Myenteric neurons of the mouse small intestine undergo significant electrophysiological and morphological changes during postnatal development

Abstract

Organized motility patterns in the gut depend on circuitry within the enteric nervous system (ENS), but little is known about the development of electrophysiological properties and synapses within the ENS. We examined the electrophysiology and morphology of myenteric neurons in the mouse duodenum at three developmental stages: postnatal day (P)0, P10–11, and adult. Like adults, two main classes of neurons could be identified at P0 and P10–11 based on morphology: neurons with multiple long processes that projected circumferentially (Dogiel type II morphology) and neurons with a single long process. However, postnatal Dogiel type II neurons differed in several electrophysiological properties from adult Dogiel type II neurons. P0 and P10–11 Dogiel type II neurons exhibited very prominent Ca(2+)-mediated after depolarizing potentials (ADPs) following action potentials compared to adult neurons. Adult Dogiel type II neurons are characterized by the presence of a prolonged after hyperpolarizing potential (AHP), but AHPs were very rarely observed at P0. The projection lengths of the long processes of Dogiel type II neurons were mature by P10–11. Uniaxonal neurons in adults typically have fast excitatory postsynaptic potentials (fEPSPs, ‘S-type' electrophysiology) mainly mediated by nicotinic receptors. Nicotinic-fEPSPs were also recorded from neurons with a single long process at P0 and P10–11. However, these neurons underwent major developmental changes in morphology, from predominantly filamentous neurites at birth to lamellar dendrites in mature mice. Unlike Dogiel type II neurons, the projection lengths of neurons with a single long process matured after P10–11. Slow EPSPs were rarely observed in P0/P10–11 neurons. This work shows that, although functional synapses are present and two classes of neurons can be distinguished electrophysiologically and morphologically at P0, major changes in electrophysiological properties and morphology occur during the postnatal development of the ENS.

Figure 1. Morphology of P0 and P10–11 myenteric neurons revealed by the injection of biocytin through the recording electrode

A, neurons with smooth cell bodies and multiple processes (DII morphology) from the duodenum of P0 (inset) and P10–11 mice. The processes of some P10–11 DII neurons projected around almost the entire circumference of the gut. Neurons with DII morphology had prolonged AHPs (see also Fig. 3A and B). B, the circumferential projection lengths of DII neurons (grey bars, LHS y-axis). DIIs had reached their mature projection lengths by P10. The circumference of the duodenum (dotted line and RHS y-axis) also increased during development, but between P0 and P10–11, the projections of DII neurons grew faster than the increase in gut circumference. C, the longitudinal projection lengths of neurons with a single long process (bars, LHS y-axis). The projection lengths increased during development. The small intestine also grew significantly in length (dashed line, RHS y-axis). However, the increases in projection lengths of these neurons did not closely parallel the increase in length of the small intestine. D, traces of long orally and anally projecting neurons at P0. The orally projecting neuron shown was the longest orally projecting neuron we encountered at P0. P0 and P10–11 neurons with a single long process were found to display S-type electrophysiology.

Figure 2. Developmental changes in the morphology of biocytin-filled neurons with a single long process

Surface rendering of projected confocal micrographs of neurons with a single long process (indicated by arrow) from the duodenum of P0, P10–11 and adult mice. At P0 and P10–11, neurons possessed only filamentous neurites (open arrow heads) (A and E), both filamentous (open arrow heads) and lamellar neurites (arrow heads) (B, D and F), or rarely, only lamellar neurites (arrow heads) (C and G). Compared to the prominent lamellar dendrites seen on adult neurons (H–K), the lamellar neurites of P0 neurons (arrow heads) were only small bumps on the soma. L, quantification of the proportions of neurons with a single long process with different morphologies. The proportion of neurons possessing only lamellar neurites increased from P0 to adult.

Figure 3. Correlation of electrophysiological properties and morphology of postnatal neurons: DII neurons with prolonged AHPs and S-neurons with a single long process were present by P0

Only one P0 neuron (A), but a group of neurons at P10–11 (B) had a prolonged AHP following a train of action potentials evoked by a 500 ms depolarizing step current, and they commonly exhibited an I_h-induced ‘sag’ in the membrane potential (arrows) response to a 500 ms hyperpolarizing pulse and anode break action potentials. These characteristics are typical of neurons exhibiting AH-type electrophysiology in the adult ENS. Neurons with prolonged AHPs at P0 and P10–11 displayed DII morphology (inverted confocal micrographs RHS). Another subgroup of neurons at P0 (C) and P10–11 (D) exhibited S-type electrophysiological properties. These neurons did not have a long AHP following action potentials, and rarely exhibited anode break action potentials and I_h-induced sag in the voltage (arrows) in response to prolonged hyperpolarizing pulse stimulus. Neurons with S-type electrophysiology at P0 and P10–11 only had one long process (inverted confocal micrographs RHS). All insets are enlarged representations of the electrophysiological traces. They reveal the action potentials evoked at the beginning of the depolarization step current stimulus.

Figure 4. DII neurons in the duodenum exhibit an ADP that is very prominent at early postnatal ages

At P10–11, prominent ADPs (arrows) were observed following action potentials that were evoked by a 500 ms long depolarizing step current (A), at the end of a 500 ms long hyperpolarizing step current (D), or stimulation of an internodal strand (single pulse, 1.8 mA) (B–C). ADPs were only exhibited by neurons with DII morphology. Small action potentials can be evoked at the rising phase of an ADP. These ADPs (arrow) were associated with a decrease in input resistance (C), and have a reversal potential of −41 mV (E). F, adult duodenal AH-type neurons also displayed an ADP (arrow) following an action potential evoked by a single pulse stimulus on an internodal strand. However adult ADPs were significantly smaller than the ADPs recorded from P10–11 neurons. All insets are enlarged representations of the electrophysiological traces and reveal the action potentials evoked at the beginning of a stimulus, as well as the initial stage of an ADP.

Figure 5. Pharmacological analysis of electrically stimulated responses recorded from P10–11 neurons

A–C, preliminary pharmacological analysis of fEPSPs recorded from P10–11 S-type neurons (n = 3). A, S-type neurons displayed a fEPSP complex in response to a single pulse stimulus on an internodal strand. This was often accompanied by an array of non-stimulus-locked fEPSPs. B, The non-stimulus-locked fEPSPs were reduced (n = 2) or abolished (n = 1) by hexamethonium (nicotinic blocker, 200 μm). The duration, but not the amplitude, of the stimulus-locked fEPSPs was reduced by hexamethonium (n = 3). C, the effects of hexamethonium were reversible with wash-out. D, AH-type neurons exhibited a prominent ADP following an action potential evoked by a single pulse stimulus on an internodal strand. E, the ADP was completely abolished by cadmium chloride (Ca²⁺ channel blocker, 100 μm; n = 6). F, cadmium chloride also abolished the long AHP displayed by some of these neurons (n = 4) causing them to convert from a phasic pattern of action potential firing to tonic firing (firing action potentials throughout the length of a 500 ms depolarizing step current). All insets are enlarged representations of the electrophysiological traces and reveal the stimulus-evoked fast EPSPs (A–C), or action potentials evoked at the beginning of a stimulus, and also the initial stage of an ADP (D–E).