We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes.

Horseradish peroxidase (HRP) was injected into the optic radiations of adult cats. With placements close to the lateral geniculate nucleus (LGN), the enzyme diffused retrogradely along the axons of geniculocortical relay neurons, entered their cell bodies, and, after reaction with diaminobenzidine, produced a Golgi-like staining of entire neurons. When the injections were made close to the visual cortex, the enzyme diffused anterogradely and filled complete axonal arborizations in area 17.In the LGN, examples of type 1 and type 2 relay neurons (Guillery, '66) were reconstructed, and their axon diameters measured. The type 1 neurons (thought to correspond to Y-cells -LeVay and Ferster, '77) possessed large diameter axons (2-3.3 pm), while the type ' 2 neurons (thought to be X-cells) had medium-sized axons (1-1.7 pm). Both types of neuron gave off axon collaterals to the perigeniculate nucleus.In the cortex, two types of afferent supplied layer IV. One distributed to the upper part of the layer (layer IVab), extending a short distance into layer 111. The parent trunks of these axons, measured in the white matter, had diameters matching those of type 1 LGN relay cells. The other type distributed to layer IVc. The diameters of these axons matched those of type 2 LGN relay cells.Most afferents of both types gave off collaterals to layer VI -there were no axons which exclusively innervated this layer.The axons supplying layer IVab had a wide lateral spread in the cortex (up to 2 mm), and the boutons were grouped into two to five clumps, whose size and arrangement were similar to ocular dominance columns. The axons supplying layer IVc had a much more restricted arborization, usually consisting of a single clump of boutons.LGN neurons with very fine axons (less than 1 pm) were found in laminae Cl-C3. They probably corresponded to Guillery's type 4 neurons. In the cortex, fine-diameter axons arborized in the upper half of layer I. These axons sometimes had collaterals in the lower part of layer I11 and in layer V.Taken together, the arborizations of the cortical afferents observed in the present study account fully for the autoradiographic labelling pattern seen after 3H-proline injections into the LGN (LeVay and Gilbert, '76).The identification of type 1 and type 2 neurons as Y-and X-cells is strengthened by the observed difference in their axon diameters, in agreement with the different axonal conduction velocities reported for Y-and X-cells. The presence of cells with very fine axons in the deeper C laminae is consistent with the reported presence of W-cells (which have slowly conducting axons) in these layers. We conclude that the different classes of geniculate relay neuron have different laminar projections in area 17.

The possible role of Ca ions in mediating the drop insensitivity associated with light adaptation in Limulus ventral photoreceptors was assessed by simultaneously measuring the sensitivity to light and the intracellular free Ca concentration (Ca i) ; the latter was measured by using Ca-selective microelectrodes . In dark-adapted photoreceptors, the mean resting Cai was 3 .5 ± 2 .5,M SD (n = 31) . No correlation was found between resting Cai and absolute sensitivity from cell to cell . Typically, photoreceptors are not uniformly sensitive to light ; the Cai rise evoked by uniform illumination was 20-40 times larger and faster in the most sensitive region of the cell (the rhabdomeral lobe) than it was away from it . In response to a brief flash, the Cai rise was barely detectable when 10 2 photons were absorbed, and it was saturated when^-10 5 photons were absorbed . During maintained illumination, starting near the threshold of light adaptation, steady Cai increases were associated with steady desensitizations over several log units of light intensity : a 100-fold desensitization was associated with a 2 .5-fold increase in Ca ; . After a bright flash, sensitivity and Cai recovered with different time courses : the cell was still desensitized by -0 .5 log units when Ca i had already recovered to the prestimulus level, which suggests that under those conditions Cai is not the rate-limiting step of dark adaptation . Ionophoretic injection of EGTA markedly decreased the light-induced Cai rise and increased the time to peak of the light response, but did not alter the resting Ca;, which suggests that the time to peak is affected by a change in the capacity to bind Ca2' and not by resting Ca i . Lowering the extracellular Ca 21 concentration (Ca .) first decreased Cai and increased sensitivity. Longer exposure to low Ca. resulted in a further decrease of Ca ; but decreased rather than increased Address reprint requests to Dr.

Drosophila transient receptor potential (TRP) is a prototypical member of a novel family of channel proteins underlying phosphoinositide-mediated Ca(2+) entry. Although the initial stages of this signaling cascade are well known, downstream events leading to the opening of the TRP channels are still obscure. In the present study we applied patch-clamp whole-cell recordings and measurements of Ca(2+) concentration by ion-selective microelectrodes in eyes of normal and mutant Drosophila to isolate the TRP and TRP-like (TRPL)-dependent currents. We report that anoxia rapidly and reversibly depolarizes the photoreceptors and induces Ca(2+) influx into these cells in the dark. We further show that openings of the light-sensitive channels, which mediate these effects, can be obtained by mitochondrial uncouplers or by depletion of ATP in photoreceptor cells, whereas the effects of illumination and all forms of metabolic stress were additive. Effects similar to those found in wild-type flies were also found in mutants with strong defects in rhodopsin, Gq-protein, or phospholipase C, thus indicating that the metabolic stress operates at a late stage of the phototransduction cascade. Genetic elimination of both TRP and TRPL channels prevented the effects of anoxia, mitochondrial uncouplers, and depletion of ATP, thus demonstrating that the TRP and TRPL channels are specific targets of metabolic stress. These results shed new light on the properties of the TRP and TRPL channels by showing that a constitutive ATP-dependent process is required to keep these channels closed in the dark, a requirement that would make them sensitive to metabolic stress.

The Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca 2+ influx sarco/ER Ca 2+ -ATPase inhibitor thapsigargin (THP). Baseline ER Ca 2+ levels measured with the ER Ca 2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca 2+ influx and decreased intracellular Ca 2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca 2+ entry regulators, with no alterations in ER Ca 2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons.