Glycine activates myenteric neurones in adult guinea-pigs

Abstract

1. We studied the effects of glycine on myenteric neurones and muscle activity in the colon and stomach of adult guinea-pigs. 2. Intracellular recordings revealed that myenteric neurones responded to local microejection of glycine (1 mM) with a fast, transient membrane potential depolarisation (57 % of 191 colonic neurones and 26 % of 50 gastric neurones). Most glycine-sensitive neurones had ascending projections and were choline acetyltransferase immunoreactive. Glycine preferentially activated neurones with a late afterhyperpolarisation (AH-neurones) and tonic spiking neurones with fast synaptic inputs (tonic S-neurones) but less frequently phasic S-neurones and inexcitable (non-spiking) neurones. The depolarisation had a reversal potential at -19 +/- 13 mV, which was increased by 18 +/- 10 % upon lowering extracellular chloride concentration and decreased by 38 +/- 14 % in furosemide (frusemide, 2 mM). 3. Strychnine (300 nM) reversibly abolished the glycine-induced depolarisation and the Cl(-) channel blocker picrotoxin (100 microM) reduced the amplitude of the depolarisation by 55 +/- 5 %. The glycine effect was a postsynaptic response because it was not changed after nerve blockade with tetrodotoxin (1 microM) or blockade of synaptic transmission in reduced extracellular [Ca(2+)]. The effect was specific since the response was not changed by the nicotinic antagonists hexamethonium (200 microM) and mecamylamine (100 microM), the GABA(A) receptor antagonist bicuculline (10 microM), the NMDA antagonist MK-801 (20 microM) or the 5-HT(3) antagonist ICS 205930 (1 microM). 4. Glycine (1 mM) induced a tetrodotoxin- and strychnine-sensitive contractile response in the colon; the contractile response in the stomach was tetrodotoxin insensitive. 5. Glycine activated myenteric neurones in the adult enteric nervous system through strychnine-sensitive mechanisms. The glycine-evoked depolarisation was caused by Cl(-) efflux and the maintenance of relatively high intracellular chloride concentrations involved furosemide-sensitive cation-chloride co-transporters.

Figure 1. Glycine evoked a rapid, transient depolarisation in colonic myenteric neurones

A, intracellular injection of neurobiotin revealed the numerous filamentous dendrites of a tonic S-neurone. This neurone responded to a 500 ms microejection of glycine (marked by arrow) with a depolarisation, which was associated with spike discharge (B). C, neurobiotin-filled AH-neurone with multipolar morphology and smooth cell body. This neurone responded to a 500 ms microejection of glycine (marked by arrow) with a depolarisation (D).

Figure 2. Dose dependency of the glycine response and its desensitisation in colonic myenteric neurones

A, the amplitude of the depolarisation and the spike discharge increased with increased duration of glycine microejection (marked by arrows) in a tonic S-neurone. The half-maximum duration and the amplitude of the depolarisation were plotted as a function of the duration of glycine microejection (•, half-maximum duration; ○, amplitude of depolarisation). B, microejections of glycine (marked by arrows) applied at relatively short intervals induced a desensitisation of the response in a gastric myenteric neurone. Increasing the application interval (Δt) induced larger amplitude responses.

Figure 3. Glycine-evoked depolarisation was associated with a decrease in membrane resistance and reversed at potentials more positive than the resting membrane potential

A, glycine (500 ms) microejection to a tonic S-neurone evoked a depolarisation. The membrane resistance decreased as indicated by the decrease in the membrane response to constant current hyperpolarising pulses (current trace not shown). B, in a different tonic S-neurone glycine was applied while clamping the membrane potential at different levels. The amplitude of the depolarisation increased with more negative membrane potential. The results from this neurone are shown in C. The amplitude was plotted against the membrane potential and a linear fit was applied. The line intersects the x-axis at −18 mV, which is the calculated reversal potential (KCl-filled intracellular electrode). Glycine applications are marked by arrows.

Figure 4. The excitatory glycine effect on colonic myenteric neurones was a postsynaptic response

A, glycine (400 ms) microejection to an AH-neurone evoked a depolarising response in normal Krebs solution. B, neither the amplitude nor the duration of the response was changed in the presence of a solution containing reduced Ca²⁺ and high Mg²⁺ concentrations, which blocked all synaptic input. C, in a tonic S-neurone, 350 ms glycine microejection evoked a depolarising response associated with spike discharge. D, blockade of nerve conduction by the Na⁺ channel blocker tetrodotoxin (0.3 μm) did not block the response. As expected in S-neurones, the sodium-carried action potentials were blocked. Glycine applications are marked by arrows.

Figure 5. Excitatory glycine response in colonic myenteric neurones was blocked by strychnine and was picrotoxin sensitive

A, a 200 ms glycine microejection evoked a depolarising response in an AH-neurone, which was totally abolished by bath application of 600 nm strychnine (2 min into strychnine application, B) and recovered 45 min after washout of strychnine (C). D, a 200 ms glycine microejection evoked a depolarising response in a tonic S-neurone which was associated with spike discharge. This response was reduced by 100 μm picrotoxin (E) and recovered 30 min after washout (F). Glycine applications are marked by arrows.

Figure 6. The excitatory glycine effect in colonic myenteric neurones was specific and not mediated by GABA_A, NMDA, nicotinic or 5-HT₃ receptors

A, C, G and I, microejection of glycine (500, 400 and 200 ms, and 1 s) evoked an excitatory response. This response was not blocked by the GABA_A receptor antagonist bicuculline (10 μm, tonic S-neurone, B), the NMDA-receptor blocker MK-801 (20 μm, phasic S-neurone, D), the nicotinic receptor blocker hexamethonium (200 μm, tonic S-neurone, H) or the 5-HT₃ receptor antagonist ICS 205930 (1 μm, AH-neurone, J). E, a 400 ms microejection of glycine evoked a depolarisation; however, 400 ms microejection of d-serine in the same neurone did not evoke any response (AH-neurone, F). Glycine applications are marked by arrows.

Figure 7. Changes in chloride conductance were involved in the glycine-evoked depolarisation in colonic myenteric neurones

A, a 500 ms microejection of glycine evoked an excitatory response in a tonic S-neurone. B, the amplitude of the depolarisation as well as the spike discharge were increased when the extracellular chloride concentration was reduced to 12.1 mm. C, 200 ms glycine microejection evoked a depolarisation with spike discharge in a tonic S-neurone. D, the spike discharge was blocked and the depolarisation was reduced after blockade of cation-chloride transporters with 2 mm furosemide. Glycine applications are marked by arrows.

Figure 8. Glycine evoked contractile responses in colonic muscle preparations

A, addition of 1 mm glycine to the organ bath evoked a biphasic contractile response which consisted of a fast and slow onset increase in muscle tone. B, in the presence of 10 μm strychnine, both components were significantly reduced. C, the glycine response almost recovered after washout of strychnine. D, in the presence of tetrodotoxin (0.3 μm) both components were abolished, leaving a very slowly developing contraction. This indicated that most of the glycine response was neurally mediated. Application of glycine is indicated by the arrows. Glycine was continuously present after addition to the bath. The muscle recordings are all from the same preparation.