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Notes: Summary The goal of the present study was to elucidate the ionic mechanisms by which cholinergic stimulation induces cell shrinkage in eccrine clear cells. Dissociated Rhesus monkey eccrine sweat clear cells were prepared by collagenase digestion of freshly isolated secretory coils and immobilized on a glass slide in a perfusion chamber at 30°C. The cell was visualized by light microscopy with differential interference contract (DIC) and was recorded with a video system (15,000× total magnification). The cell volume was calculated from the maximal cross section of the cell. Methacholine (MCh)-induced cell shrinkage, which was as much as 30% of resting cell volume, was dose dependent and pharmacologically specific. MCh-induced cell shrinkage was persistent in some cells but tended to partially wane with time in others. MCh-induced cell shrinkage was dependent on the chemical potential gradient for KCl, i.e., increasing [K] in the bath ([K] o ) from 5 to 120mm caused MCh to induce cell swelling, whereas removing [Cl] o at 120mm K partially restored the MCh-induced cell shrinkage. The interpolated null [K] o (medium [K] where the cell volume did not change by MCh) of 71mm agreed with the predicted [K] o,null. MCh-induced cell shrinkage was inhibited completely by 1mm quinidine (K-channel blocker) and partially by 1mm diphenylamine-2-carboxylic acid (DPC, a Cl-channel blocker), but not by 0.1mm ouabain or 0.1mm bumetanide, suggesting that MCh-induced cell shrinkage may be due to activation of both K and Cl channels with the resultant net KCl efflux down the chemical potential gradient. That Ca/calmodulin may be involved in cholinergic regulation of Cl and K channels is suggested because 10 μm ionomycin also induced cell shrinkage, MCh failed to induce cell shrinkage in a Ca-free medium after the endogenous Ca store was depleted, and (6-aminohexyl)-5-chlorol-naphthalenesulfonamide (W-7, a putative inhibitor of calmodulin) also inhibited MCh-induced cell shrinkage in a reversible manner.

Notes: Summary Methacholine (MCh)-induced changes in intracellular concentrations of Na, K, and Cl ([Na]i, [K]i, and [Cl]i, respectively) and in cellular dry mass (a measure of cell shrinkage) were examined in isolated monkey eccrine sweat secretory coils by electron probe X-ray microanalysis using the peripheral standard method. To further confirm the occurrence of cell shrinkage during MCh stimulation, the change in cell volume of dissociated clear and dark cells were directly determined under a light microscope equipped with differential interference contrast (DIC) optics. X-ray microanalysis revealed a biphasic increase in cellular dry mass in clear cells during continuous MCh stimulation; an initial increase of dry mass to 158% (of control) followed by a plateau at 140%, which correspond to the decrease in cell volume of 37 and 29%, respectively. The latter agrees with the MCh-induced cell shrinkage of 29% in dissociated clear cells. The MCh-induced increase in dry mass in myoepithelial cells was less than half that of clear cells. During the steady state of MCh stimulation, both [K]i and [Cl]i of clear cells decreased by about 45%, whereas [Na]i increased in such a way as to maintain the sum of [Na]i+[K]i constant. There was a small (12–15mm) increse in [Na]i and a decrease in [K]i in myoepithelial cells during stimulation with MCh. Dissociated dark cells failed to significantly shrink during MCh stimulation. The decrease in [Cl]i in the face of constant [Na]i+[K]i suggests the accumulation of unknown anion(s) inside the clear cell during MCh stimulation. While the decrease in [K]i and [Cl]i may be instrumental in facilitating influx of ions via Na−K−2Cl cotransporters, the functional significance of MCh-induced cell shrinkage remains unknown.

Notes: Summary Recent studies have shown that the interstitial nucleus of Cajal (INC) in the midbrain reticular formation is involved in the conversion of vertical semicircular canal signals into eye position during vertical vestibuloocular reflexes. Secondary vestibulo-ocular relay neurons related to the vertical canals, which constitute the majority of output neurons sending signals from the vestibular nuclei directly to the oculomotor nuclei, have been shown to project axon collaterals to the region within and near the INC. To understand how the INC is involved in the signal conversion, latencies of response of neurons in the INC region to electrical stimulaton of the vestibular nerve were examined in alert cats. The responses of 96 cells whose activity was clearly modulated by sinusoidal pitch rotation (at 0.31 Hz) were analyzed. These included 41 cells whose activity was closely correlated with vertical eye movement (38 burst-tonic and 3 tonic neurons), and 55 other cells (called pitch cells as previously). Twenty nine of the 96 cells (30%) were activated at disynaptic latencies following single shock stimulation of the contralateral vestibular nerve. Disynaptically activated cells were significantly more frequent for pitch cells than for eye movement-related cells (25/55 = 45% vs 4/41 = 10%; p 〈 0.001, Chi-square test). Conversely, cells that did not receive short-latency activation (〈 6 ms) were more frequent among eye movement-related cells than pitch cells (26/41 = 63% vs 13/55 = 24%; p 〈 0.001, Chi-square test). Pitch cells showed significantly less phase lag (re head acceleration) than eye movement-related cells during sinusoidal pitch rotation (mean ± SD 124° ± 17° vs 138° ± 14°. p 〈 0.01, t-test). These results suggest that 1) cells in the INC region other than burst-tonic and tonic neurons mainly receive direct inputs from secondary vestibulo-ocular relay neurons, and that 2) vertical canal signals reach eye movement-related neurons mainly polysynaptically.

Notes: Summary 1. Maximal activation directions of vertical burst-tonic and tonic neurons in the region of the interstitial nucleus of Cajal (INC) were examined in alert cats during vertical vestibulo-ocular reflex induced by sinusoidal rotation (at 0.11 Hz±10 deg, or 0.31 Hz±5 deg) in a variety of vertical planes using a null point analysis. The results were compared with the angles of anatomical and functional planes of vertical canals reported by Blanks et al. (1972) and Robinson (1982), and with the angles of vertical eye muscles measured in this study and by Ezure and Graf (1984). 2. Maximal activation directions of 23 cells (21 burst-tonic and 2 tonic neurons) were determined from their responses during rotation in 4 or more different vertical planes. All cells showed sinusoidal gain curves and virtually constant phase values except near the null regions, suggesting that their responses were evoked primarily by canal inputs. Phase values of 5 cells near the null regions depended on the rotation plane, suggesting additional otolith inputs. We used a measurement error range of ±10 deg for calculating the maximal activation directions from the null regions of individual cells and the values of error ranges of null calculation. Of the 23, the maximal activation directions of 7 cells were outside the measurement error ranges of vertical eye muscle angles and within the ranges of vertical canal angles (class A), those of 5 cells were within the ranges of eye muscle angles and outside the ranges of vertical canal angles (class B), and those of the remaining 11 cells were in the overlapping ranges for both angles (class C). Even if only the cells in which 5 or more measurement points were taken to determine maximal activation directions (n = 15), the results were similar. During vertical rotation with the head orientation +60 deg off the pitch plane, dissociation of cell activity and vertical compensatory eye movement was observed in 5 cells in class A or C that had null angles near +45 deg. These results suggest that the cells in class A and B carried individual vertical canal and oculomotor signals, respectively, although it is difficult to tell for the majority of cells (class C) which signals they reflected. Some cells in class A and C were antidromically activated from the medial longitudinal fasciculus at the level of abducens nucleus, suggesting that the signals carried by these cells may be sent to the lower brainstem. 3. Most burst-tonic neurons did not respond to horizontal rotation; significant responses were obtained in only 3 of 10 cells tested for which the gain was only 14–17% of their maximal vertical gain. There was no clear difference in gain or phase values of the responses to vertical rotation, or in eye position sensitivity (during spontaneous saccades) between cells whose responses coincided with individual vertical canal angles and those matching the angles of vertical recti muscles. The values of phase lag (re head acceleration during pitch rotation) and eye position sensitivity of these cells are still smaller compared to those of extraocular motoneurons reported by Delgado-Garcia et al. (1986), although they were larger than those of secondary vestibulo-ocular neurons (Perlmutter et al. 1988). All these results suggest that the signals carried by burst-tonic and tonic neurons in the INC region are different from oculomotor signals. 4. Similar analysis was done for comparison for 19 other cells that did not show close correlation with spontaneous eye movement but whose activity was clearly modulated by pitch rotation (pitch cells). More than a half (10/19) had maximal activation directions outside the measurement error ranges of individual vertical canal angles, and many shifted towards roll. Horizontal rotation produced responses with higher gain than burst-tonic neurons, suggesting a difference in the spatial response properties of burst-tonic and tonic neurons on one hand and pitch cells on the other.