Electrophysiological Features of Morphological Dogiel Type II Neurons in the Myenteric Plexus of Pig Small Intestine

Wim Cornelissen,1,2 Ann De Laet,1,2 Alfons B. A. Kroese,3 Pierre-Paul Van Bogaert,2 Dietrich W. Scheuermann,1 and Jean-Pierre Timmermans1

 1Laboratory of Cell Biology and Histology and  2Laboratory of Electrobiology, University of Antwerp (RUCA), 2020 Antwerp, Belgium; and  3Departments of Medical Physiology and Surgery, Utrecht University, 3584 CG Utrecht, The Netherlands


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cornelissen, Wim, Ann De Laet, Alfons B. A. Kroese, Pierre-Paul Van Bogaert, Dietrich W. Scheuermann, and Jean-Pierre Timmermans. Electrophysiological Features of Morphological Dogiel Type II Neurons in the Myenteric Plexus of Pig Small Intestine. J. Neurophysiol. 84: 102-111, 2000. By intracellular recording, 99 myenteric neurons with Dogiel type II morphology were electrophysiologically characterized in the porcine ileum and further subdivided into three groups based on their different types of afterhyperpolarization (AHP). In response to a depolarizing current injection, a fast AHP (fAHP; duration 34 ± 11 ms; amplitude -11 ± 6 mV; mean ± SD) immediately followed every action potential in all neurons. In 32% of the neurons, this fAHP was the sole type of hyperpolarization recorded. Statistical analysis revealed the presence of two neuronal subpopulations that displayed either a long-lasting medium AHP (mAHP; duration after a single spike 773 ± 753 ms; 51% of neurons) or a slow AHP (sAHP; 4,205 ± 1,483 ms; 17%). Slow AHP neurons also differed from mAHP neurons in the delayed onset of the AHP (mAHP 0 ms; sAHP 100-200 ms), as well as in maximum amplitude values and in the time to reach this amplitude (tmax; 148 ± 11 ms vs. 628 ± 108 ms). Medium AHP neurons further differed from the sAHP neurons in the occurrence of the AHP following subthreshold current injection and in their resting membrane potential (mAHP, -53 ± 8 mV; sAHP, -62 ± 10 mV). Medium AHP and sAHP behaved similarly in that a higher number of spikes increased their amplitude and duration, but not tmax. The majority of neurons fired multiple spikes (up to 25) in response to a 500-ms current injection (81/99) and showed a clear TTX-resistant shoulder on the repolarizing phase of the action potential (77/99), irrespective of the presence of sAHP or mAHP. These results demonstrate that the porcine Dogiel type II neurons differ in various essential electrophysiological properties from their morphological counterparts in the guinea pig ileal myenteric plexus. The most striking interspecies differences were the low occurrence of sAHP (17% vs. 80-90% in guinea pig) with relatively small amplitude (-5 vs. -20 mV), the high occurrence of mAHPs (unusual in guinea pig) and the ability to fire long spike trains (up to 25 spikes vs. 1-3 in guinea pig). In fact, Dogiel type II neurons in porcine ileum combine distinct electrophysiological features considered typical of either S-type or sAHP-type neurons in guinea pig. It can therefore be concluded that in spite of a similar morphology, Dogiel type II neurons do not behave electrophysiologically in a universal way in large and small mammals.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The use of electrophysiological recordings combined with intracellular staining and immunohistochemical techniques has contributed greatly to our understanding of enteric neuronal functioning. In the small bowel of the guinea pig, the most extensively studied experimental model in this field, this combined approach has produced increasing evidence that Dogiel type II/slow afterhyperpolarization (sAHP) neurons have a primary afferent role (Furness et al. 1998; Kunze and Furness 1999; Kunze et al. 1997, 1998). These sensory cells are believed to play a key role in the unique ability of the enteric nervous system to mediate reflex behavior independently of input from the brain or spinal cord.

Slow AHP neurons are characterized by the participation of a Ca2+ current in the upstroke of their action potentials (Baidan et al. 1992; Hirst et al. 1985a). The sAHP in these cells was found to originate from a Ca2+-activated K+ current (Hirst et al. 1985b; North 1973; North and Tokimasa 1987). Because this behavior was considered so typical of guinea pig sAHP neurons, they were named after it.

In the guinea pig small bowel, neurons displaying the above-mentioned electrophysiological features tend to have Dogiel type II morphology (Bornstein et al. 1984): i.e., a large, smooth contoured soma with long processes most often projecting in a circumferential direction and bifurcating close to the cell body (Dogiel 1899). Within the porcine myenteric plexus, such Dogiel type II neurons are preferentially located at the periphery or outside the ganglia (Scheuermann et al. 1987; Stach 1981).

The anatomical organization of the enteric nerve networks as well as the morphology and neuropeptide content of the individual neurons were found to differ significantly between large and small mammals (Gershon et al. 1994; Timmermans et al. 1990, 1992, 1997). It can be envisaged that these differences might also be reflected in the behavior of the individual neurons.

Few studies, however, performed a correlation between the morphology of enteric neurons and their electrophysiological behavior. Apart from the ileum (Bornstein et al. 1984), the proximal colon (Messenger et al. 1994), rectum (Tamura 1992), and, more recently, the duodenum (Clerc et al. 1998) and distal colon (Lomax et al. 1999) have been investigated in the guinea pig. Whereas data in other small mammals are more scarce (Browning and Lees 1996), in large mammals nothing at all was known in this respect, mainly because in these large animals studies on the electrophysiological behavior have long been hampered by practical difficulties encountered when attempting to impale individual enteric neuronal cell bodies.

This report represents, to our knowledge, the first detailed description of the electrophysiological behavior of a morphologically identified neuronal subclass in the small intestine of a large omnivorous mammal, viz. Dogiel type II neurons in myenteric plexus of the pig. Since Dogiel type II/sAHP cells have been attributed a crucial afferent role in the gastrointestinal reflexes of guinea pig small intestine, it was thought to be appropriate to investigate the electrophysiological features of neurons with a similar morphology in porcine small intestine, especially since a previous report (Cornelissen et al. 1996) indicated the absence of obvious sAHP in porcine myenteric neurons.

Part of this work was presented at the 17th International Symposium on Gastrointestinal Motility, Brugge, September 14-17, 1999, and reported in abstract form (Cornelissen et al. 1999).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue dissection

Three- to 7-wk-old pigs were stunned by a blow on the head and exsanguinated via the cervical vessels. All methods were approved by the Antwerp University Medical Ethical Committee and were carried out in accordance with the guidelines of the Belgian Ministry of Agriculture. Pieces of ileum were removed and washed with modified Krebs solution. Small segments were opened along the mesenteric border and pinned, with the mucosal surface up, to the bottom of a Petri dish lined with silicone elastomer (Sylgard).

The mucosa and submucosa were dissected away, and the circular muscle was peeled off using watchmakers' forceps and a dissection microscope. To reduce tissue thickness and improve visualization, a window of serosal tissue was removed as well. Throughout the dissection procedures the tissue was superfused with Krebs solution at room temperature.

Vital staining and selection of the neurons

As described before (Cornelissen et al. 1996), the longitudinal muscle-myenteric plexus preparations (1.5 × 1 cm) were incubated in Krebs solution (37°C) with the vital dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP) at a concentration of 4 µM for 20 min. After another 4 h in pure Krebs solution under similar conditions, the tissues were ready for electrophysiological recordings.

To this end, the preparations were pinned to Sylgard at the bottom of a recording chamber of 1 ml volume. The chamber was continuously perfused with Krebs solution (35-37°C) at a rate of 10 ml/min. Nicardipine (1 µM) was added to prevent contraction of the intestinal smooth muscle (Brookes et al. 1987; Kunze et al. 1997). Prior to impalement, myenteric ganglia and neurons were visualized with an inverted microscope (Nikon Diaphot) equipped with epi-fluorescent illumination and a B-2 A filter combination (excitation filter: 450-490 nm; dichroic mirror: 510 nm; barrier filter: 520 nm). Brief epi-fluorescent illumination of the preparation allowed to localize the neuron selected for recording, after which impalement was performed under standard conditions of transmitted light illumination.

Intracellular recordings

Recording microelectrodes were pulled from 1-mm OD glass capillaries (Clark Electromedical Instruments) with a P-97 Brown-Flaming horizontal micropipette puller (Sutter Instrument) and backfilled with a 1 M KCl solution to which either 2% biocytin or neurobiotin had been added. Electrode resistance was in the range of 60-110 MOmega . The electrode was positioned by a micromanipulator (type Narishige MO388). Intracellular recordings were made with an Axoclamp 2A current-voltage amplifier (headstage HS-2 L, gain 0.1) connected to a Labmaster TL-1 DMA interface (Axon Instruments). The amplifier bridge circuit was balanced for each electrode before impalement, and capacitance was compensated for during injection of rectangular electrical current pulses (-0.2 nA, 7 ms) through the microelectrode.

Current step commands to investigate passive and active membrane properties were created with an IBM-compatible PC/AT using the pClamp 6.0.2 software (Axon Instruments). The data were on-line low-pass filtered (3 kHz), digitized (sample rate 5 kHz), and stored on hard disk. pClamp 6.0.3, Excel 5.0, and Sigma Plot 3.02 (Jandel) software was used for subsequent analysis. Digitized electrophysiological data were additionally stored on video tape; events with slow time courses were replayed on a strip chart recorder (Gould Easygraf TA 240).

To estimate input resistance and the membrane time constant (tau ), small hyperpolarizing current pulses of variable amplitude (-0.05 to -0.3 nA) were passed through the intracellular microelectrode, and the resulting membrane potential change was measured so that current-voltage curves could be constructed. Every step was repeated three times, and the average value was used for further calculations.

Durations of action potentials in impaled neurons were measured as half-widths; i.e., the time interval between the point on the upstroke at which the amplitude of the action potential was halfway the membrane resting potential and the maximal potential and the equivalent point on the downstroke. To avoid interference of voltage changes due to depolarizing current applied, short pulses ranging between 2 and 5 ms were used to evoke a single action potential, whenever individual spike characteristics were measured.

Solutions

Modified Krebs solution contained (in mM) 118.0 NaCl, 4.75 KCl, 2.54 CaCl2, 1.2 MgSO4, 1.0 NaH2PO4, 25.0 NaHCO3, and 11.1 glucose and was gassed with 95% O2-5% CO2 (pH 7.4). The effect of tetrodotoxin (TTX) on the action potentials was studied by superfusing Krebs solution containing 1 µM TTX. Superfusion solution changes were obtained using a four-way tap, with a perfusion delay of 20 s.

Nicardipine and TTX were purchased from Sigma Chemical (St. Louis, MO); 4-Di-2-ASP from Molecular Probes (Eugene, OR).

Intracellular marking and histochemical visualization

Physiological recordings were complemented with iontophoretical injection of biocytin or neurobiotin through the impaling microelectrode. The preparation was then fixed in a modified Zamboni solution (4% paraformaldehyde, 0.2% picric acid, 0.1 M sodium phosphate buffer) for 2 h at room temperature, and biocytin/neurobiotin was visualized by the biotin-streptavidin bridge procedure using a Texas Red-conjugated streptavidin complex (Amersham RPN 1233). To allow an unambiguous identification of the impaled neuron, the corresponding ganglia showing 4-Di-2-ASP fluorescence were photographed immediately after recording and again after visualization of biocytin/neurobiotin and subsequent embedding of the whole mounts in a mounting medium for fluorescence.

Photographs were taken on a Zeiss LSM 410 confocal microscope equipped with an Argon and He/Ne-laser and a Zeiss ×40 water immersion objective (NA 1.2). Volume-rendered three-dimensional (3-D) reconstructions were performed using Imaris 2.5 software (Bitplane AG, Zürich, Switzerland) running on a Silicon Graphics Indigo II station. Shadow projections were based on a stack of 23 up to 55 confocal images taken at z-intervals of 0.33-0.54 µm.

Statistical analysis

All values are presented as means ± SD. A paired or unpaired t-test or single factor ANOVA was used for statistical comparisons of group means. On ANOVA indication of significance, post hoc multiple comparisons were made using the Newman-Keuls test modified for comparison of unequal populations. Linear multiple regression analysis was conducted to compare mAHP and sAHP features (Statistica, Statsoft). P values <0.05 were considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrical behavior

Data were obtained from 99 myenteric neurons. As described previously (Cornelissen et al. 1996), the ganglion containing the impaled neuron was photographed immediately after recording and again after visualization of biocytin/neurobiotin. This procedure allowed us to unambiguously identify all neurons used for electrophysiological characterization as having Dogiel type II morphology. Impalements were judged to be successful if the resting membrane potential was stable and more negative than -40 mV. Action potentials that were fired on direct somal depolarization had to show a clear reversal ("overshoot") of the membrane potential before the neuron was considered healthy (Browning and Lees 1996). Recordings lasted 25 min to several hours. None of the neurons displayed any fluctuations of the resting potential that could be interpreted as pacemaker activity, nor did they spontaneously discharge action potentials.

AHPs

Three different types of AHP were identified in the 99 porcine Dogiel type II neurons. The first was a fast AHP (fAHP, Fig. 1A), which was continuous with the repolarizing phase of the preceding action potential and reached its maximal amplitude (-11 ± 6 mV; mean ± SD, n = 54) at 5 ± 1 ms following the spike. Duration of the fAHP was on average 34 ± 11 ms (range, 18-58 ms). fAHP occurred exclusively in 32 (32%) of the Dogiel type II cells recorded. In the remaining 67 (68%) Dogiel type II neurons, long-lasting AHPs were observed alongside the fAHP. Detailed analysis of the characteristics of these long-lasting AHPs (see the following three subsections) led to the subclassification into sAHP and medium AHP (mAHP) neurons.



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Fig. 1. Electrophysiological features of a porcine slow afterhyperpolarization (sAHP) neuron with Dogiel type II morphology. The electrophysiological recording of this porcine Dogiel type II neuron (inset) revealed a sAHP lasting several seconds. A-D: the black arrow indicates a change in the voltage and time scales. The calibration of B also applies to C and D. A: after firing a single action potential, the sAHP (indicated with 2 white arrows) is hardly noticeable. Its amplitude was less than half that of the fast AHP (fAHP; indicated with 1 white arrow) immediately following the action potential. B-D: an increase in the number of action potentials, generated by longer current pulses, resulted in a gradual increase in the amplitude of the sAHP until it reached the apparently maximal value of -5 mV. However, it did not influence the delay of onset of the sAHP; the AHP always set in after a 100-ms interval following the 1st action potential. During this interval, the membrane voltage returned to the resting potential value. In A-D, the top trace represents transmembrane voltage, while the bottom trace is injected current. Zero potential is indicated by a dash and resting potential by a horizontal line. In this and the following figure insets, the images demonstrating cellular morphology are shadow projections based on a stack of 23 up to 55 confocal images taken at a z-interval of 0.33 up to 0.54 µm.

SAHP. sAHPs were only evoked by current pulses at amplitudes above spike firing threshold. Single action potential firing (Fig. 1A) resulted in sAHP in 3 (3%) of the 99 porcine Dogiel type II neurons, regardless of the duration of the preceding current pulse (between 2 and 200 ms). Multiple spike firing (Fig. 1, B-D) increased the sAHP amplitude, setting off this hyperpolarization in another 14 cells (14%).

It took about 628 ± 108 ms to reach the maximal amplitude (tmax) of the sAHP following the first action potential evoked with current pulses of 120 ms (n = 17). Figure 2A and Table 1 (model A) show that tmax of the sAHP is independent of the number of the preceding spikes. Eliciting a single action potential with a very short current pulse did not change tmax either (Fig. 1A). Short current pulses further revealed that the membrane resting potential was reached in between the respective hyperpolarizations of the fAHP and sAHP. This delayed onset of the sAHP (100-200 ms with respect to the 1st spike) appeared independent of the preceding number of spikes. Firing more spikes, however, did result in an increase in both duration and amplitude of the sAHP (Fig. 1, A-D), as illustrated in Fig. 2, B and C, and quantified in Table 1 (Models B and C). The values measured for these features gradually increased with increasing numbers of preceding action potentials. Multiple spike firing caused the sAHP amplitude and duration to reach levels of up to -5 mV and 7 s, respectively.



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Fig. 2. Differences in tmax, duration, and amplitude of medium AHP (mAHP) and sAHP according to the number of preceding action potentials. Dissimilar properties of sAHP () and mAHP (open circle ) are evident in the relation between the number of action potentials on the one hand, and tmax (A), duration (B), and amplitude (C) of the AHPs on the other hand. Each datapoint in the figure refers to a single measurement of AHP properties on one of the 17 sAHP or 26 mAHP neurons. For some of these neurons, AHPs were measured repeatedly in response to current pulses of different amplitudes (range 0.1-1.2 nA). The x-axis shows the number of action potentials fired during the depolarizing current pulse preceding the AHP. In A, tmax on the y-axis is the time period between the 1st action potential and the moment when the AHP reached maximal amplitude. The straight lines represent the best linear fit through each set of datapoints used for the multiple regression analysis (Table 1). A: the tmax for sAHP was substantially larger than that for mAHP, while for neither AHP tmax depended on the number of action potentials (see also Table 1, model A). From these data, the average value of tmax for each individual neuron was calculated. The histogram in the inset shows the number of sAHP and of mAHP neurons (in % of total) with a particular tmax (class size 100 ms). These data confirm that the variable tmax is a distinguishing characteristic between sAHP and mAHP neurons. B: sAHP durations significantly exceeded those of mAHP; unlike tmax the preceding number of action potentials did influence this variable (see also Table 1, model B). C: the amplitude of mAHP was larger than that of sAHP (see also Table 1, model C), and also this variable increased with the number of action potentials for both sAHP and mAHP.


                              
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Table 1. Multiple regression analysis

MAHP. mAHP was recorded in 50 porcine Dogiel type II neurons (51%). Apart from being more common, this hyperpolarization did not resemble sAHP in several aspects. To begin with, mAHP was never seen following a single action potential, when the latter was evoked by the intrasomatic injection of a short (2-5 ms) depolarizing current pulse. On the other hand, mAHP could be recorded in response to a long (200 ms) depolarizing current pulse at a level below the spike firing threshold (Fig. 3A). The mAHP seen under these circumstances was indistinguishable from that evoked with slightly stronger current pulses that went just beyond spike firing threshold and evoked firing of a single action potential (Fig. 3A).



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Fig. 3. Electrophysiological features of porcine mAHP neurons with Dogiel type II morphology. Recordings of mAHP in 2 Dogiel type II neurons (insets). In A and D, mAHP set in immediately following the current pulse (black-down-triangle ). Whenever the fAHP (down-triangle) following the last spike occurred after the cessation of the current pulse, this fAHP and the subsequent mAHP always appeared continuous (left-triangle black-triangle-left ). A: mAHP in a type II neuron, with maximal spike firing restricted to 1 action potential at the onset of a depolarizing current pulse (0.7 nA, 200 ms, ---). Following a depolarizing current pulse of minimal amplitude (0.1 nA, · · ·) no change in resting membrane potential occurred, while a near threshold current pulse (0.5 nA, - - -) evoked a mAHP of nearly identical configuration as the one following the suprathreshold current pulse. B-D: in this Dogiel type II neuron (inset) the intrasomatic application of depolarizing current pulses (0.4-0.6 nA) of 200 ms (B), 120 ms (C), and 500 ms (D) resulted in all cases in the immediate onset of the mAHP. A current pulse of 500 ms (D) also demonstrated this neuron to be restricted in repetitive spike firing. To highlight the different phenomena, voltage and time scales were changed halfway through. This switch in calibration occurred when the voltage reached resting potential value immediately following the current pulse. In C and D, calibration of the vertical scalebars is identical to that in B. In A-D, the top trace is transmembrane voltage; the bottom trace is injected current. Zero potential is indicated by a dash and resting potential by a horizontal line.

Following similar depolarizing current pulses, tmax was reached faster for the mAHP than for the sAHP, i.e., 148 ± 11 ms (tmax, n = 26). However, by changing the duration of the current pulse (Fig. 3, B-D), it was revealed that tmax of the mAHP occurred after a fixed time span following the ending of the current pulse rather than being delayed for a fixed period after the first spike, as witnessed for the sAHP. Another dissimilarity between mAHP and sAHP was that the former was continuous with the fAHP. This difference was all the more obvious when spikes were fired throughout the duration of the depolarizing current pulse and the fAHP following the last action potential appeared after the current pulse had ceased (Fig. 3, B and C). Figure 2A and Table 1 (model A) illustrate that tmax of the mAHP did not depend on the number of preceding action potentials. In contrast to tmax, both duration (Fig. 2B; Table 1, model B) and amplitude (Fig. 2C; Table 1, model C) increased in parallel with the number of preceding spikes fired.

COMPARISON OF SAHP AND MAHP. To quantify the differences in tmax of both AHPs, the tmax values for each individual neuron (obtained by averaging) were plotted in a histogram (Fig. 2A, inset). The histogram reveals two subpopulations of neuronal AHP responses. The mean values for tmax of both subpopulations were found to be significantly different (Table 1, model A). Multiple regression analysis of the data in Fig. 2 showed this difference in tmax to be accompanied by consistent differences in duration (Table 1, model B) and amplitude (Table 1, model C) of the mAHPs and sAHPs. This is also illustrated in Fig. 4, which shows a scatter plot of duration versus tmax (the 2 most distinguishing parameters) for all neurons. Since the duration of the mAHPs and sAHPs depended on the preceding number of action potentials (Fig. 2, Table 1), the worst case scenario was used for each neuron in Fig. 4, i.e., the shortest sAHP duration measured and the longest mAHP measured were selected. Statistical analysis (see Fig. 4 legend) confirmed the presence of these two clearly distinct populations of neurons and the complete absence of intermediate types. Thus sAHP and mAHP neurons form two distinct electrophysiological subpopulations that differ not only in their responses to small current pulses and onset of AHP, but also in tmax, duration and amplitude of the AHPs. Table 2 provides an overview of the average values for tmax, amplitude, and duration of the three distinct AHPs as recorded following the firing of a single action potential.



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Fig. 4. Two electrophysiological subpopulations in the porcine Dogiel type II neurons as revealed by tmax and duration of the AHPs. So as to demonstrate unequivocally the existence of 2 separate electrophysiological neuronal subgroups defined by the existence of either a mAHP or sAHP, a worst case scenario was applied. Minimal values for duration of the sAHP (; n = 17 neurons) and maximal values for the mAHP duration (open circle ; n = 26 neurons) were plotted against their mean tmax value. The median values for tmax and duration of all neurons are indicated along the axis. The symbols are unequally distributed over the 4 quadrants determined by the 2 medians (SPSS; chi 2 test, Fisher 2-tailed P < 0.0001). This leads to the conclusion that there are 2 distinct electrophysiological subpopulations of neurons.


                              
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Table 2. Distinct features of the three types of AHP

Repetitive spike firing and adaptation

Overall, most porcine Dogiel type II neurons fired multiple spikes in rapid succession on the intrasomatic application of long depolarizing current pulses. Increasing the amplitude of the current pulses gave rise to a gradually higher spike firing rate (Fig. 5). The upper limit for the discharge rate, as measured at the first spike interval during the strongest currents, was 90 spikes/s. Although repetitive spike firing sometimes lasted for several 100 ms (Fig. 5, inset), this was not interpreted as tonic firing, since the values for maximal number of spikes and duration of firing never exceeded 25 spikes and 500 ms, respectively. Furthermore, repetitive spike firing was subject to spike frequency adaptation, i.e., the interspike interval (ISI) increased with the number of spikes (Fig. 5). When few spikes were fired, adaptation set in with the first intervals. When applying stronger depolarizing currents, however, this increase in ISI was not distributed evenly among the successive intervals but manifested itself clearly in the final ones. Consequently, action potentials were fired over a longer time span at a steady high-frequency level.



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Fig. 5. Spike frequency adaptation in a porcine Dogiel type II neuron. Repetitive firing in a porcine myenteric Dogiel type II neuron was evoked by the intrasomatic application of long depolarizing currents of increasing amplitude (0.1-nA steps). At every current level the interspike interval (ISI) increased with time (spike frequency adaptation). With stronger currents the total spike train duration was extended and the firing rate increased, which is depicted as a gradual decrease in the initial ISI. Eventually these features reached saturated levels. The inset shows spike firing at threshold level (0.3 nA, · · ·) and at maximal level (0.8 nA, ---). The x/y graph shows the ISI vs. the number of intervals for a depolarizing current pulse with indicated amplitude. Plottings of the 1st and last trace are shown, respectively, as a dotted and a solid line.

Passive and active membrane properties (see Table 3)

PASSIVE MEMBRANE CHARACTERISTICS. Average resting membrane potentials were substantially more depolarized in neurons with mAHP than in neurons with sAHP and those negative for long-lasting AHP. The average membrane time constant was higher in mAHP neurons and differed from that recorded in neurons lacking any long-lasting AHP. No differences in input resistance were noted. As these passive membrane characteristics displayed small changes over long time periods, their application as distinguishing standard was considered inappropriate, which is in accordance with earlier conclusions drawn for guinea pig neurons (Bornstein et al. 1994).


                              
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Table 3. Porcine Dogiel type II neurons subdivided according to long-lasting AHP type

ACTIVE MEMBRANE PROPERTIES. The action potential half-width was not different among the different subclasses. A "shoulder" on the repolarization phase of the action potential and a double inflection in the corresponding first time derivative of the voltage (dV/dt, Fig. 6, A-C) were seen in the majority of neurons, whether they displayed sAHP (82%, 14/17) or mAHP (74%, 37/50) or were negative for long-lasting AHP (81%, 26/32).



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Fig. 6. Comparison of the action potential features in 3 distinct electrophysiological subclasses of porcine Dogiel type II neurons. Action potentials (top trace) and 1st time derivatives of the voltage record (dV/dt; bottom trace) are shown for different porcine myenteric Dogiel type II neurons displaying sAHP (A), mAHP (B), or no long-lasting AHP (C). In all neurons, action potentials revealed an obvious shoulder on the falling phase in the voltage record and 2 inflections on the 1st time derivative. Superfusion of TTX (1 µM) during 15 min blocked repetitive spike firing in sAHP neurons (D), mAHP neurons (E), and neurons with no long-lasting AHP (F). A small TTX-resistant regenerative response, however, remained at the onset of the depolarizing current pulse. In D-F, the top trace represents transmembrane voltage; the bottom trace is the injected current. Zero potential is indicated by a dash and resting potential is shown at the bottom. Scale bars of A also apply to B and C, and the scale bar of D also applies to E and F.

Superfusion of TTX (1 µM) strongly suppressed repetitive spike firing in all neurons tested (n = 13). A single regenerative response remained (Fig. 6, D-F), however, in the majority of the neurons during intrasomatic injection of suprathreshold depolarizing current pulses whether or not sAHP (2/3) or mAHP (6/6) were present or long-lasting AHP absent (4/4). The regenerative response had a slower rate of upstroke and reduced amplitude compared with spikes under control conditions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This intracellular microelectrode study was focused on neurons with Dogiel type II morphology in the porcine small intestine. To our knowledge, it represents the first attempt to correlate electrophysiological features with an identified morphological subclass in the ileal myenteric plexus of a large mammal. The electrical behavior of these Dogiel type II neurons was found to differ in many aspects from that of their guinea pig counterparts, which have been attributed a primary afferent function (Furness et al. 1998; Kunze and Furness 1999).

AHPs

Most notably, the porcine Dogiel type II neurons could be subdivided according to the mutually exclusive occurrence of sAHP and mAHP. Most common was the mAHP, which reached its largest amplitudes on the intrasomatic application of long depolarizing currents at levels that elicited a train of action potentials. In myenteric S neurons of guinea pig ileum (Bornstein et al. 1994; Morita et al. 1982; Nishi and North 1973), colon (Lomax et al. 1999), and mouse colon (Furukawa et al. 1986), a similar type of long-lasting hyperpolarization is called a posttetanic hyperpolarization.

However, already at subthreshold current levels, mAHP of small amplitudes could be discerned in porcine Dogiel type II neurons, as previously observed in myenteric neurons in the guinea pig gastric corpus (Schemann and Wood 1989) and colon (Messenger et al. 1994). Due to the fact that this hyperpolarization occurred at levels below threshold and was intermediate in duration compared with fAHP and sAHP, we opted for the term medium AHP (mAHP) rather than posttetanic hyperpolarization.

Also in such characteristics as immediate onset, rapid attainment of maximal amplitude and durations of several 100 ms, the mAHP in the myenteric porcine Dogiel type II neurons hardly differed from the above-mentioned hyperpolarizations recorded in the guinea pig (Bornstein et al. 1994; Lomax et al. 1999; Morita et al. 1982; Nishi and North 1973) and mouse myenteric neurons (Furukawa et al. 1986). Amplitudes and durations were found to depend on the number of action potentials. Since larger spike numbers are evoked by stronger depolarizing current pulses, it is difficult to say whether mAHP is dependent on the passive membrane depolarization itself, as has been demonstrated in guinea pig myenteric neurons (Schemann and Wood 1989), or whether active membrane processes are involved. There are three arguments in favor of the former hypothesis: 1) the occurrence of mAHP at subthreshold levels, 2) the impossibility to demonstrate mAHP following a single spike evoked by a 2-ms depolarizing current pulse, and 3) its continuous occurrence following a 100-ms current pulse of similar amplitude, again eliciting a single spike.

The second type of long-lasting hyperpolarization, i.e., the sAHP, was far less common. This observation is in line with a preliminary study (Cornelissen et al. 1996) presenting the apparent absence of sAHP from the entire population of porcine myenteric neurons as one of its major conclusions. However, neurons had not been selected on the basis of their morphology and neither had any special attention been paid to the detection of very small sAHPs. The present study, in which the porcine myenteric Dogiel type II neurons were considered in more detail, demonstrated a substantially lower incidence of sAHP after the firing of a single action potential than the 90% incidence rate in guinea pig (Furness et al. 1988, 1994; Pompolo et al. 1989). Similarly as in guinea pig, sAHP in porcine Dogiel type II cells could never be demonstrated following depolarizing currents at subthreshold levels, regardless of their durations. In both guinea pig and pig, an increase in the number of preceding action potentials intensified the sAHP amplitude in the myenteric neurons (Hirst et al. 1974; unpublished observations). In absolute terms, however, maximal sAHP amplitudes differed between both species. The highest values measured in guinea pig myenteric neurons, -15 and -20 mV following single and multiple spike firing, respectively (unpublished observations), largely exceeded those measured in their porcine counterparts under similar conditions. Our observations in pig are strengthened by a study carried out on human colon (Brookes et al. 1987). Even though this study was limited to 27 cells, 75% of the myenteric sAHP neurons only displayed a sAHP after a burst of spikes. Moreover, the sAHP amplitudes elicited under these conditions (-8 mV) tended to be lower than in guinea pig.

In both guinea pig and porcine myenteric neurons, the delay in onset and tmax of the sAHP are independent of the number of preceding spikes, the absolute values for these parameters being much lower in guinea pig (Hirst et al. 1974; unpublished observations) than in pig.

Repetitive spike firing

Repetitive spike firing in enteric neurons is subject to plasticity and therefore merely reflects a functional state of the neurons at a certain moment (Bornstein et al. 1994; Clerc et al. 1998; Hodgkiss and Lees 1983; Tack and Wood 1992; Tamura and Wood 1989). Yet, the fact that most of the porcine Dogiel type II neurons are capable of firing multiple spikes in rapid succession shows that in this respect they are highly different from guinea pig sAHP neurons and rather resemble guinea pig S neurons (Hirst et al. 1974; Nishi and North 1973). Spike firing in the porcine Dogiel type II neurons, however, was not regarded as tonic since spike frequency adaptation eventually limited the total spike train duration.

Furthermore, repetitive spike firing could be evoked beyond the onset of the sAHP in the majority (94%) of porcine sAHP neurons, while guinea pig sAHP neurons then enter a state of decreased excitability (Hirst et al. 1974). Due to this, as well as the relatively small amplitude of the sAHPs, it is highly unlikely that the sAHP creates a refractory state in porcine Dogiel type II neurons, as it does in the guinea pig. In this respect it is worth mentioning that in submucosal neurons in the jejunum of 1- to 2-day-old piglets, the sAHP was highly prevalent and of large amplitude while spike firing was restricted to a single action potential (Thomsen et al. 1997). In guinea pig submucous plexus, on the other hand, the majority of neurons lacked sAHP (Hirst and McKirdy 1975; Mihara 1993). These inverse observations in myenteric and submucous plexuses of large and small mammals could further strengthen the functional differentiation of the distinct nerve networks proposed earlier based on anatomical and neurochemical data (Timmermans et al. 1990, 1997).

Action potential: shoulder and TTX resistance

Slow AHP has long been considered to be the most reliable criterion by which to define the class of neurons named after it in guinea pig small intestine (Bornstein et al. 1994; Gershon and Wade 1993; Wood 1994). Recently, however, some authors have expressed doubts that sAHP is a satisfactory characteristic for defining comparable functional classes of neurons in all regions of the guinea pig gastrointestinal tract (Clerc et al. 1998; Furness et al. 1998). Action potentials displaying a so-called "shoulder" on the repolarization phase, especially in combination with a large half-width, would make a more suitable criterion (Clerc et al. 1998; Schutte et al. 1995). Most often, however, occurrence of the action potential shoulder and the sAHP tend to coincide in guinea pig neurons (Gershon and Wade 1993; Wood 1994) since the first indicates the participation of a Ca2+ current in the spike upstroke, while the second results from the subsequent activation of a Ca2+-dependent K+ current (Baidan et al. 1992; Hirst et al. 1985b).

Due to the lack of sAHP observed in porcine myenteric Dogiel type II neurons, we thought it opportune to investigate the above-mentioned action potential features and found 78% of all 99 cells to display a clear shoulder on the repolarization phase. On average, spike half-widths were found to be twice as small in porcine Dogiel type II neurons than they were in the guinea pig counterparts (Hirst et al. 1985b; unpublished observations).

Moreover, no correlation of any kind could be established between the action potential shoulder and the occurrence of sAHP, with 3 of the 17 sAHP neurons lacking a shoulder. On the other hand, large proportions of neurons with mAHP (74%) or without long-lasting AHP (81%) displayed a shoulder on their action potentials. However, this lack of correlation is not without precedent. Porcine submucous neurons are known to behave diametrically opposed to their myenteric counterparts in this respect. Notwithstanding the abundance of sAHP and its large amplitudes, a shoulder on the action potential was not recognized as a typical feature of these submucous neurons (Thomsen et al. 1997). Similarly, regional differences have been reported for rat intestine, where action potentials were observed to display a prominent shoulder in duodenal sAHP cells (Brookes et al. 1988), whereas their colonic variants lacked any indication of such a shoulder (Browning and Lees 1996). Moreover, sAHP neurons in the colon have been observed to reveal substantial interspecies differences with regard to the occurrence of the action potential shoulder, ranging from markedly present (Messenger et al. 1994) to absent (Brookes et al. 1987; Browning and Lees 1996). Although ongoing synaptic input has been shown to affect action potential shape and duration (Kaczmarek and Levitan 1987), it remains puzzling why in the same species sAHP neurons either display a clear shoulder on their action potentials or lack this feature solely depending on the gastrointestinal region or plexus investigated.

In the myenteric Dogiel type II neurons of the pig, the persistence of a TTX-resistant regenerative response provides further evidence of an inward ionic current distinct from the TTX-sensitive Na+ current. Based on a previous study on guinea pig myenteric neurons (Hirst et al. 1974), such TTX resistance could be expected in those Dogiel type II neurons displaying sAHP. Surprisingly, however, a similar TTX-resistant regenerative response was also found in neurons with mAHP and those lacking long-lasting AHP. Therefore in line with our observations regarding the action potential shoulder, no strict correlation could be established between sAHP and TTX-resistant spike firing in porcine Dogiel type II neurons of the myenteric plexus. This observation should be further investigated in light of previous findings in the submucous plexuses of the pig (Thomsen et al. 1997) and myenteric neurons of the rat colon (Browning and Lees 1996). In these studies a strong correlation was observed between the TTX resistance of the spikes and the presence of sAHP, although the action potentials in these cells, as mentioned above, obviously lacked any indication of a shoulder under control conditions. This is all the more interesting in light of the recent suggestion based on observations in guinea pig (Clerc et al. 1998; Furness et al. 1998; Schutte et al. 1995) to use the shoulder as the most reliable defining criterium of sAHP neurons.

Conclusions

The main conclusion of this study is that morphological Dogiel type II neurons in porcine ileum combine electrophysiological features considered typical of either S-type or sAHP-type neurons in guinea pig. This implies that, unlike in guinea pig, no clear-cut correlation can be established between morphology and electrophysiology in porcine Dogiel type II neurons.

The different time characteristics of mAHP and sAHP and their mutual exclusive occurrence suggest different underlying mechanisms in subpopulations of Dogiel type II neurons. In the case of sAHP, with its delayed onset and durations of many seconds, a second-messenger system is conceivable. The almost immediate onset and shorter duration witnessed in mAHP could indicate a faster underlying mechanism of shorter lasting effect. Further investigations, however, are necessary to elucidate the mechanisms of ionic currents underlying both types of long-lasting hyperpolarizations.


    ACKNOWLEDGMENTS

The authors thank W. Delnat, D. De Ryck, A. Hertog, F. Terloo, G. Sebreghts, G. Svensson, and J. Van Daele for excellent technical assistance. We also thank Dr. J. C. Van der Auwera for advice on statistical procedures.

This study was supported by Inter University Attraction Pole Grant P4/16 to J.-P. Timmermans.


    FOOTNOTES

Address for reprint requests: J.-P. Timmermans, Laboratory of Cell Biology and Histology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium (E-mail: jptimmer{at}ruca.ua.ac.be).

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

Received 8 November 1999; accepted in final form 15 March 2000.


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