Chuanyun Gao

Slow excitatory synaptic transmission mediated by P2Y1 receptors in the guinea-pig enteric nervous system.

Electrophysiological recording was used to study a type of slow excitatory postsynaptic potential (slow EPSP) that was mediated by release of ATP and its action at P2Y1 receptors on morphologically identified neurones in the submucosal plexus of guinea‐pig small intestine. MRS2179, a selective P2Y1 purinergic receptor antagonist, blocked both the slow EPSP and mimicry of the EPSP by exogenously applied ATP. Increased conductance accounted for the depolarization phase of the EPSP, which occurred exclusively in neurones with S‐type electrophysiological behaviour and uniaxonal morphology. The purinergic excitatory input to the submucosal neurones came from neighbouring neurones in the same plexus, from neurones in the myenteric plexus and from sympathetic postganglionic neurones. ATP‐mediated EPSPs occurred coincident with fast nicotinic synaptic potentials evoked by the myenteric projections and with noradrenergic IPSPs evoked by sympathetic fibres that innervated the same neurones. The P2Y1 receptor on the neurones was identified as a metabotropic receptor linked to activation of phospholipase C, synthesis of inositol 1,4,5‐trisphosphate and mobilization of Ca2+ from intracellular stores.

Immunoreactivity of Hu proteins facilitates identification of myenteric neurones in guinea-pig small intestine.

Hu proteins, together with neurone‐specific enolase (NSE), protein gene product 9.5 (PGP‐9.5), microtubule‐associated protein‐2 (MAP‐2) and tubulin beta III isoform, were evaluated immunohistochemically as neuronal markers in whole‐mount preparations and cultures obtained from the myenteric plexus of guinea‐pig small intestine. Anti‐Hu immunostaining marked the ganglion cell somas and nuclei without staining of the neuronal processes in the whole‐mounts and cultures. The ganglion cell bodies were not obscured by staining of multiple neuronal fibres and this facilitated accurate counting of the neurones. MAP2 immunostaining also provided clear images of individual neurones in both whole mounts and cultures. Immunoreactivity for NSE, PGP‐9.5 and tubulin beta III isoform provided sharp images of the ganglion cells in culture, but not in whole‐mount preparations. Strong staining of the neuronal processes in the whole‐mount preparations obscured the profiles of the ganglion cell bodies to such an extent that accurate counting of the total neuronal population was compromised. Anti‐Hu immunostaining was judged to be an acceptable method for obtaining reliable estimates of total numbers of myenteric neurones in relation to other specific histochemical properties such as histamine binding.

P2X7 receptors in the enteric nervous system of guinea-pig small intestine

The P2X7 purinergic receptor subtype has been cloned and emphasized as a prototypic P2Z receptor involved in neurotransmission in the central nervous system and ATP‐mediated lysis of macrophages in the immune system. Less is known about the neurobiology of P2X7 receptors in the enteric nervous system (ENS). We studied the distribution of the receptor with indirect immunofluorescence and used selective agonists and antagonists to analyze pharmacologic aspects of its electrophysiologic behavior as determined with intracellular “sharp” microelectrodes and patch‐clamp recording methods in neurons identified morphologically by biocytin injection in the ENS. Application of ATP or 2`‐ (or‐3`‐) O‐(4‐benzoylbenzoyl) adenosine 5`‐triphosphate (BzBzATP) activated an inward current in myenteric neurons. Brilliant blue G, a selective P2X7 antagonist, suppressed the responses to both agonists. Potency of the antagonist was greatest (smaller IC50) for the current evoked by BzBzATP. The P2X7 antagonists 1‐[N,O‐bis (1,5‐isoquinolinesulfonyl)‐N‐methyl‐l‐tyrosyl]‐4‐piperazine (KN‐62) and oxidized ATP also suppressed the BzBzATP‐activated current. Micropressure application of BzBzATP evoked rapidly activating depolarizing responses in intracellular studies with “sharp” microelectrodes. Oxidized‐ATP suppressed these responses in both myenteric and submucosal neurons. Rapidly activating depolarizing responses evoked by application of nicotinic, serotonergic 5‐HT3, or γ‐aminobutyric acid A (GABAA) receptor agonists were unaffected by brilliant blue G. Immunoreactivity for the P2X7 receptor was widely distributed surrounding ganglion cell bodies and associated with nerve fibers in both myenteric and submucous plexuses. P2X7 immunoreactivity was colocalized with synapsin and synaptophysin and surrounded ganglion cells that contained either calbindin, calretinin, neuropeptide Y, substance P, or nitric oxide synthase. The mucosa, submucosal blood vessels, and the circular muscle coat also showed P2X7 receptor immunoreactivity. J. Comp. Neurol. 440:299–310, 2001.

Histamine H3 receptor-mediated suppression of inhibitory synaptic transmission in the submucous plexus of guinea-pig small intestine.

Abstract Conventional intracellular microelectrodes and marker injection techniques were used to study the actions of histamine on inhibitory synaptic transmission in the submucous plexus of guinea-pig small intestine. Bath application of histamine (1–300 μM) reversibly suppressed both noradrenergic and non-adrenergic slow inhibitory postsynaptic potentials in a concentration-dependent manner. These effects of histamine were mimicked by the selective histamine H3 receptor agonist R(−)-α-methylhistamine but not the selective histamine H1 receptor agonist, 6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl) heptanecarboxamide (HTMT dimaleate), or the selective histamine H2 receptor agonist, dimaprit. The histamine H3 receptor antagonist, thioperamide, blocked the effects of histamine. Histamine H1 and H2 receptor antagonists did not change the action of histamine. Hyperpolarizing responses to focal application of norepinephrine or somatostatin by pressure ejection from micropipettes were unaffected by histamine and R(−)-α-methylhistamine. The results suggest that histamine acts at presynaptic histamine H3 receptors on the terminals of sympathetic postganglionic fibers and intrinsic somatostatinergic nerves in the small intestine to suppress the release of the inhibitory neurotransmitters, norepinephrine and somatostatin.

The P2Y1 purinergic receptor expressed by enteric neurones in guinea-pig intestine

Abstract  Electrophysiological recording methods provided evidence for presynaptic release of ATP from enteric neurones and postganglionic sympathetic fibres in the enteric nervous system (ENS) of guinea‐pig intestine (J Physiol Lond 2003; 550: 493–504). The released ATP acted at postsynaptic P2Y1 receptors to evoke slow synaptic excitation in neurones in the submucosal division of the ENS. Here, we report the cloning and characterization of the P2Y1 receptor, which was found in the guinea‐pig submucosal layer. A 1178 bp cDNA clone was isolated from guinea‐pig submucosal RNA by reverse transcription polymerase chain reaction (RT–PCR). The cDNA contained an open‐reading frame of 1119 bp, encoding a 373 amino acid polypeptide of the same length and with 95% identity to the human P2Y1 receptor. Stable expression of the guinea‐pig cDNA in human embryonic kidney (HEK)293 cells was accompanied by a marked increase in sensitivity for elevation of free intracellular calcium evoked by ATP or related nucleotides. The potency order for ATP and its analogues was: 2‐methio‐adenosine diphosphate > 2‐methio‐adenosine triphosphate > ADP > ATP‐γ‐S > ATP. The selective P2Y1 receptor antagonist, MRS2179, was a competitive antagonist for the receptor with a pA2 value of 6.5. The results add to existing evidence for expression of a functional P2Y1 purinergic receptor in neurones of the submucosal division of the ENS.

Functional group I metabotropic glutamate receptors in submucous plexus of guinea‐pig ileum

Intracellular recording methods and immunostaining revealed the existence of functional group I metabotropic glutamate receptors (mGluRs) in submucous plexus neurons of guinea‐pig ileum. Selective group I, but not groups II or III metabotropic glutamate receptor agonists induced concentration‐dependent, slowly‐activating depolarizing responses. Group I metabotropic glutamate receptor antagonism observed with (S)‐4‐carboxyphenylglycine (S‐4‐CPG) (100–600 μM) was competitive as determined by Schild analysis (pA2=3.81±0.02). Neither the group II and III metabotropic nor ionotropic glutamate receptor antagonists altered responses evoked by group I receptor agonists. Immunoreactivities for metabotropic glutamate 1α and 5 receptors were found to locate exclusively in neurons in the submucous plexus of guinea‐pig ileum with the highest density around the cell bodies. The results suggest that group I metabotropic glutamate receptors are functionally expressed in the submucous plexus of guinea‐pig small intestine.

Chemical coding and electrophysiology of enteric neurons expressing neurofilament 145 in guinea pig gastrointestinal tract.

Electrophysiologic recording and indirect immunofluorescence were combined to study localization of the medium‐sized neurofilament 145 (NF145) component of the cytoskeleton in morphologically identified neurons in the myenteric and submucosal plexuses of the guinea pig enteric nervous system. Neuronal localization of chemical markers, including calbindin DK28, calretinin, nitric oxide synthase, choline‐acetyltransferase, neuropeptide Y, serotonin, neurokinin 1 receptor protein, and somatostatin, was integrated with electrophysiologic and morphologic results for a more complete assessment. NF145 immunoreactivity (‐IR) was present in ganglion cells with Dogiel type I morphology in the myenteric plexus of the stomach and small and large intestine. NF145‐IR was not found in myenteric ganglion cells with Dogiel type II morphology. NF145‐IR was not present in any of the ganglion cells in the submucosal plexus. NF145 was expressed in nerve fibers in both myenteric and submucosal plexuses. The majority of these fibers were identified as sympathetic postganglionic axons based on their disappearance in organotypic culture and on their expression of tyrosine hydroxylase. The myenteric ganglion cells with NF145‐IR had electrophysiologic properties of S‐type enteric neurons. NF145‐IR was found in neurons with vasoactive intestinal peptide, serotonin, nitric oxide synthase, somatostatin, and neurokinin 1 receptor but not with neuropeptide Y or calbindin. The results in general suggest that NF145 is localized to distinct subsets of myenteric motor neurons and interneurons. Absence of NF145 from ganglion cells in the submucosal plexus is an example of differences between myenteric and submucosal components of the enteric nervous system. J. Comp. Neurol. 442:189–203, 2002.

Actions of galanin on neurotransmission in the submucous plexus of guinea pig small intestine

Electrophysiologic recording methods were used to study the actions of galanin on synaptic transmission in the submucous plexus of guinea pig ileum. Exposure to galanin resulted in concentration-dependent suppression of slow noradrenergic inhibitory postsynaptic potentials and fast nicotinic excitatory postsynaptic potentials in the majority of neurons. Failure of galanin to suppress nicotinic depolarizing responses to micropressure pulses of acetylcholine and failure to suppress hyperpolarizing responses to micropressure pulses of norepinephrine suggested that galanin acted at presynaptic inhibitory receptors to suppress release of acetylcholine and norepinephrine. Galanin suppressed slow excitatory postsynaptic potentials in eight of eight neurons with AH (after-hyperpolarization) type electrical behavior and in none of 26 neurons with S (synaptic) type electrical behavior. Suppression of excitatory neurotransmission in AH neurons was always associated with membrane hyperpolarization. Excitatory responses caused by experimentally applied substance P were also inhibited by galanin. Galanin-(1-16) and galanin-like peptide mimicked the inhibitory actions of galanin on neurotransmission. The selective galanin GAL2 receptor agonist [D-Trp(2)]galanin was inactive. The chimeric peptides, galanin-(1-13)-spantide I, galantide, galanin-(1-13)-neuropeptide Y(25-36) amide, galanin-(1-13)-bradykinin-(2-9)amide and galanin-(1-13)-Pro-Pro-Ala-Leu-Ala-Leu-Ala amide all produced varying degrees of suppression of the synaptic potentials. The evidence suggests that the galanin GAL1 receptor, but not the galanin GAL2 receptor, mediated the presynaptic and postsynaptic inhibitory actions of galanin.