W. A. A. Kunze

INTRINSIC PRIMARY AFFERENT NEURONS OF THE INTESTINE

After a long period of inconclusive observations, the intrinsic primary afferent neurons of the intestine have been identified. The intestine is thus equipped with two groups of afferent neurons, those with cell bodies in cranial and dorsal root ganglia, and these recently identified afferent neurons with cell bodies in the wall of the intestine. The first, tentative, identification of intrinsic primary afferent neurons was by their morphology, which is type II in the terminology of Dogiel. These are multipolar neurons, with some axons that project to other nerve cells in the intestine and other axons that project to the mucosa. Definitive identification came only recently when action potentials were recorded intracellularly from Dogiel type II neurons in response to chemicals applied to the lumenal surface of the intestine and in response to tension in the muscle. These action potentials persisted after all synaptic transmission was blocked, proving the Dogiel type II neurons to be primary afferent neurons. Less direct evidence indicates that intrinsic primary afferent neurons that respond to mechanical stimulation of the mucosal lining are also Dogiel type II neurons. Electrophysiologically, the Dogiel type II neurons are referred to as AH neurons. They exhibit broad action potentials that are followed by early and late afterhyperpolarizing potentials. The intrinsic primary afferent neurons connect with each other at synapses where they transmit via slow excitatory postsynaptic potentials, that last for tens of seconds. Thus the intrinsic primary afferent neurons form self-reinforcing networks. The slow excitatory postsynaptic potentials counteract the late afterhyperpolarizing potentials, thereby increasing the period during which the cells can fire action potentials at high rates. Intrinsic primary afferent neurons transmit to second order neurons (interneurons and motor neurons) via both slow and fast excitatory postsynaptic potentials. Excitation of the intrinsic primary afferent neurons by lumenal chemicals or mechanical stimulation of the mucosa appears to be indirect, via the release of active compounds from endocrine cells in the epithelium. Stretch-induced activation of the intrinsic primary afferent neurons is at least partly dependent on tension generation in smooth muscle, that is itself sensitive to stretch. The intrinsic primary afferent neurons of the intestine are the only vertebrate primary afferent neurons so far identified with cell bodies in a peripheral organ. They are multipolar and receive synapses on their cell bodies, unlike cranial and spinal primary afferent neurons. They communicate with each other via slow excitatory synaptic potentials in self reinforcing networks and with interneurons and motor neurons via both fast and slow EPSPs.

Intracellular recording from myenteric neurons of the guinea-pig ileum that respond to stretch.

1 Isolated longitudinal muscle‐myenteric plexus preparations from guinea‐pig ileum were used to investigate the activity of myenteric neurons when the tissue was stretched in the circumferential direction. Membrane potentials were recorded via flexibly mounted intracellular recording electrodes containing Neurobiotin in 1 M KCl. The preparations were stretched to constant widths (+20 % and +40 % beyond slack width). 2 Multipolar neurons (Dogiel type II morphology) discharged spontaneous action potentials and proximal process potentials during maintained stretching, three of twenty‐one at +20 % stretch and seven of nine at +40 % stretch. At the maximum extent of stretch tried, +40 % beyond slack tissue width, action potentials in Dogiel type II neurons occurred at 10‐33 Hz. Neurons with other morphologies were all uniaxonal. Some displayed spontaneous fast EPSPs or action potentials, three of forty‐one at +20 % stretch and seven of nineteen at +40 % stretch. 3 In seven of eight Dogiel type II neurons, action potentials or proximal process potentials persisted when membrane hyperpolarization was imposed via the recording electrode. Action potential discharge was abolished by hyperpolarization in seven of nine uniaxonal neurons; the exceptions were two orally projecting neurons. 4 Dogiel type II and uniaxonal neurons were classified as rapidly accommodating if they discharged action potentials only at the beginning of a 500 ms intracellular depolarizing pulse and slowly accommodating if they discharged for more than 250 ms. For Dogiel type II neurons, three of thirteen were slowly accommodating at +20 % stretch and two of four at 40 % stretch. For uniaxonal neurons the corresponding data were twelve of twenty‐six and fifteen of nineteen neurons. The slowly accommodating state was associated with increased cell input resistance in uniaxonal neurons. 5 The spontaneous action potential discharge in Dogiel type II and uniaxonal neurons ceased when the muscle was relaxed pharmacologically by nicardipine (3 μM) or isoprenaline (1 μM), although the applied stretch was maintained. At the same time, evoked spike discharge became rapidly accommodating 6 We conclude that many Dogiel type II neurons, and possibly some orally projecting uniaxonal neurons, are intrinsic, stretch‐sensitive, primary afferent neurons that respond to muscle tension with sustained action potential discharge.

Identification of sensory nerve cells in a peripheral organ (the intestine) of a mammal

It is commonly believed that the cell bodies of mammalian sensory neurons are contained within spinal and cranial sensory ganglia associated with the central nervous system or within the central nervous system itself. However, strong circumstantial evidence implies that some sensory neurons are contained entirely within the gastrointestinal tract. We have investigated this possibility by using intracellular methods to record the responses of myenteric neurons in the guinea-pig small intestine to physiological stimuli applied to the neighbouring mucosa. The results show that the myenteric plexus contains a population of chemosensitive sensory neurons and that these neurons correspond to neurons with AH electrophysiological properties and Dogiel type II morphology. This is the first direct evidence that some sensory neurons are contained entirely within the peripheral nervous system.

The terminals of myenteric intrinsic primary afferent neurons of the guinea-pig ileum are excited by 5-hydroxytryptamine acting at 5-hydroxytryptamine-3 receptors

The aim of this study was to identify the receptor type(s) by which 5-hydroxytryptamine applied to the intestinal mucosa excites the terminals of myenteric AH neurons. The AH neurons have been identified as the intrinsic primary afferent (sensory) neurons in guinea-pig small intestine and 5-hydroxytryptamine has been identified as a possible intermediate in the sensory transduction process. Intracellular recordings were taken from AH neurons located within 1mm of intact mucosa to which 5-hydroxytryptamine was applied. Trains of action potentials and/or slow depolarizing responses were recorded in AH neurons in response to mucosal application of 5-hydroxytryptamine (10 or 20microM) or the 5-hydroxytryptamine-3 receptor agonist, 2-methyl-5-hydroxytryptamine (1 or 3mM), and to electrical stimulation of the mucosa. The 5-hydroxytryptamine-2 receptor agonist, alpha-methyl-5-hydroxytryptamine (100microM), and the 5-hydroxytryptamine-1,2,4 receptor agonist, 5-methoxytryptamine (10microM), did not elicit such responses. The 5-hydroxytryptamine-3 receptor-selective antagonist, granisetron (typically 1microM), and the 5-hydroxytryptamine-3,4 receptor antagonist, tropisetron (typically 1microM), each reduced or abolished the responses to 5-hydroxytryptamine, while the selective 5-hydroxytryptamine-4 receptor antagonist, SB 204070 (1microM), did not. It is concluded that application of 5-hydroxytryptamine to the mucosa activates a 5-hydroxytryptamine-3 receptor that triggers action potential generation in the mucosal nerve terminals of myenteric AH neurons.

Simultaneous intracellular recordings from enteric neurons reveal that myenteric AH neurons transmit via slow excitatory postsynaptic potentials.

Simultaneous intracellular electrical recordings were made from pairs of neurons separated circumferentially by 100-200 microns of the myenteric plexus of the guinea-pig ileum in vitro. The recording electrodes were filled with the dye neurobiotin which was injected into impaled nerve cells, and later revealed histochemically. Intracellular current pulses were used to evoke action potentials via the recording electrode in one type of myenteric neuron, in most cases an AH neuron, while a second electrode was used to record from a simultaneously impaled S neuron or AH neuron. AH neurons are thought to be primary sensory neurons, whereas S neurons are interneurons and motor neurons. Ninety pairs of neurons were adequately tested for interaction. From these, 17 S neurons and three AH neurons that responded to AH neuron stimulation were detected. In each case, the response was a slow depolarization that was seen only in response to a train of stimuli at 10 Hz. The slow depolarizations were enhanced by passing depolarizing current and diminished by hyperpolarization. Responses were also diminished by lowering external Ca.2+ and elevating Mg2+. In all cases in which intracellular recording indicated communication between neurons, morphological evidence of connection was seen. In no case was there communication without connection, but in four instances, morphological connections appeared to exist, although no physiological evidence of communication was obtained.

The soma and neurites of primary afferent neurons in the guinea‐pig intestine respond differentially to deformation

1 Intrinsic primary afferent neurons in the small intestine are exposed to distortion of their processes and of their cell bodies. Recordings of mechanosensitivity have previously been made from these neurons using intracellular microelectrodes, but this form of recording has not permitted detection of generator potentials from the processes, or of responses to cell body distortion. 2 We have developed a technique to record from enteric neurons in situ using patch electrodes. The mechanical stability of the patch recordings has allowed recording in cell‐attached and whole cell configuration during imposed movement of the neurons. 3 Pressing with a fine probe initiated generator potentials (14 ± 9 mV) from circumscribed regions of the neuron processes within the same myenteric ganglion, at distances from 100 to 500 μm from the cell body that was patched. Generator potentials persisted when synaptic transmission was blocked with high Mg2+, low Ca2+ solution. 4 Soma distortion, by pressing down with the whole cell recording electrode, inhibited action potential firing. Consistent with this, moderate intra‐electrode pressure (10 mbar; 1 kPa) increased the opening probability of large‐conductance (BK) potassium channels, recorded in cell‐attached mode, but suction was not effective. In outside‐out patches, suction, but not pressure, increased channel opening probability. Mechanosensitive BK channels have not been identified on other neurons. 5 The BK channels had conductances of 195 ± 25 pS. Open probability was increased by depolarization, with a half‐maximum activation at a patch potential of 20 mV and a slope factor of 10 mV. Channel activity was blocked by charybdotoxin (20 nM). 6 Stretch that increased membrane area under the electrode by 15 % was sufficient to double open probability. Similar changes in membrane area occur when the intestine changes diameter and wall tension under physiological conditions. Thus, the intestinal intrinsic primary afferent neurons are detectors of neurite distortion and of compression of the soma, these stimuli having opposite effects on neuron excitability.

Contractile activity in intestinal muscle evokes action potential discharge in guinea‐pig myenteric neurons

1 The process by which stretch of the external muscle of the intestine leads to excitation of myenteric neurons was investigated by intracellular recording from neurons in isolated longitudinal muscle‐myenteric plexus preparations from the guinea‐pig. 2 Intestinal muscle that was stretched by 40% beyond its resting size in either the longitudinal or circular direction contracted irregularly. Both multipolar, Dogiel type II, neurons and uniaxonal neurons generated action potentials in stretched tissue. Action potentials persisted when the membrane potential was hyperpolarized by passing current through the recording electrode for 10 of 14 Dogiel type II neurons and 1 of 18 uniaxonal neurons, indicating that the action potentials originated in the processes of these neurons. For the remaining four Dogiel type II and 17 uniaxonal neurons, the action potentials were abolished, suggesting that they were the result of synaptic activation of the cell bodies. 3 Neurons did not fire action potentials when the muscle was paralysed by nicardipine (3 μm), even when the preparations were simultaneously stretched by 50% beyond resting length in longitudinal and circular directions. Spontaneous action potentials were not recorded in unstretched (slack) tissue, but when the L‐type calcium channel agonist (‐)‐Bay K 8644 (1 μm) was added, the muscle contracted and action potentials were observed in Dogiel type II neurons and uniaxonal neurons. 4 The proteolytic enzyme dispase (1 mg ml−1) added to preparations that were stretched 40% beyond slack width caused the myenteric plexus to lift away from the muscle, but did not prevent muscle contraction. In the presence of dispase, the neurons ceased firing action potentials spontaneously, although action potentials could still be evoked by intracellular current pulses. After the action of dispase, (‐)‐Bay K 8644 (1 μm) contracted the muscle but did not cause neurons to fire action potentials. 5 Gadolinium ions (1 μm), which block some stretch activated ion channels, stopped muscle contraction and prevented action potential firing in tissue stretched by 40%. However, when (‐)‐Bay K 8644 (1 μm) was added in the presence of gadolinium, the muscle again contracted and action potentials were recorded from myenteric neurons. 6 Stretching the tissue 40% beyond its slack width caused action potential firing in preparations that had been extrinsically denervated and in which time had been allowed for the cut axons to degenerate. 7 The present results lead to the following hypotheses. The neural response to stretching depends on the opening of stretch activated channels in the muscle, muscle contraction in response to this opening, and mechanical communication from the contracting muscle to myenteric neurons. Distortion of sensitive sites in the processes of the neurons opens channels to initiate action potentials that are propagated to the soma, where they are recorded. Neurons are also excited indirectly by slow synaptic transmission from neurons that respond directly to distortion.

Analysis of whole‐cell currents by patch clamp of guinea‐pig myenteric neurones in intact ganglia

Whole‐cell patch‐clamp recordings taken from guinea‐pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of −47 ± 6 mV and input resistances (Rin) of 713 ± 49 MΩ at voltages ranging from −90 to −40 mV. At more negative levels, activation of a time‐independent, caesium‐sensitive, inward‐rectifier current (IKir) decreased Rin to 103 ± 10 MΩ. AH neurones had resting potentials of −57 ± 4 mV and Rin was 502 ± 27 MΩ. Rin fell to 194 ± 16 MΩ upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, Ih, and to IKir. Resting potential and Rin exhibited a low sensitivity to changes in [K+]o in both AH and S neurones. This indicates that both cells have a low background K+ permeability. The cationic current, Ih, contributed about 20 % to the resting conductance of AH neurones. It had a half‐activation voltage of −72 ± 2 mV, and a voltage sensitivity of 8.2 ± 0.7 mV per e‐fold change. Ih has relatively fast, voltage‐dependent kinetics, with on and off time constants in the range of 50–350 ms. AH neurones had a previously undescribed, low threshold, slowly inactivating, sodium‐dependent current that was poorly sensitive to TTX. In AH neurones, the post‐action‐potential slow hyperpolarizing current, IAHP, displayed large variation from cell to cell. IAHP appeared to be highly Ca2+ sensitive, since its activation with either membrane depolarization or caffeine (1 mm) was not prevented by perfusing the cell with 10 mm BAPTA. We determined the identity of the Ca2+ channels linked to IAHP. Action potentials of AH neurones that were elongated by TEA (10 mm) were similarly shortened and IAHP was suppressed with each of the three Ω‐conotoxins GVIA, MVIIA and MVIIC (0.3–0.5 μm), but not with Ω‐agatoxin IVA (0.2 μm). There was no additivity between the effects of the three conotoxins, which indicates the presence of N‐ but not of P/Q‐type Ca2+ channels. A residual Ca2+ current, resistant to all toxins, but blocked by 0.5 mm Cd2+, could not generate IAHP. This patch‐clamp study, performed on intact ganglia, demonstrates that the AH neurones of the guinea‐pig duodenum are under the control of four major currents, IAHP, Ih, an N‐type Ca2+ current and a slowly inactivating Na+ current.

Correlation of electrophysiological and morphological characteristics of myenteric neurons of the duodenum in the guinea-pig

Intracellular recording, dye filling and immunohistochemistry were used to investigate neurons of the proximal duodenum of the guinea-pig. Recordings were made from neurons of the myenteric plexus in the presence of nicardipine to quell muscle contractions, using microelectrodes that contained the marker substance Neurobiotin. Preparations were subsequently processed histochemically to reveal nerve cell shapes and immunoreactivity for calbindin, calretinin or nitric oxide synthase. Neurons were distinguished by their shapes and axonal projections as Dogiel type II, Dogiel type I, filamentous descending interneurons and small filamentous neurons. Dogiel type II cells had large cell bodies and multiple axon processes. They each had a broad action potential (mean half-width, 2.9 ms) and a prominent inflection (hump) on the falling phase of the action potential. The majority (70%) of Dogiel type II cells were AH neurons, defined by their having a prolonged hyperpolarizing potential that followed a soma action potential and lasted more than 2 s. Fast excitatory postsynaptic potentials were not recorded from Dogiel type II neurons. Two thirds of Dogiel type II neurons fired phasically in response to intracellularly injected 500 ms depolarizing current pulses and one-third fired tonically. Calbindin immunoreactivity occurred in 70% of Dogiel type II neurons. Dogiel type I neurons had lamellar dendrites and a single axon. They had brief action potentials (mean half-width, 1.7 ms) with no, or a slight hump. They responded to fibre tract stimulation with fast excitatory postsynaptic potentials. Only 2/21 exhibited a prolonged hyperpolarization following action potentials. The majority of Dogiel type I neurons thus belong to the S neuron category. Nine Dogiel type I neurons fired phasically in response to 500 ms depolarizing current pulses, while 12 fired tonically. Filamentous descending interneurons had long, branching filamentous dendrites and a single anally-projecting axon which gave rise to varicose branches in myenteric ganglia. Action potential characteristics of filamentous interneurons ranged between those of Dogiel type II and type I neurons. Small neurons. Small neurons with short filamentous, or few simple dendrites were also characterized. They had single axons, which could be traced either locally to the circular muscle, or to the longitudinal muscle. None of 12 filamentous interneurons or of 10 small filamentous neurons exhibited a prolonged post-spike hyperpolarization, whereas fast excitatory postsynaptic potentials were recorded from a majority. It is concluded that the morphological types of neuron that are encountered in the ileum also occur in the duodenum, but the electrophysiological characteristics of the neurons are more variable for each morphological class. Thus, it is not always possible to predict the morphology of myenteric neurons in the duodenum from their electrophysiological properties. Part of the electrophysiological variability appears to be due to duodenal neurons being more excitable than ileal neurons.

The circuitry of the enteric nervous system

Abstract A brief account of the aquisition of knowledge of the enteric nervous system and the ways in which technological developments have contributed to analysis of the reflex circuits is presented. The review concentrates on the motility controlling circuits in the small intestine of the guinea‐pig, where much more is known than for any other region or species. In this region, the basic circuit is known. Primary sensory neurons connect monosynaptically to motor neurons, and also make connections via chains of interneurons, which in turn provide outputs to the motor neurons. The ascending excitatory and descending inhibitory reflexes are manifested through these circuits. Sufficient details of the functions and connections of all neuron classes are available to permit activity in the reflex pathways to be realistically simulated in a computer model, which is briefly described.