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Neurotransmitter receptors are considered an important class of membrane proteins that are involved in plasticity-induced changes underlying learning and memory. Recent studies, which demonstrated that the mRNAs encoding for various receptor proteins are localized to specific dendritic domains, allude toward the possibility that these membrane bound molecules may be synthesized locally. However, direct evidence for the local axonal or dendritic synthesis and functional integration of receptor proteins in either vertebrates or invertebrates is still lacking. In this study, using an invertebrate model system we provide the first direct evidence that isolated axons (in the absence of the soma) can intrinsically synthesize and functionally integrate a membrane-bound receptor protein from an axonally injected mRNA. Surgically isolated axons from identified neurons were injected with mRNA encoding a G-protein–coupled conopressin receptor. Immunocytochemical and electrophysiological techniques were used to demonstrate functional integration of the receptor protein into the membrane of the isolated axon. Ultrastructural analysis of axonal compartments revealed polyribosomes, suggesting that some components of the protein synthesizing machinery are indeed present in these extrasomal compartments. Such axonal propensity to locally synthesize and functionally insert transmitter receptors may be instrumental in plasticity induced changes, for instance those that underlie learning and memory. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 72–81, 2000

1. In the present study we tested the ability of the general anesthetic, halothane, to affect synaptic transmission at in vivo and in vitro reconstructed peptidergic synapses between identified neurons of Lymnaea stagnalis. 2. An identified respiratory interneuron, visceral dorsal 4 (VD4), innervates a number of postsynaptic cells in the central ring ganglia of Lymnaea. Because VD4 has previously been shown to exhibit immunoreactivity for FMRFamide-related peptides, it was hypothesized that these peptides may be utilized by VD4 during synaptic transmission. In the intact, isolated CNS of Lymnaea, we have identified novel connections between VD4 and the pedal A (PeA) cells. We demonstrate that VD4 makes inhibitory connections with the PeA neurons, in particular PeA4, and that these synaptic responses are mimicked by exogenous application of FMRFamide. 3. The synaptic transmission between VD4 and the PeA cells in an intact, isolated CNS preparation was completely blocked in 2%, but not 1% halothanc. Interestingly, the postsynaptic responses (PeA) to exogenous FMRFamide were maintained in the presence of both 1 and 2% halothane. 4. To determine the specificity of the observed responses and to determine the precise synaptic site of anesthetic action, we reconstructed the VD4/PeA synapses in vitro. After isolation from their respective ganglia, both cell types extended processes and established neuritic contact. We demonstrated that not only did the presynaptic neuron reestablish the appropriate inhibitory synapses with the PeA neurons, but that the PeA cells also maintained their responsiveness to exogenous FMRFamide. 5. Superfusion of the in vitro synaptically connected VD4 and PeA cells with 2% halothane completely abolished the synaptic transmission between these cells. However, even higher concentrations of 4% halothane failed to block the responsiveness of the PeA neurons to exogenous FMRFamide. Moreover, both 1 and 2% halothane enhanced the duration of the postsynaptic response to exogenously applied FMRFamide. These data suggest that the halothane-induced depression of synaptic transmission most likely occurred at the presynaptic level. 6. This study provides the first direct evidence that peptidergic transmission in the nervous system may also be susceptible to the actions of general anesthetics. In addition, we utilized a novel approach of in vitro reconstructed synapses for studying the effects of general anesthetics on monosynaptic transmission in the absence of other synaptic influences.

1. Transmucosal electrical resistance (Rt) and short‐circuit current (Isc) were determined in rabbit isolated fundic mucosa. Under basal conditions, with a HCO(3‐)‐free HEPES‐buffered solution (pH 7.4) bathing both sides of the mucosae, Rt was 161.5 + 5.0 omega cm2 and Isc 41.8 +/‐ 1.8 microA cm‐2, and these values were not significantly different to values observed in HCO(3‐)‐buffered Krebs‐Hensleit solution. 2. The basal Isc was inhibited by the Cl‐ channel blocker diphenylamine‐2‐carboxylate, and ouabain, but unaffected by the Na+ channel blocker amiloride (10(‐5) M), consistent with electrogenic chloride secretion dependent upon a sodium gradient. Prostaglandin E2 (10(‐7) M) stimulated an increase in Isc which was susceptible to inhibition by diphenylamine‐2‐carboxylate, but not amiloride, again consistent with Cl‐ secretion. 3. Stepwise acidification of the mucosal solution to pH 2.8 resulted in an increase in Rt of 43%, as compared with that measured with mucosal pH 7.4. Isc did not change during the acidification to pH 2.8, indicating retention of tissue viability. Increased Rt while Isc remained constant is consistent with an acid‐induced decrease in the shunt (paracellular) conductance in this Cl(‐)‐secreting tissue. At pH less than 2.8, Rt declined rapidly and Isc declined and reversed, consistent with H+ back‐diffusion. Scanning electron microscopic investigation of tissue exposed to mucosal pH 2.8 revealed little difference from control (pH 7.4) tissue, but there was considerable evidence of cellular damage and membrane disruption in tissue exposed to pH 1.8. 4. Acidification of the serosal solution did not increase Rt, which was maintained until pH 3.7, and then rapidly declined at pH less than 3.7. Bilateral acidification produced a mixed response; Rt increased, as for mucosal acidification, down to pH 2.8, after which there was a rapid decline in Rt following the pattern observed for serosal acidification. 5. Compared at a mucosal pH approximately 2.8, DIDS (4 x 10(‐4) M) and amiloride (10(‐3) M) inhibited the acid‐induced increase in Rt, suggesting a role for both Cl(‐)‐HCO3‐ and Na(+)‐H+ exchange in the response. In contrast, the acid‐induced increase in Rt was unaffected by a lower concentration of amiloride (10(‐5) M), acetazolamide and ouabain. Therefore, neither Na+ channels nor a Na+ gradient appear to be involved in the acid‐induced increase in Rt.(ABSTRACT TRUNCATED AT 400 WORDS)