The present results show that 45% (32/71) of Dogiel type I neurons from inflamed ileum had an electrophysiological phenotype that was distinctly different from that normally exhibited by these neurons. The phenotypically changed neurons exhibited prominent late AHPs and in many respects their electrophysiological phenotype resembled that of Dogiel type II neurons. In addition, neurons with Dogiel type II morphology became more excitable following inflammation, but retained the major characteristics that define their electrophysiological phenotype: a large amplitude action potential with a hump on the falling phase and a prominent, long-lasting, late AHP. In both control and inflamed ileum very few Dogiel type II neurons exhibited fast excitatory synaptic potentials.
Phenotypically changed Dogiel type I neurons
The prolonged AHPs in transformed Dogiel type I neurons were associated with a conductance increase and were blocked by TRAM-34 (10 μm), which is highly selective for intermediate conductance, Ca2+-activated potassium (IKCa) channels over other classes of K+ channel (Wulff et al. 2000). In enteric Dogiel type II neurons, IKCa channels are activated following entry of Ca2+ during the action potential, augmented by Ca2+-activated Ca2+ release from intracellular stores (Hillsley et al. 2000; Vogalis et al. 2002a). Dogiel type I neurons from control intestine, in earlier work identified by their S electrophysiological phenotype, do not appear to have a Ca2+ contribution to the action potential, which is completely blocked by tetrodotoxin (North, 1973; Hirst et al. 1974; Wood, 1987). We found that Dogiel type I neurons with late AHPs had a TTX-resistant component of their action potential. Thus, the transformation of Dogiel type I neurons could be due to the activation of pre-existing IKCa channels following the emergence of a Ca2+ component of the action potential. Whether there is de novo or augmented expression of IKCa channels in these Dogiel type I neurons remains to be determined.
In normal intestine, Dogiel type II neurons have broader action potentials compared with those of Dogiel type I neurons and a prominent inflection (hump) on their repolarizing phase due to the Ca2+ component. Previously, the inflection on the repolarizing phase of an action potential and the presence of a TTX-resistant component were considered to be reliable features for identification of AH/Dogiel type II neurons (Hirst et al. 1985; Schutte et al. 1995; Furness et al. 1998). Our results show that this cannot be applied to the classification of neurons from the inflamed intestine, as 30% of the phenotypically changed Dogiel type I neurons had action potentials with inflections. The absence of inflection in some of the neurons may be due to differences in the kinetics of Ca2+ currents activated during the action potential, or to other differences, such as in the properties of delayed rectifier and/or large conductance (BK) potassium channels that are involved in repolarization. The amplitudes and durations of action potentials in Dogiel type I neurons with late AHPs were similar to those in unchanged Dogiel type I cells.
Without their morphological identification by dye filling at the time of recording, the transformed Dogiel type I neurons could easily be misidentified as AH cells, which are normally neurons with Dogiel type II morphology. However, one distinguishing electrophysiological characteristic did remain: these Dogiel type I neurons with late AHPs, but very rarely Dogiel type II cells from the control or inflamed ileum, had fast synaptic inputs. The amplitudes and durations of fast EPSPs in Dogiel type I neurons with late AHPs were not significantly different compared with amplitudes and durations of fast EPSPs in Dogiel type I/S neurons from the inflamed or control ileum. This is an interesting observation, because inflammatory mediators, applied acutely to myenteric ganglia, reduce the amplitudes of fast EPSPs (Xia et al. 1999; Gao et al. 2002). Our observations suggest that this reduction, if it occurs in vivo, does not persist. Our observations are consistent with those of Krauter et al. (2007), who found that fast EPSPs had greater amplitude than control 6 days after TNBS was injected into the lumen of the colon.
Late AHPs have been previously reported in a small subpopulation of uniaxonal neurons from non-inflamed small intestine or colon. Late AHPs in these neurons were less prominent than in Dogiel type II neurons, and the AHP was sometimes only observed after a volley of action potentials (Song et al. 1997; Clerc et al. 1998; Lomax et al. 1999; Tamura et al. 2001; Nurgali et al. 2003). The late AHPs in transformed Dogiel type I neurons were significantly briefer in duration compared with Dogiel type II neurons from both the inflamed and control ileum, but they were of similar amplitude (about 8 mV) and occurred after a single action potential. A late AHP following a burst of action potentials has been reported in highly excitable Dogiel type I neurons which are located at the corners of the ganglia close to internodal strands in the myenteric plexus of the guinea-pig ileum (Smith et al. 1999). The AHP in these cells was reduced by apamin, which blocks SK but not IKCa channels, and does not affect the AHP in other enteric neurons (Tack & Wood, 1992; Vogalis et al. 2002a). The uniaxonal neurons in which we found AHPs after inflammation were not these neurons: they differed in size, shape and projections (see below).
The majority of Dogiel type I neurons with changed phenotype (31/32) had descending axons. The other neuron was deduced to be a local circular muscle motor neuron, from its shape and the projection of its axon to the muscle. In the non-inflamed small intestine, three classes of Dogiel type I neurons with descending axons have been identified in the myenteric plexus: inhibitory circular muscle motor neuron that are immunoreactive for nitric oxide synthase (NOS) and comprise 16% of all neurons in the myenteric plexus; NOS-immunoreactive descending interneurons (5%); and 5-HT-immunoreactive descending interneurons (2%, Furness, 2006). Except in rare cases, we were not able to distinguish between the circular muscle motor neurons and interneurons, due to the removal of the circular muscle layer during preparation for electrophysiology. Three neurons (1 local, 2 descending) were traced to the circular muscle, and none to the longitudinal muscle. The cell capacitance of Dogiel type I neurons with late AHPs was not different from the cell capacitance in unchanged Dogiel type I neurons, indicating that these cells have the same size as other Dogiel type I neurons, on average.
Dogiel type I neurons with late AHPs from the inflamed ileum had low thresholds for depolarizing current pulses to evoke action potentials, they fired more action potentials to depolarization, but only 1/32 exhibited spontaneous action potentials. The lower threshold for generation of action potentials might be contributed by a reduced threshold for activation of voltage-activated Na+ channels in these neurons after inflammation. In addition, a higher incidence of anodal break action potentials was observed in Dogiel type I neurons with AHPs (47%), compared with Dogiel type I/S neurons from the inflamed ileum (24%).
Changes in Dogiel type II neurons
Dogiel type II neurons from the inflamed ileum were hyperexcitable, but their electrophysiological phenotype was very similar to that of Dogiel type II neurons from control ileum. In both cases the neurons had large amplitude action potentials with an inflection on the falling phase and a component of the action potential was resistant to tetrodotoxin. The action potentials were followed by prolonged AHPs. After inflammation, the neurons had higher input resistances, reduced thresholds for depolarizing current pulses to evoke action potentials, fired more action potentials to depolarization and exhibited spontaneous action potentials. This conversion from a low excitability or unexcitable state to a hyperexcitable state is typical of AH/Dogiel type II neurons that are exposed to inflammatory mediators, for example, histamine (Tamura & Wood, 1992), interleukins (Xia et al. 1999) or leukotrienes (Liu et al. 2003). Enhancement of neuronal excitability and phenotypic changes in myenteric neurons suggest a plasticity of enteric neurons that is initiated through the actions of various inflammatory mediators released during TNBS-induced ileitis. Further mechanisms may be involved in the maintenance of the changes.
Spontaneous action potentials are not observed in Dogiel type II neurons from control ileum under the conditions used in this study. Despite the fact that spontaneous action potentials occurred in the neurons from inflamed ileum, they were not depolarized relative to Dogiel type II neurons of control ileum. This, and the lower threshold for generation of action potentials by intracellular current injection, suggests that there was a reduced threshold for activation of voltage-sensitive Na+ currents in Dogiel type II neurons after inflammation.
Action potentials that follow hyperpolarizing voltage steps in Dogiel type II neurons (anodal break action potentials) have been attributed to the Cs+-sensitive hyperpolarization-activated cation current, Ih (Galligan et al. 1990; Rugiero et al. 2002; Xiao et al. 2004). The incidence of anodal break action potentials in Dogiel type II neurons was 39% in the inflamed ileum, compared with 23% in the control ileum. It has been reported that Ih is increased during TNBS-induced inflammation in AH myenteric neurons in the distal colon (Linden et al. 2003). However, the present study indicated that the increase in the prominence of anodal break action potentials was not simply due to a change in Ih. Although Ih was blocked by CsCl in the Dogiel type II neurons, we continued to observe anodal break action potentials in some neurons. The resistance of these events to CsCl suggests that activation of other current(s) was involved in the generation of anodal break action potentials in AH neurons from the inflamed intestine. The other current that can cause anodal break firing is the low-voltage-activated T-type Ca2+ current (Jahnsen & Llinas, 1984; Huguenard, 1996). T currents have not been previously reported in enteric neurons and we are currently investigating the properties of this current and its channels in myenteric neurons.
The Ih current has been implicated in limiting the amplitude and duration of the AHP (Galligan et al. 1990), and its enhancement has been suggested to decrease the magnitude of the late AHP in myenteric AH neurons of the distal colon following inflammation (Linden et al. 2003). However, in the present study there were no differences in the amplitudes and durations of the late AHPs in Dogiel type II neurons from the control and inflamed ileum. Consistent with our results, Lomax et al. (2005) did not detect any difference in the effects of 2 mm CsCl on the AHP of submucosal AH neurons from the inflamed and control distal colon. Moreover, these authors showed, by quantitative analysis of the amplitude of the Ih current (measured as the amplitude of the ‘sag’ that occurred during 500 ms hyperpolarization current injection), that there is no difference in the Ih amplitude between inflamed and control AH neurons. Nevertheless, a reduction of AHP magnitude was observed in submucosal AH neurons, which was associated with a reduced duration of action potential, and presumably a smaller influx of Ca2+ (Lomax et al. 2005).
Dogiel type I neurons without phenotypic change
The electrophysiological phenotype of the majority of Dogiel type I neurons from the inflamed guinea-pig ileum was not different from the Dogiel type I neurons of the control ileum; that is, these neurons had the S type phenotype, and accordingly we refer to these as Dogiel type I/S neurons. Although the majority of Dogiel type I/S neurons from the inflamed ileum (36/39) were not detectably different from Dogiel type I/S neurons from the control ileum, three Dogiel type I/S neurons from inflamed intestine were hyperexcitable. They had low thresholds for depolarizing current pulses to evoke action potentials (53 ± 10 pA) and fired on average 34 ± 11 action potentials in response to 300 pA depolarizing current pulses.
Hyperexcitable S neurons have been previously reported in the non-inflamed guinea-pig ileum by Smith et al. (1999). The authors described tonic S neurons characterized by low resting membrane potentials, high input resistance (about 500 MΩ), due to a small size of their cell bodies, low threshold for action potential generation, firing of action potentials throughout a depolarizing pulse and spontaneous fast EPSPs. Morphologically they were identified as motor neurons and ascending interneurons located at the corners of large ganglia close to internodal strands. In our experiments the hyperexcitable Dogiel type I/S neurons had cell capacitance of 33.5 ± 3.5 pF and input resistance of 196.3 ± 27 MΩ; these parameters were not statistically different from those in other Dogiel type I/S neurons from the inflamed ileum. In all other respects these three neurons were not different from the majority of Dogiel type I/S neurons. Morphologically they were identified as one ascending Dogiel type I and two descending Dogiel type I neurons. A hyperexcitable filamentous neuron was also encountered. These neurons were localized in the middles of their ganglia.
In a study in non-inflamed intestine with intact mucosa, Kunze et al. (1997) reported that some S neurons that received ongoing input from Dogiel type II neurons were hyperexcitable. We encountered spontaneously active Dogiel type II neurons in our experiments in inflamed ileum which could feasibly have provided slow excitatory inputs to the Dogiel type I/S neurons and contributed to their hyperexcitability.
Other phenotypic changes
In the present work, we have investigated phenotypic changes in the electrophysiological properties of enteric neurons. It is probable that other changes occur, for example in neurochemistry, including neuropeptide and receptor expression. However, there is no evidence of substantial morphological change. All the filled neurons could be classified into the morphological types that have been documented in the normal intestine, and it seems unlikely that the neurons switch their shapes and projections. Future studies will be needed to carefully examine which other changes occur in the neurons.
Comparison with previous studies
The only previous electrophysiological study of post-inflammatory changes in myenteric neurons of the small intestine was following infection with Trichinella spiralis (Palmer et al. 1998). In that study, an enhanced excitability of neurons with the AH electrophysiological phenotype was reported, but whether the AH-type neurons after inflammation included both Dogiel type II and Dogiel type I neurons was not determined, because cells were not identified by morphology. Palmer et al. (1998) found that 53% of AH neurons from Trichinella spiralis-infected guinea-pigs had fast EPSPs. In our study, all 32 Dogiel type I neurons with AHPs had fast EPSPs, but only 1 of 93 Dogiel type II neurons from the inflamed ileum had a fast EPSP. This suggests that the AH cell population identified by Palmer et al. included both Dogiel type II and Dogiel type I neurons. In other regions also, the most prominent change observed in electrophysiological properties was the enhanced excitability of AH neurons. This has been reported in both myenteric (Linden et al. 2003) and submucosal neurons (Lomax et al. 2005, 2006; O'Hara et al. 2007) from the guinea-pig distal colon. The majority of myenteric neurons (61%) were identified morphologically in the study of Linden et al. (2003), although no illustrations were included. They reported that changed excitability occurred in AH/Dogiel type II neurons.
In each place, different mechanisms have been uncovered. As is discussed above, we have found that increased excitability of Dogiel type II neurons was associated with a lowered voltage threshold for action potential generation and possibly the de novo appearance of a T-like current, whereas in myenteric neurons of the distal colon an enhanced h current was implicated (Linden et al. 2003) and in submucosal neurons there was a reduced Ca2+ influx during the action potential that had the consequence of reducing the AHP (Lomax et al. 2005, 2006).
Further studies are required to define what impact the phenotypic changes in neurons found in this study have on changes in motility or secretion that occur in the gastrointestinal tract after inflammation (Kellow, 2002; De Giorgio et al. 2004; Lomax et al. 2004; Wood, 2004). Modulation of the AHP by compounds affecting IKCa channels have profound effects on the motility of the non-inflamed intestine (Ferens et al. 2007), so it is feasible that the de novo appearance of the AHP in descending Dogiel type I interneurons or inhibitory motor neurons will affect motility. Whether the changes in currents and channels are due to the direct effects of inflammatory mediators or are secondary responses in the cascade of events following inflammation remains open, and a difficult problem to resolve.