to 0.5 �M Ca2�. Eb: all-points–amplitude histogramsmade from unitary current recordings in Ea. Slope of dashed line connecting single-channel open states give single-channel conductance of 27 pS. F: P0 vs.transpatch voltage. With 0.5 �M Ca2� bathing the cytoplasmic side of the inside-out patch the open channel activity altered very little with voltage.

1003MOUSE MYENTERIC SENSORY NEURONS

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

P0 was plotted against V in Fig. 5D and the curve fitted with theBoltzmann equation, giving V1/2 � �77 mV and k � 10 mVfor this neuron. Mean values (n � 12) of P0 are plotted againstcommand voltage in Fig. 6A, giving V1/2 � �78 � 7 mV anda slope factor k � 11 � 4 mV.

The Ih reversal potential (Eh) was measured from the inter-section of two instantaneous I–V relations (Lamas 1998). Weused Ii from the “ON” current response for the protocol shownin Fig. 5A (Vhold � �50 mV) for one curve, and instantaneoustails for the protocol shown in Fig. 5E (hold � �130 mV) forthe other curve. For the experiment shown in Fig. 5G, Eh was�26 mV, and the average of 10 AH cells was Eh � �28 � 3mV. The maximal conductance (g�) was calculated using therelation I � g�P0�V � Eh�. From the Boltzmann fits describedabove, when P0 � 1, V � �140 mV and Imax � 205 � 96 pA,and thus g� � 2 nS. Because P0 � 0.1 at V � �55 mV (Fig.6A), Ih conductance near rest would be about 0.2 nS. Thevariable step and prestep protocols shown in Fig. 5, A and Eproduced activation and deactivation traces, respectively, andthese were well fitted with single-exponential functions yield-

ing time constants (�) at various potentials (Fig. 6B). Theequation (Willms et al. 1999)

� ��0e

��V�V0�/k

1 � e�V�V0�/k

was simultaneously fitted to the combined activation and de-activation �–V plots of Fig. 6B, where �0, V0, k, and � are freeto vary during the fitting process. The fitting program gave�0 � 593 � 30 ms, V0 � �73 � 7 mV, k � 9 � 1 mV, and� � 0.4 � 0.1. The location of the peak of the �–V plot did notdiffer from that for V1/2 in the Boltzmann fit to the steady-stateactivation curve (P � 0.1, t-test, two-tailed). This suggests thatthe assumptions made in fitting the time constant data werereasonable.

This current was reversibly blocked by addition of 2 mMCsCl to the extracellular saline (middle trace compared withtop trace in Fig. 6C). The inward rectifier (IKir) is manifest asa deviation from linearity in the instantaneous I–V plot whenV � �90 mV, and this was also blocked by extracellular CsCl(Fig. 6D). The current that remained over the voltage range

FIG. 5. Hyperpolarization-activated current (Ih) in Dogiel type II neuron. A, top: activation of Ih current, arrows point to instantaneous (Ii) and steady-state(Iss) currents; bottom: step voltage commands. Tails (ti) were measured at �50 mV and used to calculate fractional activation. B: single exponentials (E) werefitted to the evolving current and extrapolated to onset of voltage step to give values for Ii; �(s) for Ih activation were taken from exponential fits. C: steady-stateIh for each step command was given by the difference between Iss and Ii. Ih–V plot was fitted with the Boltzmann equation and the upper limit of the fit takenas maximal Ih (Imax). D: steady-state activation (P0 � Ih/Imax) was plotted against voltage and fitted with the Boltzmann equation yielding k � 10 mV andV1/2 � �77 mV for this neuron. E, top: deactivation was measured from tail currents (ti) after maximally activating Ih with �130-mV presteps. Voltage protocolgiven in bottom panel. F: single exponentials (F) were fitted to tails and extrapolated to offset of prestep voltage commands to give instantaneous tail currents(ti) and time constants (�) for Ih deactivation. G: Ih reversal potential (Eh) was estimated from intersection of linear fits to Ii and ti currents. Eh was �26 mV forthis neuron.

1004 Y. MAO, B. WANG, AND W. KUNZE

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

from �140 to �40 mV was an essentially linear leak currentof 3.9 nS, as indicated by the line joining the filled circles.

PERSISTING, TTX-INSENSITIVE NA� CURRENT. All 32 cells had aninflection in the quasi-steady-state I–V curves generated by thefirst of the pair of voltage-ramp commands applied to each. Insome cases, the inflection was sufficiently marked to produce aregion of negative conductance just positive to Vrest (e.g., trace1 in Fig. 7). The inflection was comparable to that produced bythe INa,P expressed in guinea pig AH cells (Rugiero et al.2002). For 12 cells we extracted INa,P by blocking Ih withextracellular Cs�. It was still present with 2 �M extracellularTTX present, but was eliminated when 130 mM of NaCl in theextracellular Krebs saline was replaced 1:1 with NMDG-Cl.The difference current (Fig. 7B) before and after the substitu-tion showed that INa,P had an onset of �56 � 2 mV, n � 12(i.e., close to Vrest). During 25 mV s�1 depolarizing voltageramp commands the current had a peak amplitude of �150 �60 pA at �25 � 3 mV (n � 12).

Direct (sensory) and synaptic activation of AH cells

To determine whether the processes of AH cells are acti-vated by mechanical stimulation, we pressed with a fine hair onthe ganglion containing the neuron being monitored (Kunze etal. 2000). Pressing circumferentially from patched AH cellselicited depolarizations (Fig. 8A) that matched the generator-like potentials previously recorded from guinea pig myentericneurons (Kunze et al. 2000). Equivalent responses were seen in17/18 AH cells tested, 13 of which were filled with Neurobi-otin and confirmed to be Dogiel type II neurons. The amplitudeof the potentials ranged from 5 to 21 mV between cells but wasconsistent for repeat pressings on the same cell; for 17 AH cellsthey were 12 � 7 mV. To ascertain whether responses werecaused by activation of synapses or whether they resulted from

direct transduction, we blocked all synaptic transmission byswitching the extracellular saline to a low-Ca2�, high-Mg2�

one (Kunze et al. 1993). In each case, repeated pressing elicitedconsistent responses and synaptic blockade failed to lessenresponses to mechanical distortion (e.g., Fig. 8, A and B).Receptive loci were confined to the myenteric plexus; pressing

FIG. 6. Summary kinetics and cesium sensitivity of Ih cur-rent. A: steady-state activation (P0) of Ih determined as de-scribed in RESULTS. Data were taken from 12 AH cells andfitted with the Boltzmann equation (solid curve). Dashed linesshow that P0 � 0.1 when V � �55 mV. B: voltage dependencyof �h for Ih activation and deactivation. Solid curve was fittedaccording to the equation given in RESULTS. C, top: Ih evokedby hyperpolarizing voltage step commands shown in bottomtrace. Middle: 2 mM CsCl blocked Ih and IKir. D: I–V plots ofinstantaneous and steady-state currents measured from C; �,control instantaneous current; ‚, control steady-state current;F, instantaneous currents in the presence of CsCl. The Slope ofa straight line fit to current measurements in the presence ofCsCl gave a leak conductance of 3.9 nS for this neuron.

FIG. 7. Tetrodotoxin (TTX)-resistant, persisting Na� current in AH cell. A:I–V traces of currents evoked by slowly (25 mV s�1) depolarizing voltageramp command with 2 mM CsCl and 2 �M TTX added to standard extracel-lular saline. Negative inflection in trace 1 was abolished when 90% ofextracellular NaCl was replaced by NMDG-Cl (trace 2). B: persistent sodiumcurrent (INa,P), revealed as the difference current (1 � 2), was first activatednear Vrest (�55 mV).

1005MOUSE MYENTERIC SENSORY NEURONS

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

directly onto adjacent longitudinal muscle, even to a degreethat the patched soma moved relative to the pipette, neverelicited excitatory responses. Moreover, the receptive loci weredirectly in the path of circumferentially running processes ofthe neuron being tested (see Fig. 8C).

Synaptic activation was also tested by stimulating one of theinternodal strands connected circumferentially to the ganglionbeing studied. No AH cells (0/29) received fast excitatorypostsynaptic potentials (EPSPs) after single-pulse (0.1-ms du-ration) cathodal stimuli, although brief stimulus volleys at 20Hz of 20–30 pulses evoked a prolonged slow EPSP in 9/11 AHcells tested. The slow EPSP was associated with a reduction inbackground conductance as is evident from the increase in theamplitude of voltage transients evoked by 10 pA hyperpolar-izing-current pulses (Fig. 9A). In a simple attempt to detect theconductance that was reduced, we recorded a quasi-steady-state I–V curve just before (control) and during (test) a slowexcitatory postsynaptic current (EPSC) evoked by internodalstrand stimulation at 20 Hz (Fig. 9, B and C). When thejunction potential was taken into account, the difference cur-rent (Fig. 9D) had a null current potential at EK (�90 mV).Gating for this current was voltage insensitive because it waswell fitted by the GHK current equation for potassium, yieldingPK � 0.019 � 10�9 cm3 s�1, and [K�]i � 151 mM.

D I S C U S S I O N

Overall, the present work produced two main findings. First,as might have been expected by analogy with previous resultsfrom guinea pig—but frankly was not known—mouse smallintestine myenteric Dogiel type II, AH cells are mechanosen-sory neurons. Second, voltage- and current-clamp properties ofmouse AH cells matched those of homologous cells in guineapig to a noteworthy extent.

Basic whole cell properties

Values of Vrest, membrane Rin, and �, and AP parameters inmouse small intestine AH cells obtained by our standard patch

pipette solution recordings are equivalent to those of guinea pigsmall intestine myenteric AH cells (Rugiero et al. 2002). Thiscomparison is particularly cogent because both results wereacquired with the same LMMP patch-clamp technique usingidentical pipette filling solutions. Conversely, MeSO4 patchand intracellular sharp recordings produced Vrest, Rin, and APfiring adaptation comparable to those reported for guinea pigsmall intestine (Furness et al. 1998). Therefore differences inthe small intestine resting behavior between these species(Bornstein et al. 2002) are unlikely to be attributable to thebasic properties of their sensory neurons when these are in anunstimulated state.

The single-spike slow AHP in mouse AH cells

All 32 AH cells (standard KCl patch pipette solution) ex-hibited an outward current evoked by the depolarizing ramp.

FIG. 8. Direct excitation of AH by mechanical stimulation during synapticblockade. A: depolarization and APs in AH cell evoked by pressing on theganglion with a fine hair about 60 �m from the soma (see C). Onset of pressingat downward and offset at upward arrowhead. B: responses from same neuronas in A were not attenuated after synaptic blockade with high (10 mM) Mg2�

and low (0.25 mM) Ca2�. C: digital image of Texas Red fluorescence from AHcell filled with Neurobiotin to reveal multiaxonal (Dogiel type II) shape;dashed line indicates edge of ganglion. Symbols indicate sites where pressingon the ganglion evoked (filled circle) or failed to evoke (open circles)excitatory responses. Responses to pressing at F shown in A and B.

FIG. 9. Synaptic input to AH cells. A: slow excitatory postsynaptic poten-tial evoked in AH cell by 20 Hz volley of 0.1-ms cathodal pulses (S) appliedto internodal strand. Downward deflections are electrotonic responses to 50 msduration constant current pulses applied by the patch pipette. Increase inelectrotonic response amplitudes after stimulation indicates decrease wholecell conductance. B: slow excitatory postsynaptic current (EPSC) evoked inanother AH cell by 20-Hz stimulus volley (S) applied to internodal strand.Slow (25 mV s�1) voltage command ramps were applied to the neuron before(1) and during (2) the slow EPSC. C: I–V curves elicited by ramp commandbefore (1) and during (2) slow EPSC shown in B. Trace (2 � 1) is thedifference current. D: inverted difference current from C was fitted with theGHK equation for K� currents. Resulting permeability value quantifies thereduction in a voltage-independent K� current.

1006 Y. MAO, B. WANG, AND W. KUNZE

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

This current was not gated by membrane voltage, reversed atEK, and required the presence of extracellular calcium. Theseproperties as well as its permeability at 0.13 � 0.03 � 10�9

cm3 s�1 are commensurate with that of an identical current inguinea pig [0.17 � 10�9 cm3 s�1 (Rugiero et al. 2002)], whichis responsible for the slow AHP. Probably the least invasiveway to obtain information on whole cell currents is to record inthe cell-attached mode. This can be done either by forming amacropatch containing enough channels to generate a substan-tial part of the whole cell current or by generating an ensembleaverage of unitary currents to reproduce the macroscopiccurrent. Because we have not yet successfully made macro-patches from the in situ myenteric plexus preparation, we usedthe latter method. Intermediate conductance calcium-depen-dent K� channels (IKCa) have long been known to be presentin dorsal root ganglion (DRG) sensory (Hay and Kunze 1994)and enteric neurons (Shen et al. 1992). In guinea pig AH cells,their opening generates the slow AHP current, which is charyb-dotoxin sensitive but apamin and voltage insensitive (Greffrathet al. 1998; Kunze and Mueller 2002; Kunze et al. 1994;Vogalis et al. 2002a). In mouse colon as in guinea pig, IKCa(KCa3.1) channel immunoreactivity has been localized toDogiel type II neurons (Neylon et al. 2004), so it was expectedthat the IKCa channel would also be expressed in mouse smallintestine AH cells. In fact, evidence for the slow AHP–generating IKCa channels was found in four of nine cellspatched with KMeSO4 solution pipettes. For each neuron, asingle AP evoked a prolonged increase in IKCa channel open-ing (P0) (Fig. 4). Before the AP, P0 was extremely low, whichmeans that the slow AHP–IKCa channel is unlikely to make amajor contribution to the resting background conductance.Ensemble averages of post-AP channel activity formed simu-lacra of the single-spike slow AHP (see Fig. 4), demonstratingthe role that IKCa has in slow AHP generation (Kunze andMueller 2002; Vogalis et al. 2002a). Further evidence that theIKCa channels significantly contribute to the slow AHP camefrom the block of the slow AHP by extracellular charybdotoxinand clotrimazole. However, neither of these substances isspecific for IKCa channels. Charybdotoxin also blocks large-conductance KCa and some delayed rectifier channels, andclotrimazole blocks cytochrome P450 and calcium-release–activated Ca2� channels (Jensen et al. 1999). The combinedsingle channel and whole cell data at least support the suppo-sition that a large part of the slow AHP is generated by IKCachannel opening. Also, IKCa immunoreactivity has now beenreported in rat (Furness et al. 2003) and human (Furness et al.2004b) Dogiel type II cells; it therefore seems likely that theIKCa channel and slow AHP current are highly conservedacross several species.

The presence of a slow AHP after a single AP of �2-sduration has been the standard requirement for electrophysio-logical identification of AH/Dogiel type II cells (Bornstein etal. 1994; Hirst et al. 1974) in guinea pig. It is now apparentthat, on its own, the slow AHP can be a somewhat fickleidentifier of AH cells. Homologous neurons in pig smallintestine (Cornelissen et al. 2000) and mouse large intestine(Nurgali et al. 2004) often do not exhibit a slow AHP, evenwhen they are recorded with sharp intracellular electrodes andin the absence of sensory stimulation. It is known that record-ing conditions can influence the magnitude of the slow AHP; inparticular, patch-clamp compared with sharp intracellular re-

cording has been associated with decreased leak conductance,decreased postspike slow AHP, and increased electrorespon-siveness (Gola and Niel 1993; Zhang et al. 1994; see alsoFurness et al. 2004a). Ren et al. (2003), recording with sharpelectrodes from mouse small intestine myenteric neurons (pre-sumed to be Dogiel II cells because each had a TTX-resistantAP), reported that the cells expressed pronounced single-spikeslow AHPs. On the other hand, no slow AHPs were reported inpatch-clamp recordings taken from any of 43 cultured mousesmall intestine myenteric neurons (Liu et al. 2002). Suchdifferences, and similar ones reported for guinea pig (Furnesset al. 2004a), raise the question as to whether a sharp-elec-trode–impalement Ca2� leak (Georgiou et al. 1987; Kudo andOgura 1986) causes artifactually high background conductanceand Ca2� priming of the slow AHP. Alternatively, dialysiswith patch pipette solutions, including Ca2� chelators such asEGTA might reduce Ca2�-dependent K� currents (Staley et al.1992; Velumian and Carlen 1999). Therefore we expected thatsharp electrodes might have artifactually facilitated the record-ing of single-spike AHPs because the slow AHP appears todepend on the priming of intracellular Ca2� stores (Hillsley etal. 2000) and because impalement produces shunt currents thatcan load the cell with calcium (Clements and Redman 1989;Spruston and Johnston 1992; Staley et al. 1992; Thurbon et al.1998). Furthermore, measurements made using the Ca2� indi-cator fura-2 in hippocampal neurons have demonstrated anincrease of �1 �M in intracellular [Ca2�] that was caused byimpalement with a fine microelectrode (Kudo and Ogura1986). Yet our results argue that this is not the correct inter-pretation; sharp electrodes recorded a natural single-spike slowAHP, but this was inhibited by the standard EGTA-containingKCl-rich patch pipette saline. This was probably attributable todirect action of the anion on the AHP channel and to inappro-priate buffering of intracellular free Ca2� (Velumian andCarlen 1999; Zhang et al. 1994). When sharp intracellularpipettes were used, all nine Dogiel type II cells tested hadprominent single-spike AHPs. Furthermore, a slow AHP-fa-voring patch pipette solution of Velumian and Carlen (1999)produced recordings of slow AHPs equivalent to those re-corded with sharp electrodes. In addition, the backgroundconductance was also comparable with that from sharp record-ings (see RESULTS and Table 1). The most parsimonious expla-nation for these outcomes would be that the standard patchsolution reduced a physiological background conductance andsharp electrode recording did not cause substantial impalementleakage.

Inward currents active near Vrest

The Ih and TTX-resistant Na� current (INa,P) are presentmainly in AH cells in guinea pig and rat small intestine(Rugiero et al. 2002, 2003). Our results demonstrate that theyare well represented in mouse AH cells, without excluding thepossibility that they are expressed in S cells. For example,hyperpolarization-activated nucleotide-gated channel isoformshave been localized to some S and to AH cells in guinea pig,rat, and mouse myenteric plexuses (Xiao et al. 2004).

Apart from the slow AHP currents, the Ih has probably beenstudied in more detail than any other current in Dogiel type IIneurons. Electrophysiological recording has shown that func-tional somatic Ih channels are well expressed in guinea pig AH

1007MOUSE MYENTERIC SENSORY NEURONS

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

cells (Galligan et al. 1990; Rugiero et al. 2002; Xiao et al.2004). We found that the Ih was present in all 32 AH cells andwe studied its kinetic properties in 24 of these cells. Remark-ably, the Hodgkin–Huxley activation and deactivation param-eters as well as their time constants closely match those takenfrom guinea pig (Galligan et al. 1990; Rugiero et al. 2002).However, the Ih reversal potential (Eh) seemed to differ be-tween species; it was �28 mV for mouse but �40 mV for theguinea pig myenteric neurons (Rugiero et al. 2002). Reversalpotential measurements can be inaccurate because other un-known overlapping voltage-gated currents might contaminatethem. We tried to minimize such problems by using the methodof Lamas (1998) to estimate Eh (see RESULTS), but this was notdone for the previous guinea pig work. Nevertheless, if theapparent difference in reversal potentials is sustained by furtherexperiments this would suggest that the Ih Na�:K� permeabil-ity ratio is larger in mouse than in guinea pig, This possiblyreflects the species differences between AH cells in Ih channelisoform expression (Xiao et al. 2004).

The second major inward current, active near Vrest, was aTTX-resistant Na� current (INa,P). INa,P is a relative newcomeramong identified currents in AH cells but it has already beenrecorded in rat (Coste et al. 2004) and in guinea pig (Rugieroet al. 2002, 2003). For this current also, basic parameters suchas voltage of first activation and maximal current were com-parable between mouse and guinea pig. Based on gatingcharacteristics and the presence of mRNA and positive immu-nostains it has been argued that INa,P in AH cells is carried bythe Nav1.9 Na channel isoform (Delmas and Coste 2003;Rugiero et al. 2003). Analogous currents are proposed tomodulate the electroresponsiveness of small dorsal root ganglia(Herzog et al. 2001) and spinal motoneurons (Lee and Heck-man 2001), especially by amplifying small depolarizations(Dib-Hajj et al. 2002) and they may similarly influence intrin-sic enteric sensory neurons.

Sensory responses

One of the advantages of the LMMP patch-clamp recordingtechnique is that it provides mechanical stability and thusdistortion of the ganglion from which the recording is beingmade is possible without loss of seal or signal (Kunze et al.2000). This is why direct identification of mechanosensorymyenteric neurons by recording the sensory response was notachievable using sharp intracellular recording; close mechani-cal stimulation dislodged the electrode (Smith et al. 1992). Forthe mouse, as for the guinea pig (Kunze et al. 2000), we foundthat pressing on the ganglia containing the neuron beingpatched evoked excitatory responses (see RESULTS) that couldbe recorded at the soma for each of 14 AH cells tested. Thiswas a direct (“sensory”) response because it persisted duringsynaptic blockade. The depolarization and discharge are notlikely to be related to axonal injury because they could berepeatedly evoked from the same receptive locus (see RESULTS

and Kunze et al. 2000) and because the distribution of recep-tive loci is punctuate (see Fig. 6 in Kunze et al. 2000). Incontrast, injury discharge would be expected to be elicitedalong the entire length of the path of the neurite; also, neuritedamage ought to interfere with successive responses from thesame locus.

Our recordings are consistent with the deduction made forguinea pig that AH/Dogiel type II cells are mechanosensoryneurons that respond to tension (Kunze et al. 1998, 2000;Spencer and Smith 2004). They do not preclude the possibilitythat some mouse S cells might also have a sensory role, as isthe case for guinea pig (Kunze et al. 1998; Spencer and Smith2004). The present results are from the first recording of AHcell sensory responses in the mouse and thus set the stage forthe study of the mechano-transducing mechanisms involved.

Synaptic input

When presynaptic fibers were electrically stimulated at 20Hz, AH cells responded with slow EPSPs, although fast EPSPswere never discerned in any of 29 cells. Because we tested forsynaptic input from only one internodal strand per cell, it wasnot possible to absolutely rule out fast synaptic input to Dogieltype II neurons without more extensive and systematic stimu-lus–response mapping. Nonetheless, the present results agreewith Bian et al. (2003), who also found that, in mouse smallintestine, fast EPSP input is confined to S cells. Slow EPSPsseem to be a highly conserved AH cell property. They havebeen recorded in guinea pig small intestine AH/Dogiel type IIcells (Hodgkiss and Lees 1984; Kunze et al. 1993; Takaki andNakayama 1988; Wells and Mawe 1993; Wood and Mayer1979), in rat Dogiel type II cells (Brookes et al. 1988; Brown-ing and Lees 1996), and in the one human AH cell recorded byBrookes et al. (1987). Furukawa et al. (1986) also reportedslow EPSPs in AH cells of the mouse colon myenteric plexus.

In our experiments the slow EPSP was associated with adecrease in whole cell conductance, indicating that a back-ground current had been reduced (Bertrand and Galligan 1995;Johnson et al. 1980). The difference current between quasi-steady-state I–V curves made before and during the slowdepolarization suggests that the slow depolarizing potentialwas caused by a reduction in a background K� current.Because it did not appear to be voltage dependent the slowEPSP current was probably analogous to the K� current(s)whose reduction underlies the slow EPSP in guinea pig smallintestine AH cells (Bertrand and Galligan 1995; Johnson et al.1980).

Functional implications and conclusion

We recorded from AH/Dogiel type II cells because thenetwork of reciprocally connected intrinsic sensory neurons isthought to exert a critical influence over ENS processing(Bertrand and Thomas 2004; Kunze and Furness 1999) andmay be the component where ENS functional plasticity andmemory are expressed (Furness et al. 2000). A significantfeature of our results was the extent to which mouse AH cellcurrents were quantitatively like those in guinea pig, attestingto a high degree of conservation of ion channels between thesespecies. Direct mechanosensory responses were recorded frommouse AH cells that were similar to previous recordings fromguinea pig, which had been, up to now, the only species wherethis was done. This result increases the likelihood that AH cellswill be found to be intrinsic sensory neurons in other vertebratespecies, including human.

1008 Y. MAO, B. WANG, AND W. KUNZE

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

A C K N O W L E D G M E N T S

We thank Professor Jan D. Huizinga for helpful discussion and commentaryon the manuscript.

G R A N T S

Part of this work was performed under the auspices of the Centre forMedical Research, Department of General Surgery, University of Tubingen,Tubingen, Germany.

R E F E R E N C E S

Baidan LV, Zholos AV, Shuba MF, and Wood JD. Patch-clamp recordingin myenteric neurons of guinea pig small intestine. Am J Physiol Gastro-intest Liver Physiol 262: G1074–G1078, 1992.

Bers DM, Patton CW, and Nuccitelli R. A practical guide to the preparationof Ca2� buffers. Methods Cell Biol 40: 3–29, 1994.

Bertrand PP and Galligan JJ. Signal-transduction pathways causing slowsynaptic excitation in guinea pig myenteric AH neurons. Am J PhysiolGastrointest Liver Physiol 269: G710–G720, 1995.

Bertrand PP, Kunze WA, Bornstein JC, Furness JB, and Smith ML.Analysis of the responses of myenteric neurons in the small intestine tochemical stimulation of the mucosa. Am J Physiol Gastrointest Liver Physiol273: G422–G435, 1997.

Bertrand PP and Thomas EA. Multiple levels of sensory integration in theintrinsic sensory neurons of the enteric nervous system. Clin Exp PharmacolPhysiol 31: 745–755, 2004.

Bian X, Ren J, DeVries M, Schnegelsberg B, Cockayne DA, Ford AP, andGalligan JJ. Peristalsis is impaired in the small intestine of mice lacking theP2X3 subunit. J Physiol 551: 309–322, 2003.

Bornstein JC, Furness JB, and Kunze WA. Electrophysiological character-ization of myenteric neurons: how do classification schemes relate? J AutonNerv Syst 48: 1–15, 1994.

Bornstein JC, Furness JB, Kunze WAA, and Bertrand PP. Enteric reflexesthat influence motility. In: Nervous Control of the Gastrointestinal Tract,edited by Costa M and Brookes SJH. London: Taylor & Francis, 2002, p.1–55.

Brookes SJ. Classes of enteric nerve cells in the guinea-pig small intestine.Anat Rec 262: 58–70, 2001.

Brookes SJ, Ewart WR, and Wingate DL. Intracellular recordings frommyenteric neurones in the human colon. J Physiol 390: 305–318, 1987.

Brookes SJ, Ewart WR, and Wingate DL. Intracellular recordings from cellsin the myenteric plexus of the rat duodenum. Neuroscience 24: 297–307,1988.

Browning KN and Lees GM. Myenteric neurons of the rat descending colon:electrophysiological and correlated morphological properties. Neuroscience73: 1029–1047, 1996.

Bullard DC and Weaver CT. Cutting-edge technology. IV. Genomic engi-neering for studies of the gastrointestinal tract in mice. Am J PhysiolGastrointest Liver Physiol 283: G1232–G1237, 2002.

Clements JD and Redman SJ. Cable properties of cat spinal motoneuronesmeasured by combining voltage clamp, current clamp and intracellularstaining. J Physiol 409: 63–87, 1989.

Clerc N, Furness JB, Bornstein JC, and Kunze WA. Correlation of elec-trophysiological and morphological characteristics of myenteric neurons ofthe duodenum in the guinea-pig. Neuroscience 82: 899–914, 1998.

Cole KS. Squid axon membrane: impedance decrease to voltage clamp. AnnuRev Neurosci 5: 305–323, 1982.

Cornelissen W, De Laet A, Kroese AB, Van Bogaert PP, ScheuermannDW, and Timmermans JP. Electrophysiological features of morphologicalDogiel type II neurons in the myenteric plexus of pig small intestine.J Neurophysiol 84: 102–111, 2000.

Costa M, Hennig GW, and Brookes SJ. Intestinal peristalsis: a mammalianmotor pattern controlled by enteric neural circuits. Ann NY Acad Sci 860:464–466, 1998.

Coste B, Osorio N, Padilla F, Crest M, and Delmas P. Gating andmodulation of presumptive NaV1.9 channels in enteric and spinal sensoryneurons. Mol Cell Neurosci 26: 123–134, 2004.

Delmas P and Coste B. Na� channel Nav1.9: in search of a gating mecha-nism. Trends Neurosci 26: 55–57, 2003.

Der T, Bercik P, Donnelly G, Jackson T, Berezin I, Collins SM, andHuizinga JD. Interstitial cells of Cajal and inflammation-induced motordysfunction in the mouse small intestine. Gastroenterology 119: 1590, 2000.

Dib-Hajj S, Black JA, Cummins TR, and Waxman SG. NaN/Nav1.9: asodium channel with unique properties. Trends Neurosci 25: 253–259, 2002.

Franklin JL and Willard AL. Voltage-dependent sodium and calcium cur-rents of rat myenteric neurons in cell culture. J Neurophysiol 69: 1264–1275, 1993.

Furness JB, Clerc N, and Kunze WA. Memory in the enteric nervous system[Discussion]. Gut 47, Suppl. 4: iv60–iv62, 2000.

Furness JB and Costa M. The Enteric Nervous System. New York: ChurchillLivingstone, 1987.

Furness JB, Jones C, Nurgali K, and Clerc N. Intrinsic primary afferentneurons and nerve circuits within the intestine. Prog Neurobiol 72: 143–164,2004a.

Furness JB, Kearney K, Robbins HL, Hunne B, Selmer IS, Neylon CB,Chen MX, and Tjandra JJ. Intermediate conductance potassium (IK)channels occur in human enteric neurons. Auton Neurosci 112: 93–97,2004b.

Furness JB, Kunze WA, Bertrand PP, Clerc N, and Bornstein JC. Intrinsicprimary afferent neurons of the intestine. Prog Neurobiol 54: 1–18, 1998.

Furness JB, Robbins HL, Selmer IS, Hunne B, Chen MX, Hicks GA,Moore S, and Neylon CB. Expression of intermediate conductance potas-sium channel immunoreactivity in neurons and epithelial cells of the ratgastrointestinal tract. Cell Tissue Res 314: 179–189, 2003.

Furukawa K, Taylor GS, and Bywater RA. An intracellular study ofmyenteric neurons in the mouse colon. J Neurophysiol 55: 1395–1406,1986.

Galligan JJ, Tatsumi H, Shen KZ, Surprenant A, and North RA. Cationcurrent activated by hyperpolarization (IH) in guinea pig enteric neurons.Am J Physiol Gastrointest Liver Physiol 259: G966–G972, 1990.

Georgiou P, Bountra C, and House CR. Intracellular and whole-cell record-ings from zona-free hamster eggs: significance of leak impalement artifact.Q J Exp Physiol 72: 105–118, 1987.

Gershon MD. Lessons from genetically engineered animal models. II. Disor-ders of enteric neuronal development: insights from transgenic mice. Am JPhysiol Gastrointest Liver Physiol 277: G262–G267, 1999.

Gola M and Niel JP. Electrical and integrative properties of rabbit sympa-thetic neurones re-evaluated by patch clamping non-dissociated cells.J Physiol 460: 327–349, 1993.

Greffrath W, Martin E, Reuss S, and Boehmer G. Components of after-hyperpolarization in magnocellular neurones of the rat supraoptic nucleus invitro. J Physiol 513: 493–506, 1998.

Hanani M, Francke M, Hartig W, Grosche J, Reichenbach A, andPannicke T. Patch-clamp study of neurons and glial cells in isolatedmyenteric ganglia. Am J Physiol Gastrointest Liver Physiol 278: G644–G651, 2000.

Haschke G, Schafer H, and Diener M. Effect of butyrate on membranepotential, ionic currents and intracellular Ca2� concentration in cultured ratmyenteric neurones. Neurogastroenterol Motil 14: 133–142, 2002.

Hay M and Kunze DL. An intermediate conductance calcium-activatedpotassium channel in rat visceral sensory afferent neurons. Neurosci Lett167: 179–182, 1994.

Herzog RI, Cummins TR, and Waxman SG. Persistent TTX-resistant Na�

current affects resting potential and response to depolarization in simulatedspinal sensory neurons. J Neurophysiol 86: 1351–1364, 2001.

Hillsley K, Kenyon JL, and Smith TK. Ryanodine-sensitive stores regulatethe excitability of AH neurons in the myenteric plexus of guinea-pig ileum.J Neurophysiol 84: 2777–2785, 2000.

Hirning LD, Fox AP, and Miller RJ. Inhibition of calcium currents incultured myenteric neurons by neuropeptide Y: evidence for direct receptor/channel coupling. Brain Res 532: 120–130, 1990.

Hirst GD, Holman ME, and Spence I. Two types of neurones in themyenteric plexus of duodenum in the guinea pig. J Physiol 236: 303–326,1974.

Hodgkiss JP and Lees JS. Slow intracellular potentials in AH-neurons of themyenteric plexus evoked by repetitive activation of synaptic inputs. Neuro-science 11: 255–261, 1984.

Holzer P. Gastrointestinal afferents as targets of novel drugs for the treatmentof functional bowel disorders and visceral pain. Eur J Pharmacol 429:177–193, 2001.

Holzer P, Schicho R, Holzer-Petsche U, and Lippe IT. The gut as aneurological organ. Wien Klin Wochenschr 113: 647–660, 2001.

International Union of Basic and Clinical Pharmacology (IUPHAR).IUPHAR ion channel compendium. http://wwwiuphar-dborg/iuphar-ic/indexhtml. Accessed 2002.

Jensen BS, Odum N, Jorgensen NK, Christophersen P, and Olesen SP.Inhibition of T cell proliferation by selective block of Ca(2�)-activatedK(�) channels. Proc Natl Acad Sci USA 96: 10917–10921, 1999.

1009MOUSE MYENTERIC SENSORY NEURONS

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from

Johnson SM, Katayama Y, and North RA. Slow synaptic potentials inneurones of the myenteric plexus. J Physiol 301: 505–516, 1980.

Kudo Y and Ogura A. Glutamate-induced increase in intracellular Ca2�

concentration in isolated hippocampal neurones. Br J Pharmacol 89: 191–198, 1986.

Kunze W, Mueller MH, Jiang GJ, and Grundy D. On the enteric sensoryneuron in the mouse. Proc Neurogastroenterol Motil, Tuebingen, Germany,2002, p. 566.

Kunze WA, Bornstein JC, and Furness JB. Identification of sensory nervecells in a peripheral organ (the intestine) of a mammal. Neuroscience 66:1–4, 1995.

Kunze WA, Bornstein JC, Furness JB, Hendriks R, and Stephenson DS.Charybdotoxin and iberiotoxin but not apamin abolish the slow after-hyperpolarization in myenteric plexus neurons. Pfluegers Arch 428: 300–306, 1994.

Kunze WA, Clerc N, Furness JB, and Gola M. The soma and neurites ofprimary afferent neurons in the guinea-pig intestine respond differentially todeformation. J Physiol 526: 375–385, 2000.

Kunze WA and Furness JB. The enteric nervous system and regulation ofintestinal motility. Annu Rev Physiol 61: 117–142, 1999.

Kunze WA, Furness JB, Bertrand PP, and Bornstein JC. Intracellularrecording from myenteric neurons of the guinea-pig ileum that respond tostretch. J Physiol 506: 827–842, 1998.

Kunze WA, Furness JB, and Bornstein JC. Simultaneous intracellularrecordings from enteric neurons reveal that myenteric AH neurons transmitvia slow excitatory postsynaptic potentials. Neuroscience 55: 685–694,1993.

Kunze WA and Mueller MH. Kinetics and functions of calcium modulatedpotassium channels in enteric sensory neurons. Proc DDW Conf Gastroen-terol, San Francisco, CA, 2002, p. A-163.

Lamas JA. A hyperpolarization-activated cation current (Ih) contributes toresting membrane potential in rat superior cervical sympathetic neurones.Pfluegers Arch 436: 429–435, 1998.

Lee RH and Heckman CJ. Essential role of a fast persistent inward currentin action potential initiation and control of rhythmic firing. J Neurophysiol85: 472–475, 2001.

Li WC, Soffe SR, and Roberts A. A direct comparison of whole cell patchand sharp electrodes by simultaneous recording from single spinal neuronsin frog tadpoles. J Neurophysiol 92: 380–386, 2004.

Liu MT, Rayport S, Jiang Y, Murphy DL, and Gershon MD. Expressionand function of 5-HT3 receptors in the enteric neurons of mice lacking theserotonin transporter. Am J Physiol Gastrointest Liver Physiol 283: G1398–G1411, 2002.

Neylon CB, Nurgali K, Hunne B, Robbins HL, Moore S, Chen MX, andFurness JB. Intermediate-conductance calcium-activated potassium chan-nels in enteric neurones of the mouse: pharmacological, molecular andimmunochemical evidence for their role in mediating the slow afterhyper-polarization. J Neurochem 90: 1414–1422, 2004.

Nurgali K, Stebbing MJ, and Furness JB. Correlation of electrophysiolog-ical and morphological characteristics of enteric neurons in the mouse colon.J Comp Neurol 468: 112–124, 2004.

Picciotto MR and Wickman K. Using knockout and transgenic mice to studyneurophysiology and behavior. Physiol Rev 78: 1131–1163, 1998.

Ren J, Bian X, DeVries M, Schnegelsberg B, Cockayne DA, Ford AP, andGalligan JJ. P2X2 subunits contribute to fast synaptic excitation in myen-teric neurons of the mouse small intestine. J Physiol 552: 809–821, 2003.

Rugiero F, Gola M, Kunze WA, Reynaud JC, Furness JB, and Clerc N.Analysis of whole-cell currents by patch clamp of guinea-pig myentericneurones in intact ganglia. J Physiol 538: 447–463, 2002.

Rugiero F, Mistry M, Sage D, Black JA, Waxman SG, Crest M, Clerc N,Delmas P, and Gola M. Selective expression of a persistent tetrodotoxin-

resistant Na� current and NaV1.9 subunit in myenteric sensory neurons.J Neurosci 23: 2715–2725, 2003.

Schutte IW, Kroese ABA, and Akkermans LMA. Somal size and locationwithin the ganglia for electrophysiologically identified myenteric neurons ofthe guinea pig ileum. J Comp Neurol 355: 563–572, 1995.

Shen KZ, North RA, and Surprenant A. Potassium channels opened bynoradrenaline and other transmitters in excised membrane patches ofguinea-pig submucosal neurones. J Physiol 445: 581–599, 1992.

Smith TK, Bornstein JC, and Furness JB. Convergence of reflex pathwaysexcited by distension and mechanical stimulation of the mucosa onto thesame myenteric neurons of the guinea pig small intestine. J Neurosci 12:1502–1510, 1992.

Spencer NJ. Control of migrating motor activity in the colon. Curr OpinPharmacol 1: 604–610, 2001.

Spencer NJ and Smith TK. Mechanosensory S-neurons rather than AH-neurons appear to generate a rhythmic motor pattern in guinea-pig distalcolon. J Physiol 558: 577–596, 2004.

Spruston N and Johnston D. Perforated patch-clamp analysis of the passivemembrane properties of three classes of hippocampal neurons. J Neuro-physiol 67: 509–529, 1992.

Staley KJ, Otis TS, and Mody I. Membrane properties of dentate gyrusgranule cells: comparison of sharp microelectrode and whole cell record-ings. J Neurophysiol 67: 1346–1358, 1992.

Takaki M and Nakayama S. Effects of mesenteric nerve stimulation on theelectrical activity of myenteric neurons in the guinea pig ileum. Brain Res442: 351–353, 1988.

Tatsumi H, Hirai K, and Katayama Y. Measurement of the intracellularcalcium concentration in guinea-pig myenteric neurons by using fura-2.Brain Res 451: 371–375, 1988.

Thurbon D, Luescher H, Hofstetter T, and Redman SJ. Passive electricalproperties of ventral horn neurons in rat spinal cord slices. J Neurophysiol79: 2485–2502, 1998.

Velumian AA and Carlen PL. Differential control of three after-hyperpolar-izations in rat hippocampal neurones by intracellular calcium buffering.J Physiol 517: 201–216, 1999.

Vogalis F, Harvey JR, and Furness JB. TEA- and apamin-resistant K(Ca)channels in guinea-pig myenteric neurons: slow AHP channels. J Physiol538: 421–433, 2002a.

Vogalis F, Harvey JR, Neylon CB, and Furness JB. Regulation of K�

channels underlying the slow afterhyperpolarization in enteric afterhyper-polarization-generating myenteric neurons: role of calcium and phosphory-lation. Clin Exp Pharmacol Physiol 29: 935–943, 2002b.

Wells DG and Mawe GM. Physiological and morphological properties ofneurons in sphincter of Oddi region of the guinea pig. Am J PhysiolGastrointest Liver Physiol 265: G258–G269, 1993.

Willms AR, Baro DJ, Harris-Warrick RM, and Guckenheimer J. Animproved parameter estimation method for Hodgkin–Huxley models.J Comput Neurosci 6: 145–168, 1999.

Wood JD and Mayer CJ. Intracellular study of tonic-type enteric neurons inguinea pig small intestine. J Neurophysiol 42: 569–581, 1979.

Xiao J, Nguyen TV, Ngui K, Strijbos PJ, Selmer IS, Neylon CB, andFurness JB. Molecular and functional analysis of hyperpolarisation-acti-vated nucleotide-gated (HCN) channels in the enteric nervous system.Neuroscience 129: 603–614, 2004.

Zhang L, Weiner JL, Valiante TA, Velumian AA, Watson PL, JahromiSS, Schertzer S, Pennefather P, and Carlen PL. Whole-cell recording ofthe Ca(2�)-dependent slow afterhyperpolarization in hippocampal neu-rones: effects of internally applied anions. Pfluegers Arch 426: 247–253,1994.

1010 Y. MAO, B. WANG, AND W. KUNZE

J Neurophysiol • VOL 96 • SEPTEMBER 2006 • www.jn.org

by guest on June 3, 2013http://jn.physiology.org/

Dow

nloaded from