Ionic currents and current-clamp depolarisations of type I and type II hair cells from the developing rat utricle.

Ionic currents and the voltage response to injected currents were studied in an acutely dissected preparation of the rat utricle between birth and postnatal day 12 (PN12). Based upon morphological criteria, the sensory cells examined were divided into two classes, "type I" and "type 2 category," the latter of which may include some immature type I cells. The former group comprises a clearly defined electrophysiological population, with one large outwardly rectifying potassium conductance that is sensitive to 4-aminopyridine (4-AP), insensitive to tetraethylammonium (TEA) and displays voltage-dependent activation kinetics. In the absence of enzymatic dissociation procedures, and with the epithelium left largely intact, the mean half activation of this conductance was -30.3 mV at PN3, and -37.5 mV at PN12. At both stages it was almost entirely turned off at -74 mV. Omission of ATP from the intracellular solution appeared to prevent rundown of this conductance. Type II category hair cells formed a more heterogeneous population, exhibiting a distinct TEA-sensitive delayed rectifier potassium conductance; the rapidly activating and inactivating IA; an inward rectifier; and inward sodium currents at around PN3. Both cell types depolarised strongly in response to injected currents, with time courses reflecting the activation kinetics of their major outward conductances.

Examination of the cupula and stereocilia of the horizontal semicircular canal in the toadfish Opsanus tau.

We imaged the horizontal semicircular canal (HSCC) crista and cupula of toadfish, Opsanus tau, by using a) confocal light microscopy of isolated vital HSCC; b) serial sections of fixed, trichrome-stained HSCC; and c) scanning electron microscopy of fixed HSCCs. HSCC were dissections which included an ampulla and an attached canal tube (long and slender canal portion), and, in some cases, a small portion of the utricular wall. Cupulae were seen as multipartite mucus connective tissue shells rising from the crista and extending toward the ampullary roof. They were composed of several refractile bands traversing the cupulae perpendicular to longitudinal fibers extending from the cupular base to its apex. Alcian green-stained cupulae showed an asymmetric alcianphilic, dark, X-shaped structure, indicating that the pillar is rich in mucin and carbohydrate, an interpretation supported by images of trichrome-stained sections. The cupular antrum is devoid of prominent refractile fibers. No tubes or channels were observed in the cupula or antrum of vital preparations. Cupular shell fibers cover the surface of the crista, are roughly parallel, and are associated with a translucent material having a refractive index greater than the surrounding endolymph. Stereocilia were thin, 100-microm-long structures, with little longitudinal curvature, which end with no end bulb. No strands extend from stereocilia to the roof or other portions of the cupular antrum. Gross movements of stereocilia were not seen in mechanically quiescent preparations. Within the cupular antrum, stereocilia were parallel to connective tissue fibers, all embedded in an isotropic gel. This fiber-reinforced gel and cupular matrix are sensitive to N-acetlyneuraminidase and beta-N-acetyl glucosaminidase, and minimally sensitive to beta-N-acetyl hexosaminidase. Connective tissue fibers may serve to stiffen the gel, whose matrix would restrict lateral motion of embedded fibers and stereocilia thereby providing mechanical support for stereocilia.

Electrophysiology and pharmacology of outward potassium currents in semicircular canal hair cells of toadfish, Opsanus tau.

Outward currents from hair cells from the horizontal semicircular canal (HSCC) of the toadfish were investigated using whole cell patch clamp methods. Two classes of hair cells are found. One class (approx. 10% of cells) showed only a non-inactivating current (IKCa) which was blocked by 2 mM TEA. A second class had both inactivating and non-inactivating currents. The former (IA) was blocked by 4-AP (1 mM) and the latter (IKCa) by TEA (2-20 mM) . While the majority of the cells expressed both these outward currents, due to IA inactivation both currents are functionally present in the same cell only between -60 and -40 mV. At more depolarized membrane potentials, IA was inactivated, suggesting that a single hair cell may have two distinct signalling modes, one dominated by IA at more hyperpolarized membrane potentials and the other by IKCa at depolarized values where ICa is beginning to grow, increasing both amplitude and activation rate of IKCa. The switch between modes will be determined by the amplitude and frequency characteristics of the stimulus and possibly also by actions of efferent transmitters. In current clamp mode, 10% of the HSCC hair cells showed high Q and high frequency resonance, from 44 to 360 Hz at 12 degrees C. These cells expressed only one outward calcium dependent, non-inactivating, TEA sensitive current, characteristic of IKCa. A suggested role for high frequency resonance is as positive feedback to produce a high frequency updating of the stereociliary compliance to most faithfully transduce angular acceleration.

Voltage-gated potassium current and resonance in the toadfish saccular hair cell.

Resonance of the membrane potential in response to a perturbing current has been demonstrated in sensory hair cells of many acoustico-lateralis systems and modelled as the result of the interaction of passive membrane properties and the magnitude and kinetics of activation and deactivation of an outward calcium-activated potassium current (IKCa) and an inward calcium current (ICa). However, the majority of the hair cells of the toadfish saccule have, in addition to IKCa, a voltage-gated potassium current (IK) active in the same membrane potential range as IKCa but with considerably slower activation and deactivation kinetics. Additionally, some of these cells have an A current (IA). In the present work, the resonance of cells with these three outward potassium currents were compared with those from cells containing only IKCa. Hair cells with only IKCa produced a high-quality factor (Q) resonance with symmetrical ringing at current onset and termination. In many cells having the IK, resonance could be evoked as a high Q ringing only at the onset of the current pulse. The resonance at command onset was dependent on the presence of IKCa and could be converted into a spike by blocking the IKCa with TEA. Some hair cells with IKCa and IK produced spikes rather than resonance at all holding potentials tested. This spiking was seen in cells with low levels of IKCa or slowly activating IKCa and with cells with IA. The presence of cells with such different response modes implies a difference between hair cells in their role in sensory coding.

Characterization of voltage-gated and calcium-activated potassium currents in toadfish saccular hair cells.

Patch clamp methods were used to study calcium activated (IKCa) and voltage-gated (IK) potassium currents in enzymatically disassociated hair cells from the saccule of the toadfish Opsanus tau. In one population of hair cells, tetraethylammonium bromide (TEA) blocked all outward current, leaving only an inward calcium current (ICa). This current blocked by TEA was also blocked by barium (5 mM) and cadmium (0.2 mM) but only partially blocked by zero external calcium. In the majority of the cells, after TEA (25 mM) was used to block IKCa, a second outward current remained. This current was resistant to block by apamin, barium (5 mM) and cadmium (0.2 mM). Its kinetics of activation and deactivation were considerably slower than those of IKCa. Because of the current/voltage characteristics, its resistance to block by the above agents and voltage-gated activation, this current was termed IK. Study of the rates of activation and deactivation of the two currents in hair cells exhibiting either fast or slow total outward current activation showed that these two kinetic parameters were linked in a cell, i.e., cells with fast IKCa kinetics exhibit faster IKCa kinetics than cells with slower IKCa kinetics. Cell attached and inside out recordings showed a high conductance channel with short open times and a lower conductance channel with longer open times active over the same voltage ranges as those seen in whole cell recordings. Since these two currents with quite different but linked kinetics are active over the same voltage range, their co-existence may be of some importance to sensory coding in the hair cells.

Acetylcholine receptor channel gating and conductance involve extracellular disulfide bond(s).

Disulfide bonds are critical determinants of the function of the acetylcholine receptor at the vertebrate neuromuscular junction. In the present study, the role of these bonds in acetylcholine receptor channel gating and conductance was investigated at the single channel level. Disulfide bond reducing agents decreased the single channel conductance of both ligand-gated and spontaneously opening acetylcholine receptor channels, indicating that the observed decrease in conductance is not due to blockade of the channel lumen by agonist molecules. In addition, the reducing agents increased the opening frequency of both liganded and unliganded acetylcholine receptor channels. Use of inside-out patches and both membrane permeant and impermeant reducing agents demonstrated that the disulfide bonds involved are all extracellular. These findings indicate that both channel gating and conductance involve conformational changes in extracellular regions of the acetylcholine receptor.

Toadfish saccular hair cell bundle has a preferred orientation in the otolithic membrane.

The macula of the saccule of the toadfish, Opsanus tau, is covered by an otolithic membrane containing sockets into which the stereocilia and kinocilia of the hair cells project. We have found that the hair cell bundle has a distinct eccentric orientation within this space of the otolithic socket. Although the sockets of the otolithic membrane are irregular in shape, all kinocilia are located closet to the same border of the sockets. Since the socket is a fluid or gel filled space through which the hair cell bundle moves, this orientation may have some significance for transduction since it leaves a larger space in the on direction for stereociliary movement.

Acetylcholine modulated potassium channel in the hair cell of the toadfish saccule.

Sensory transduction in the acoustic-lateralis system is modulated by an efferent synapse on the sensory hair cells and afferent fibers. Acetylcholine has been implicated as a neuro-transmitter at this synapse. This work addresses the ionic mechanism of action of acetylcholine on the hair cell of the toadfish saccule using path clamp methods and single channel recording. Acetylcholine and oxotremorine, a muscarinic agonist, were found to increase the open time and opening rate of a high conductance K+ channel in the basolateral walls of the hair cell. This effect is not dependent on the presence of Ca2+ in the extracellular media. The involvement of an intracellular mediator is implicated since bath applied agonist opens K+ channels isolated under the recording pipette.

Further kinetic analysis of the chemically modified acetylcholine receptor.

The acethylcholine receptor was chemically modified using bisulfite to add a sulfonate group to a disulfide bond on the alpha subunit, and diamide, an oxidizing agent, to form an interchain disulfide bond between beta subunits of adjacent receptors. In previous work, both reagents increased mepc decay times but produced no change in mean channel open time or conductance as measured by spectral analysis of endplate current fluctuations (Steinacker and Zuazaga 1981). In the current work, we show that, while both chemical modifications increase the decay time of the miniature endplate current, only sulfonation increases the time to peak. Sulfonation also produced an effect on voltage jump current relaxation time, which parallels the increase in miniature endplate current decay time, and an increase in the ratio of the current relaxation amplitudes. Diamide had no effect on voltage jump current relaxation amplitudes or time constants. These data are analyzed in an attempt to correlate changes in specific rate constants to changes in the macroscopic current measurements.

Presynaptic effects of sodium bisulfite at the frog neuromuscular junction.

Both spontaneous and evoked transmitter release from the frog neuromuscular junction can be modified by application of sodium bisulfite, a reagent specific for disulfide bonds. An increase in miniature endplate frequency is produced that is not dependent on external calcium, sodium, or presynaptic terminal depolarization. The increased release can be halted by application of the sulfhydryl oxidizing agent DTNB. The response of bisulfite can be prevented by prior treatment of the endplate with acetylcholine or an anticholinesterase. It is concluded that bisulfite produces its effects by acting on a protein in the presynaptic membrane that is involved in regulation of transmitter release.

Changes in neuromuscular junction endplate current time constants produced by sulfhydryl reagents.

The acetylcholine receptor is a protein that contains certain critical disulfide bonds. Experiments were designed to determine the role such bonds might play in the physiological activity of the receptor. Modification of the receptor with sodium bisulfite and diamide produced an increase in the time constants of the miniature endplate current without changes in the single-channel properties of the receptor. Controls were done to determine that this change in the miniature endplate current was not due to an effect on acetylcholinesterase at the endplate. These data are interpreted to mean that the reagents increase the time acetylcholine is bound to the receptor before the channel opens and is most probably due to a change in receptor affinity brought about by chemical modification of the receptor protein.

Staining PO2 measurement sites in the rat brain cortex and quantitative morphometry of the surrounding capillaries.

The exact positions of microelectrodes used to measure the PO2 in the cerebral cortex of the rat were determined by staining the tissue with Alcian Blue. The measurement sites were subsequently located under a light microscope and correlated with the capillary and cellular arrangement of the cortex. The microelectrodes used for the PO2 measurements were made of gold glass fibers; the Alcian Blue was injected hydrostatically through a micropipette attached to the PO2 microelectrode. The sites where dye had been deposited were seen under a light microscope as green blue spots about 100 micrometers in diameter. The capillaries were visualized by silver nitrate perfusion. Differences between the local PO2 values in the neo- and the archeocortex were found. In the neocortex the mean PO2 was 31 mm Hg, capillary volume 1.6%, capillary surface area 980/mm2, capillary length 13.5/mm; whereas in the archeocortex these values where 21 mm Hg, 0.9%, 820/mm2 and 9.4/mm respectively. These data indicate a relationship between the microcirculatory transport system and the local oxygen tension and provide further evidence that the mean PO2 level tends to decrease when moving from the surface into the archeocortex.

The anatomy of the decapod crustacean auxiliary heart.

The anatomy of an auxiliary heart found in many decapod crustaceans is described. This heart is found at the anterior end of the dorsal median artery before the artery divides to supply the cerebral nervous system. The heart is essentially two strips of modified somatic muscle located inside a sinus in the dorsal median artery. These muscles are innervated by four motoneurons located in the supraesophageal ganglion. Sensory innervation and possible neurosecretory elements are also described.