Martin J. Stebbing

Correlation of electrophysiological and morphological characteristics of enteric neurons in the mouse colon

We report on the first correlative study of the electrophysiological properties, shapes, and projections of enteric neurons in the mouse. Neurons in the myenteric plexus of the mouse colon were impaled with microelectrodes containing biocytin, their passive and active electrophysiological properties determined, and their responses to activation of synaptic inputs investigated. Biocytin, injected into the neurons from which recordings were made, was converted to an optically dense product and used to determine the shapes of neurons. By electrophysiological properties, almost all neurons belonged to one of two classes, AH neurons or S neurons. AH neurons had a biphasic repolarization of the action potential, and slow afterhyperpolarizing potentials usually followed the action potentials. S neurons had monophasic repolarizations, no slow afterhyperpolarization, and fast excitatory postsynaptic potentials in response to fibre tract stimulation. By shape, neurons were divided into Dogiel type II (28/136 neurons) and uniaxonal neurons. Dogiel type II neurons had large, smooth‐surfaced cell bodies and several long processes that supplied branches within myenteric ganglia. All Dogiel type II neurons had AH electrophysiology; conversely, most AH neurons had Dogiel type II morphology. The majority of uniaxonal neurons had lamellar dendrites, i.e., Dogiel type I morphology. They projected to the circular muscle (circular muscle motor neurons), to the longitudinal muscle (longitudinal muscle motor neurons), and to other myenteric ganglia (interneurons) and in some cases could not be traced to target cells. All S neurons were uniaxonal. A small proportion of uniaxonal neurons (3/70) had AH electrophysiology. Fast excitatory synaptic potentials were only recorded from uniaxonal neurons and were in most cases blocked by nicotinic receptor antagonists. A small component of fast excitatory transmission in some neurons was antagonized by the purine receptor antagonist PPADS. Slow excitatory postsynaptic potentials were observed in both AH and S neurons. Slow inhibitory postsynaptic potentials were recorded from S neurons. We conclude that the major classes of neurons are Dogiel type II neurons with AH electrophysiological properties and Dogiel type I neurons with S electrophysiological properties. The S/Dogiel type I neurons include circular muscle motor neurons, longitudinal muscle motor neurons, and interneurons. J. Comp. Neurol. 468:112–124, 2004.

Projections and chemistry of Dogiel type II neurons in the mouse colon

The physiological properties, shapes, projections and neurochemistries of Dogiel type II neurons have been thoroughly investigated in the guinea-pig intestine in which these neurons have been identified as intrinsic primary afferent neurons. Dogiel type II neurons in the myenteric ganglia of mice have similar physiological properties to those in guinea-pigs but whether other features of the neurons are similar is unknown. We have used intracellular dye-filling, retrograde tracing, immunohistochemistry and nerve lesions to determine salient features of Dogiel type II neurons of the mouse colon. Dye-filling showed that the neurons provide profuse terminal networks in the myenteric ganglia and also have axons that project towards the mucosa. Retrograde tracing and lesion studies showed that these axons provide direct innervation to the mucosa. High proportions of the neurons had immunoreactivity for calretinin, calbindin, choline acetyltransferase, the purine P2X2 receptor and calcitonin gene-related peptide (CGRP). CGRP was the most selective marker of the neurons. Following surgery to remove an area of myenteric plexus, the CGRP-immunoreactive nerve fibres in the mucosa degenerated. Thus, Dogiel type II neurons in mice have similar shapes and projections but some differences in chemistry from those in guinea-pigs. The close similarities between the two species in the shapes, projections and electrophysiology of these neurons suggest that they serve the same functions in both species.

Changes in the action potential in sensory neurones after peripheral axotomy in vivo

Following nerve injury, modified somatic ion channels may underlie ectopic activity in axotomized A-type neurones in dorsal root ganglia (DRGs) leading to abnormal pain signalling. Using intracellular microelectrodes both in vivo and in vitro, action potentials (APs) were recorded in rat DRG neurones classified by axonal conduction velocity. After lesions to L5 spinal or sciatic nerves, APs in both A alpha/beta and A delta cells were wider, and those in A alpha/beta neurones more frequently showed inflections during repolarization, than APs in cells in undamaged ganglia. AP amplitudes and dV/dt(max) were not significantly altered by axotomy. These results confirm previous observations in intact ganglia in vitro but differ from those reported for dissociated neurones using patch recording techniques.

Microglia activation in the hypothalamic PVN following myocardial infarction

Following a myocardial infarction (MI), inflammatory cytokines are elevated in the brain, as well as in plasma, indicating that inflammation is occurring in the brain in addition to the periphery. Microglia are the immune cells in the central nervous system and can produce cytokines when they are activated by an insult or injury. In the present study, we investigated whether MI in rats induces activation of microglia in the brain. We used immunohistochemistry to detect CD11b (clone OX-42) and morphological changes to identify activated microglia. Compared to control rats that had undergone sham surgical procedures, there was a significant increase in activated microglia in the hypothalamic paraventricular nucleus (PVN) following myocardial infarction. Activated microglia were not observed in the ventral hypothalamus, adjacent to the PVN, nor in the cortex, indicating the response was not the result of a generalized inflammatory reaction in the brain. Echocardiography and haemodynamic parameters after myocardial infarction indicated that reduced left ventricular function but congestive heart failure had not developed. In conclusion, microglia are activated in the PVN but not in the adjacent hypothalamus following myocardial infarction. The activated microglia may contribute to the increased local production of pro-inflammatory cytokines observed in the PVN after myocardial infarction and resulting in reduced left ventricular function.

Are there functional P2X receptors on cell bodies in intact dorsal root ganglia of rats

P2X purinoceptors have been suggested to participate in transduction of painful stimuli in nociceptive neurons. In the current experiments, ATP (1-10 mM), alpha,beta-methylene-ATP (10-30 microM) and capsaicin (10 nM-1 microM) were applied to neurons impaled with high resistance microelectrodes in rat dorsal root ganglia (L4 and L5) isolated in vitro together with the sciatic nerve and dorsal roots. The agonists were either bath applied or focally applied using a picospritzer. GABA (100 microM) and 40-80 mM K+ solutions gave brisk responses when applied by either technique. Only three of 22 neurons with slowly conducting axons (C cells) showed evidence of P2X-purinoceptor-mediated responses. Only two of 13 cells which responded to capsaicin (putative nociceptors), and none of 29 cells with rapidly conducting axons (A cells), responded to the purinergic agonists. When acutely dissociated dorsal root ganglion cells were studied using patch-clamp techniques, all but four of 30 cells of all sizes responded with an inward current to either ATP or alpha,beta-methylene-ATP (both 100 microM). Our data suggest that few sensory cell bodies in intact dorsal root ganglia express functional purinoceptors.

Differential effects of SOCS2 on neuronal differentiation and morphology

Neuronal differentiation of neural progenitor cells is regulated by a variety of growth and transcription factors, that not only regulate cell fate of the progenitor cells but that can also regulate neuronal morphology. Suppressor of cytokine signaling-2 (SOCS2) is an intracellular regulator of Growth Hormone (GH) signaling that is expressed in neural stem cells and neurons during development and is required to overcome the inhibitory effects of GH on neuronal differentiation. SOCS2 also promotes neurite outgrowth, however, whether the mechanism by which SOCS2 regulates neuronal differentiation and neurite outgrowth is the same is not clear. Furthermore, whether the over-expression of SOCS2 has physiological in addition to morphological effects is unknown. To address these questions, we differentiated adult neural progenitor cells derived from wildtype C57BL/6 or SOCS2 over-expressing transgenic mice (SOCS2Tg) in the presence or absence of GH and determined effects on neuronal differentiation and morphology. Compared to wildtype cells, differentiation of SOCS2Tg neurospheres resulted in increased neurogenesis, which was not inhibited by GH. The neurons derived from these cells appeared more complex, with increased neurite outgrowth and number. GH did not, however, have any effect on neurite outgrowth of wildtype or SOCS2Tg neurons. Furthermore, basic electrophysiological analysis of wildtype and SOCS2Tg neurons derived from the neurospheres showed that they were both of an immature electrophysiological neuronal phenotype, indicating that although SOCS2 expression can regulate neuronal morphology, it appears to have little effect on neuronal ion channel expression.

Role of α2‐adrenoceptors in the sympathetic inhibition of motility reflexes of guinea‐pig ileum

1 Sympathetic regulation of the motility of guinea‐pig ileum was investigated using mesenteric nerve (MN) stimulation to inhibit motility reflexes, in vitro. 2 Transmural electrical stimulation (5 Hz, 1 s) in intact intestinal segments, or inflation of a balloon against the mucosa in opened segments, evoked contractions of the circular and longitudinal muscles oral to the stimulus. 3 MN stimulation (10 Hz, 5 s) usually abolished contractions of the longitudinal and circular muscles evoked by either electrical or mechanical stimuli. 4 The inhibition was mimicked by UK14,304 (70‐100 nm) and abolished by idazoxan (100 nm), revealing an enhancement of circular muscle contractions. There was no evidence for α2‐receptors on the muscle, suggesting sympathetic inhibition was via the myenteric plexus. 5 Possible sites of action of noradrenaline released from sympathetic nerves were investigated using intracellular recordings from the circular muscle in a multichambered organ bath. 6 When in the stimulation chamber, UK14,304 depressed (by 50 %) excitatory junction potentials (EJPs) recorded oral to a distension stimulus, but did not affect inhibitory junction potentials (IJPs) recorded anal to the stimulus. When added to a chamber between the stimulus and recording chambers, UK14,304 depressed EJPs by 40 %, but did not alter IJPs. When in the recording chamber, UK14,304 depressed EJPs by 20 %, but had no effect on IJPs. IJPs were inhibited, however, when UK14,304 was applied to the whole bath. 7 It is concluded that sympathetic activity inhibits intestinal motility mainly via α2‐adrenoceptors on ascending interneurons and intrinsic sensory neurons of the orally directed reflex pathway.

Electrophysiological mapping of fast excitatory synaptic inputs to morphologically and chemically characterized myenteric neurons of guinea-pig small intestine

Neurons within the myenteric plexus of the guinea-pig ileum were impaled using conventional intracellular electrodes. Points of stimulation within the surrounding ganglia and connectives which gave rise to fast excitatory synaptic potentials were mapped using a movable monopolar stimulating electrode. Cells were then injected with the intracellular marker, biocytin, and processed for multiple label immunohistochemistry to reveal their morphologies, chemical contents and, hence, their functional classes. Of 65 neurons belonging to the S electrophysiological class, 53 received fast excitatory synaptic inputs from stimulation at sites at least 2 mm away in a directly circumferential direction. These inputs almost certainly arise from stimulation of the circumferentially-directed axons of the Dogiel type II/AH-neurons, which are thought to be intrinsic sensory neurons. The majority of cells which projected anally and were immunoreactive for nitric oxide synthase (19/25), all neurons which ramified in the tertiary plexus and were identified as longitudinal muscle motor neurons (6/6) and all neurons identified as excitatory motor neurons innervating the circular muscle (12/12) received inputs from these circumferentially-directed pathways. However only one of six descending filamentous interneurons impaled received such inputs, suggesting they may be differentially innervated. The conduction velocities of circumferentially-directed axons giving rise to fast excitatory post synaptic potentials were estimated to be 0.41 +/- 0.10 m/s (mean +/- standard deviation, n = 21). The conduction velocities estimated for longitudinally-directed pathways were 0.55 +/- 0.25 m/s (n = 29). Thus, the majority of myenteric neurons receive fast excitatory synaptic input from putative intrinsic sensory neurons which project circumferentially around the intestine.

Electrophysiological analysis of the convergence of peripheral inputs onto neurons of the coeliac ganglion in the guinea pig

The convergence of intestinofugal axons from different intestinal regions onto individual neurons in the coeliac ganglion of the guinea pig was investigated using intracellular recording methods in vitro. Peripheral nerve trunks from the distal ileum, the most proximal colon and the colon near the colonic flexure were electrically stimulated along with preganglionic fibres running in the splanchnic nerve. Fast cholinergic excitatory synaptic potentials (EPSPs) were seen in ganglion cells in response to stimulation of each nerve trunk. Roughly half of 78 neurons impaled received inputs from stimulation of peripheral nerves, and almost all of these received input from the proximal colon. Most cells responded to stimulation of more than one peripheral nerve indicating that coeliac neurons receive converging inputs from intestinofugal neurons located in more than one intestinal region. In a second series of experiments, segments of intestine were left attached to the ganglion and distended with saline to stimulate peripheral mechanosensory input to the coeliac ganglion. In each experiment, two segments were stimulated. A subgroup of ganglion cells exhibited spontaneous fast EPSPs and the frequency of these potentials was increased by distension of one or other of the attached intestinal segments. However, few neurons responded to distension of both of the attached intestinal segments suggesting that some of the intestinofugal inputs to the coeliac ganglion identified by electrical stimulation may be sensitive to sensory modalities other than distension. Hexamethonium (0.5 mM) applied to the intestine, and not to the coeliac ganglion, reduced the frequency of the spontaneous synaptic potentials seen in coeliac ganglion cells, but did not abolish the response to distension of the colon (n = 8). When the Ca2+ concentration of the solution bathing the proximal colon was reduced to block all synaptic transmission in the enteric plexuses the background synaptic input was further depressed, but again the response to distension was little changed (n = 4). This suggests that at least some of the neurons projecting from the colon to the coeliac ganglion are first order mechanosensory neurons.

Effects of substance P on excitability and ionic currents of normal and axotomized rat dorsal root ganglion neurons

Substance P (SP) may act within dorsal root ganglia (DRG) to modulate the transmission of nociceptive information. Because peripheral nerve injury (axotomy) alters the peptide content of sensory neurons, we used whole‐cell recording to examine the effects of sciatic nerve section on the sensitivity of rat lumbar DRG neurons to SP (0.3–1 µm). At 1 µm, SP increased the excitability of ‘small’, putative nociceptive neurons but had little effect on the excitability of ‘large’ neurons. Two‐four weeks after sciatic nerve section, however, the effect of SP on ‘large’ axotomized neurons was increased and its effect on ‘small’ neurons was decreased. SP did not affect Ca2+ channel currents in control or axotomized neurons. The effects of SP on the current‐voltage (I–V) relationship of 77% of neurons involved increased inward current at potentials below −30 mV and suppressed outward current at potentials above −20 mV. The effects of SP on the I–V relationship were similar in control and in axotomized neurons and the altered sensitivity of ‘small’ and ‘large’ cells could not be attributed to axotomy‐induced changes in input resistance or membrane potential. The possible relevance of alterations in sensitivity, of ‘large’ DRG neurons to SP, to the generation of neuropathic pain is discussed.