Characteristics of GABAergic and cholinergic neurons in perinuclear zone of mouse supraoptic nucleus

The perinuclear zone (PNZ) of the supraoptic nucleus (SON) contains some GABAergic and cholinergic neurons thought to innervate the SON proper. In mice expressing enhanced green fluorescent protein (eGFP) in association with glutamate decarboxylase (GAD)65 we found an abundance of GAD65-eGFP neurons in the PNZ, whereas in mice expressing GAD67-eGFP, there were few labeled PNZ neurons. In mice expressing choline acetyltransferase (ChAT)-eGFP, large, brightly fluorescent and small, dimly fluorescent ChAT-eGFP neurons were present in the PNZ. The small ChAT-eGFP and GAD65-eGFP neurons exhibited a low-threshold depolarizing potential consistent with a low-threshold spike, with little transient outward rectification. Large ChAT-eGFP neurons exhibited strong transient outward rectification and a large hyperpolarizing spike afterpotential, very similar to that of magnocellular vasopressin and oxytocin neurons. Thus the large soma and transient outward rectification of large ChAT-eGFP neurons suggest that these neurons would be difficult to distinguish from magnocellular SON neurons in dissociated preparations by these criteria. Large, but not small, ChAT-eGFP neurons were immunostained with ChAT antibody (AB144p). Reconstructed neurons revealed a few processes encroaching near and passing through the SON from all types but no clear evidence of a terminal axon arbor. Large ChAT-eGFP neurons were usually oriented vertically and had four or five dendrites with multiple branches and an axon with many collaterals and local arborizations. Small ChAT-eGFP neurons had a more restricted dendritic tree compared with parvocellular GAD65 neurons, the latter of which had long thin processes oriented mediolaterally. Thus many of the characteristics found previously in unidentified, small PNZ neurons are also found in identified GABAergic neurons and in a population of smaller ChAT-eGFP neurons.

Anionic leak currents through the Na+/monocarboxylate cotransporter SMCT1

SMCT1 is a Na-coupled cotransporter of short chain monocarboxylates, which is expressed in the apical membrane of diverse epithelia such as colon, renal cortex, and thyroid. We previously reported that SMCT1 cotransport was reduced by extracellular Cl− replacement with cyclamate− and that the protein exhibited an ostensible anionic leak current. In this paper, we have revisited the interaction between small monovalent anions and SMCT cotransport and leak currents. We found that the apparent Cl− dependence of cotransport was due to inhibition of this protein by the replacement anion cyclamate, whereas several other replacement anions function as substrates for SMCT1; a suitable replacement anion (MES−) was identified. The observed outward leak currents represented anionic influx and favored larger anions (NO3−>I−>Br−>Cl−); currents in excess of 1 μA (at +50 mV) could be observed and exhibited a quasilinear relationship with anion concentrations up to 100 mM. Application of 25 mM bicarbonate did not produce measurable leak currents. The leak current displayed outward rectification, which disappeared when external Na+ was replaced by N-methyl-d-glucamine+. More precisely, external Na+ blocked the leak current in both directions, but its Ki value rose rapidly when membrane potential became positive. Thus SMCT1 possesses a anionic leak current that becomes significant whenever external Na+ concentration is reduced. The presence of this leak current may represent a second function for SMCT1 in addition to cotransporting short chain fatty acids, and future experiments will determine whether this function serves a physiological role in tissues where SMCT1 is expressed.

AMRP Peptides Modulate a Novel K+ Current in Pleural Sensory Neurons of Aplysia

Modulation of Aplysia mechanosensory neurons is thought to underlie plasticity of defensive behaviors that are mediated by these neurons. In the past, identification of modulators that act on the sensory neurons and characterization of their actions has been instrumental in providing insight into the functional role of the sensory neurons in the defensive behaviors. Motivated by this precedent and a recent report of the presence of Aplysia Mytilusinhibitory peptide-related (AMRP) neuropeptides in the neuropile and neurons of the pleural ganglia, we sought to determine whether and how pleural sensory neurons respond to the AMRPs. In cultured pleural sensory neurons under voltage clamp, AMRPs elicited a relatively rapidly developing, then partially desensitizing, outward current. The current exhibited outward rectification; in normal 10 mM K+, it was outward at membrane potentials more positive than −80 mV but disappeared without reversing at more negative potentials. When external K+ was elevated to 100 mM, the AMRP-elicited current reversed around −25 mV; the shift in reversal potential was as expected for a current carried primarily by K+. In the high-K+ solution, the reversed current began to decrease at potentials more negative than −60 mV, creating a region of negative slope resistance in the I-V relationship. The AMRP-elicited K+ current was blocked by extremely low concentrations of 4-aminopyridine (4-AP; IC50= 1.7 × 10−7 M) but was not very sensitive to TEA. In cell-attached patches, AMRPs applied outside the patch—thus presumably through a diffusible messenger—increased the activity of a K+ channel that very likely underlies the macroscopic current. The single-channel current exhibited outward rectification, and the open probability of the channel decreased with hyperpolarization; together, these two factors accounted for the outward rectification of the macroscopic current. Submicromolar 4-AP included in the patch pipette blocked the channel by reducing its open probability without altering the single-channel current. Based on the characteristics of the AMRP-modulated K+ current, we conclude that it is a novel current that has not been previously described in Aplysia mechanosensory neurons. In addition to this current, two other AMRP-elicited currents, a slow, 4-AP-resistant outward current and a Na+-dependent inward current, were occasionally observed in the cultured sensory neurons. Responses consistent with all three currents were observed in sensory neurons in situ in intact pleural ganglia.

Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFTR

A mutation in the fifth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel (V317E) resulted in whole cell currents that exhibited marked outward rectification on expression in Xenopus oocytes. However, the single-channel unitary current ( i)-voltage ( V) relationship failed to account for the rectification of whole cell currents. In excised patches containing one to a few channels, the time-averaged single-channel current ( I)- V relationship ( I = N × P o × i, where N is the number of active channels and P o is open probability) of V317E CFTR displayed outward rectification, whereas that of wild-type CFTR was linear, indicating that the P o of V317E CFTR is voltage dependent. The decrease in P o at negative potentials was due to both a decreased burst duration and a decreased opening rate that could not be ameliorated by a 10-fold increase in ATP concentration. This behavior appears to reflect a true voltage dependence of the gating process because the P o- V relationship did not depend on the direction of Cl− movement. The results are consistent with the introduction, by a point mutation, of a novel voltage-dependent gating mode that may provide a useful tool for probing the portions of the protein that move in response to ATP-dependent gating.