Endoplasmic reticulum sorting and kinesin-1 command the targeting of axonal GABAB receptors.

In neuronal cells the intracellular trafficking machinery controls the availability of neurotransmitter receptors at the plasma membrane, which is a critical determinant of synaptic strength. Metabotropic γ amino-butyric acid (GABA) type B receptors (GABA(B)Rs) are neurotransmitter receptors that modulate synaptic transmission by mediating the slow and prolonged responses to GABA. GABA(B)Rs are obligatory heteromers constituted by two subunits, GABA(B)R1 and GABA(B)R2. GABA(B)R1a and GABA(B)R1b are the most abundant subunit variants. GABA(B)R1b is located in the somatodendritic domain whereas GABA(B)R1a is additionally targeted to the axon. Sushi domains located at the N-terminus of GABA(B)R1a constitute the only difference between both variants and are necessary and sufficient for axonal targeting. The precise targeting machinery and the organelles involved in sorting and transport have not been described. Here we demonstrate that GABA(B)Rs require the Golgi apparatus for plasma membrane delivery but that axonal sorting and targeting of GABA(B)R1a operate in a pre-Golgi compartment. In the axon GABA(B)R1a subunits are enriched in the endoplasmic reticulum (ER), and their dynamic behavior and colocalization with other secretory organelles like the ER-to-Golgi intermediate compartment (ERGIC) suggest that they employ a local secretory route. The transport of axonal GABA(B)R1a is microtubule-dependent and kinesin-1, a molecular motor of the kinesin family, determines axonal localization. Considering that progression of GABA(B)Rs through the secretory pathway is regulated by an ER retention motif our data contribute to understand the role of the axonal ER in non-canonical sorting and targeting of neurotransmitter receptors.

ATP- and ACh-induced responses in isolated cat petrosal ganglion neurons.

Chemoreceptor (glomus) cells of the carotid body are synaptically connected to the sensory nerve endings of petrosal ganglion (PG) neurons. In response to natural stimuli, the glomus cells release transmitters, which acting on the nerve terminals of petrosal neurons increases the chemosensory afferent discharge. Among several transmitter molecules present in glomus cells, acetylcholine (ACh) and adenosine 5'-triphosphate (ATP) are considered to act as excitatory transmitter in this synapse. To test if ACh and ATP play a role as excitatory transmitters in the cat CB, we recorded the electrophysiological responses from PG neurons cultured in vitro. Under voltage clamp, ATP induces a concentration-dependent inward current that partially desensitizes during 20-30 s application pulses. The ATP-induced current has a threshold near 100 nM and saturates between 20-50 muM. ACh induces a fast, inactivating inward current, with a threshold between 10-50 muM, and saturates around 1 mM. A large part of the population of PG neurons (60%) respond to both ATP and ACh. Present results support the hypothesis that ACh and ATP act as excitatory transmitters between cat glomus cells and PG neurons.

Electrophysiological characterization of nicotinic acetylcholine receptors in cat petrosal ganglion neurons in culture: effects of cytisine and its bromo derivatives.

Petrosal ganglion neurons are depolarized and fire action potentials in response to acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic acetylcholine receptors involved, although alpha4 and alpha7 subunits have been identified in petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic acetylcholine receptors containing alpha4 or alpha7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and alpha-bungarotoxin in isolated petrosal ganglion neurons. Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days. Using patch-clamp technique, we recorded whole-cell currents evoked by 5-10 s applications of acetylcholine, cytisine or its bromo derivatives. Agonists and antagonists were applied by gravity from a pipette near the neuron surface. Neurons responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast inward currents that desensitized during application of the stimuli and were reversibly blocked by 1 microM hexamethonium or 10 nM alpha-bungarotoxin. The order of potency of the agonists was 3-bromocytisine >> acetylcholine approximately = cytisine >> 5-bromocytisine, suggesting that homomeric alpha7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture.

Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms.

We tested the hypothesis that nitric oxide (NO) produced within the carotid body is a tonic inhibitor of chemoreception and determined the contribution of neuronal and endothelial nitric oxide synthase (eNOS) isoforms to the inhibitory NO effect. Accordingly, we studied the effect of NO generated from S-nitroso-N-acetylpenicillamide (SNAP) and compared the effects of the nonselective inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and the selective nNOS inhibitor 1-(2-trifluoromethylphenyl)-imidazole (TRIM) on chemosensory dose-response curves induced by nicotine and NaCN and responses to hypoxia (Po(2) approximately 30 Torr). CBs excised from pentobarbitone-anesthetized cats were perfused in vitro with Tyrode at 38 degrees C and pH 7.40, and chemosensory discharges were recorded from the carotid sinus nerve. SNAP (100 microM) reduced the responses to nicotine and NaCN. l-NAME (1 mM) enhanced the responses to nicotine and NaCN by increasing their duration, but TRIM (100 microM) only enhanced the responses to high doses of NaCN. The amplitude of the response to hypoxia was enhanced by l-NAME but not by TRIM. Our results suggest that both isoforms contribute to the NO action, but eNOS being the main source for NO in the cat CB and exerting a tonic effect upon chemoreceptor activity.