Carbonic anhydrase and neuronal enzymes in cultured glomus cells of the carotid body of the rat

Summary The cellular localization of carbonic anhydrase (CAH) in the carotid body of the rat was investigated by means of Hansson's cobalt-precipitation technique in cultures of dissociated cells. In both young (2-day-old) and old (77-day-old) cultures, the parenchymal glomus (type-I) cells were selectively stained by this technique, and in addition expressed tyrosine hydroxylase and neuron-specific enolase as revealed by immunofluorescence. Enzymic reaction product of CAH appeared to be predominantly intracellular since staining was more intense and occurred more rapidly following permeabilization of the cell membranes with Triton X-100; its formation was inhibited by the CAH-inhibitor acetazolamide (1–10 M) or by increasing the pH from 5.8 to 7.5. Cryostat sections of the carotid bifurcation revealed intense CAH-reaction product in cell clusters of the carotid body, in a few cells of the nodose ganglion, and in red blood cells. Neuronal cell bodies of the petrosal ganglion and superior cervical ganglion (SCG) were largely non-reactive. The SCG is known to contain clusters of small intensely fluorescent (SIF) cells, which were also non-reactive when grown in dissociated cell culture. Thus, although glomus and SIF cells are often considered to be similar cell types, functional CAH-activity appears unique to glomus cells, and this may be important for the physiological response of the carotid body to certain chemosensory stimuli.     

An ultrastructural study of the carotid body of horse and dog

Summary Carotid body tissue from horse and dog has been investigated ultrastructurally. Several cell types are recognized: glomus cells which are regarded as chemoreceptors, sustentacular cells which enclose the glomus cells, and nerve fibers.The glomus cells contain electron dense granules which are interpreted as packages of biogenic monoamines. There are both dark and light glomus cells, the former containing more granules and ribosomes. Invaginations of the plasma membranes as well as free coated vesicles are often seen in the cytoplasm of glomus cells. Nerves within the glomus lobules are generally wrapped by sustentacular cells, but nerve endings are also seen in close contact with the glomus cells. Some endings contain synaptic vesicles as well as a great concentration of mitochondria. The corresponding fibers are thought to be efferent. Another type of contact is interpreted as en passant synapses of afferent fibers.The author wishes to express his gratitude to Professor L. Nicander who initiated this project and took most of the micrographs and to Professor Nils Obel and associate Professor Gustav Björk at the Royal Veterinary College for their valuable help with the surgical procedure and to Dr. Martin Ritzén of the Royal Medical College for making the tests for biogenic monoamines.     

大鼠颈动脉体球细胞缺氧时膜电位变化与球细胞大小的关系

用细胞内记录法测定了８５个分离培养的大鼠颈动脉体球细胞的膜电位,并由显微照相法记录球细胞的形态进而以测微器测量球细胞的直径。由连二亚硫酸钠（Ｎａ２Ｓ２Ｏ４）造成缺氧（ＰＯ２,１．３－８．０ｋＰａ）。不同直径的球细胞对缺氧有两种不同反应：直径为８．０４±１．０９μｍ的球细胞对缺氧的反应均为去极化,直径为１４．３８±４．２１μｍ的球细胞对缺氧反应为超极化。因此似可认为,球细胞存在功能不同的亚型。缺氧程度不同对球细胞膜电位的改变也有一定影响,缺氧程度严重可使小型球细胞的去极化程度增加,但缺氧程度的高低不能改变两型球细胞对缺氧反应的固有型式。     

Carotid body chemosensory responses in mice deficient of TASK channels

Background K⁺ channels of the TASK family are believed to participate in sensory transduction by chemoreceptor (glomus) cells of the carotid body (CB). However, studies on the systemic CB-mediated ventilatory response to hypoxia and hypercapnia in TASK1- and/or TASK3-deficient mice have yielded conflicting results. We have characterized the glomus cell phenotype of TASK-null mice and studied the responses of individual cells to hypoxia and other chemical stimuli. CB morphology and glomus cell size were normal in wild-type as well as in TASK1^−/− or double TASK1/3^−/− mice. Patch-clamped TASK1/3-null glomus cells had significantly higher membrane resistance and less hyperpolarized resting potential than their wild-type counterpart. These electrical parameters were practically normal in TASK1^−/− cells. Sensitivity of background currents to changes of extracellular pH was drastically diminished in TASK1/3-null cells. In contrast with these observations, responsiveness to hypoxia or hypercapnia of either TASK1^−/− or double TASK1/3^−/− cells, as estimated by the amperometric measurement of catecholamine release, was apparently normal. TASK1/3 knockout cells showed an enhanced secretory rate in basal (normoxic) conditions compatible with their increased excitability. Responsiveness to hypoxia of TASK1/3-null cells was maintained after pharmacological blockade of maxi-K⁺ channels. These data in the TASK-null mouse model indicate that TASK3 channels contribute to the background K⁺ current in glomus cells and to their sensitivity to external pH. They also suggest that, although TASK1 channels might be dispensable for O₂/CO₂ sensing in mouse CB cells, TASK3 channels (or TASK1/3 heteromers) could mediate hypoxic depolarization of normal glomus cells. The ability of TASK1/3^−/− glomus cells to maintain a powerful response to hypoxia even after blockade of maxi-K⁺ channels, suggests the existence of multiple sensor and/or effector mechanisms, which could confer upon the cells a high adaptability to maintain their chemosensory function.     

Hypoxia induces production of nitric oxide and reactive oxygen species in glomus cells of rat carotid body

The carotid body is an arterial chemoreceptor organ that senses arterial pO(2) and pH. Previous studies have indicated that both reactive oxygen species (ROS) and nitric oxide (NO) are important potential mediators that may be involved in the response of the carotid body to hypoxia. However, whether their production by the chemosensitive elements of the carotid body is indeed oxygen-dependent is currently unclear. Thus, we have investigated their production under normoxic (20% O(2)) and hypoxic (1% O(2)) conditions in slice preparations of the rat carotid body by using fluorescent indicators and confocal microscopy. NO-synthesizing enzymes were identified by immunohistochemistry and histochemistry, and the subcellular localization of the NO-sensitive indicator diaminofluorescein was determined by a photoconversion technique and electron microscopy. Glomus cells of the carotid body responded to hypoxia by increases in both ROS and NO production. The hypoxia-induced increase in NO generation required (to a large extent, but not completely) extracellular calcium. Glomus cells were immunoreactive to endothelial NO synthase but not to the neuronal or inducible isoforms. Ultrastructurally, the NO-sensitive indicator was observed in mitochondrial membranes after exposure to hypoxia. The data show that glomus cells respond to exposure to hypoxia by the enhanced production of both ROS and NO. NO production by glomus cells is probably mediated by endothelial NO synthase, which is activated by calcium influx. The presence of NO indicator in mitochondria suggests the hypoxic regulation of mitochondrial function via NO in glomus cells.     

Diagnosis of gastric glomus tumor by endoscopic ultrasound-guided fine needle aspiration biopsy. A case report with cytologic,histologic and immunohistochemical studies

BACKGROUND: The majority of glomus tumor are small, benign neoplasms that arise from modified smooth muscle cells. They usually occur in the dermis or subcutis of the extremities. However, rare cases have been reported in the visceral locations, most often in the stomach. CASE: A 32-year-old woman presented with episodes of right upper quadrant pain. She was found to have a gastric tumor that was biopsied at another hospital, where the diagnosis of gastrointestinal stromal tumor (GIST) was made. Endoscopic ultrasound (EUS) performed at our institution revealed a gastric submucosal tumor that was then biopsied by fine needle aspiration (FNA). Cytology revealed cohesive clusters of uniform, round, small cells with ill-defined cytoplasmic borders and scanty, amphophilic cytoplasm. Nuclei were round, with smooth nuclear membranes and evenly distributed, dusty chromatin. Intermingled with those epithelioid cells were small, short, spindled, normal endothelial cells. Immunohistochemical studies performed on cell block showed that the tumor cells were negative for CD34, CD117, chromogranin, synaptophysin, desmin and AE1/AE3 and were strongly positive for SMA, HHF-35 and collagen type IV. Glomus tumor was diagnosed and later confirmed by histology. CONCLUSION: EUS-guided FNA biopsy is efficient and permits adequate sampling for accurate diagnosis of gastric glomus tumor. Although rare, glomus tumor should be in the differential diagnosis among other gastric lesions, such as well-differentiated adenocarcinoma, epithelioid GIST and carcinoid tumor.     

Glutamate in the glomus cells of the cat carotid body: Immunocytochemistry and in vitro release

The identity of the postulated excitatory transmitter released by glomus cells is not known. Since our preliminary work on paraffin sections of the cat carotid body indicated that most glomus cells were intensely immunoreactive to glutamate, we decided to investigate whether glutamate might be such a transmitter, using two approaches. One approach was to make a quantitative immunogold analysis of ultrathin sections to assess the level of glutamate immunoreactivity of glomus cells relative to glia and to afferent axon terminals. The other approach was to measure the potassium-induced release of glutamate from carotid bodies superfused in vitro. We consistently found that glomus cell profiles had 50% more immunogold particles per unit of area than glial cell or axonal profiles. However, the levels of glutamate immunoreactivity of glomus cells were lower than those expected for glutamatergic terminals. We also found that glutamate was not released from in vitro carotid bodies stimulated with high concentrations of potassium. These findings indicate that the oxygen-sensitive glomus cells have a high concentration of glutamate, which is not released by superfusion with high potassium. Thus, glutamate is not the excitatory transmitter released by glomus cells. We speculate that the high concentrations of glutamate might instead be related to the known dependence of the “in vitro” chemosensory activity on metabolic substrates.     

Histochemically demonstrable increase in the catecholamine content of the carotid body in adult rats treated with methylprednisolone or hydrocortisone

Synopsis Carotid bodies of adult albino rats were examined using the formaldehyde-induced fluorescence (FIF) method for the demonstration of fluorogenic monaomines and staining with I% Toluidine Blue for morphological observations.In the carotid body of normal controls, most glomus (principal or type I) cells exhibited a FIF presumably due to catecholamines. The intensity of the fluorescence was weak in most cells, while some glomus cells were non-fluorescent and others exhibited a moderate or intense FIF. The sustentacular (satellite, supporting or type II) cells were essentially non-fluorescent.One week after the administration of a single intraperitoneal injection of the long-acting glucocorticoid 6-methylprednisolone sypionate (400 mg/kg) or after seven intraperitoneal injections of the water-soluble glucocorticoid hydrocortisone sodium succinate (40 mg/kg daily for a week), a distinct increase was observed in the FIF of the glomus cells. No non-fluorescent glomus cells were observed after treatment with either glucocorticoid, and the intensity of most fluorescent glomus cells was moderate or intense.It is concluded that glucocorticoids cause an increased storage of catecholamines in the glomus cells of the carotid body of the adult rat, an observation of interest in view of the fact that such changes due to glucocorticoids have as yet been reported only in catecholamine-storing cells of newborn rats.     

Dense granule-containing cells in arterial chemoreceptor areas of the tortoise (Testudo hermanni)

Light- and electron-microscopic observations of the chemosensory areas of the arteries of the tortoise (Testudo hermanni) reveal that clusters of nonmuscular cells are found in the adventitial layer of restricted regions of the carotid artery, aortic arch, and pulmonary artery. In these clusters, three types of cells are complexly interwoven: the G-cell closely resembles the glomus cell, which has been found in the arterial chemoreceptor area of several animal species; the LG-cell has very large electron-dense granules; the third type is a G- and LG-cell supporting cell. Membrane specializations are often observed at apposing membranes between G-cells. Two kinds of nerve endings synapse with G-cells, one with numerous clear synaptic vesicles, the other without vesicles. Some G-cells are in membrane-to-membrane contact with smooth-muscle cells (g-s connection), and here a membrane thickening is visible. Nerve terminals with numerous synaptic vesicles synapse with the LG-cells. The G-cell in the carotid artery, the aorta, and the pulmonary artery is a chemoreceptor element ultrastructurally the same as the glomus cell in the arterial chemoreceptor area of various vertebrate species.     

The effect of polymyxin B nonapeptide (PMBN) on transformation

The effect of the outer membrane permeabilizing polycation, polymyxin B nonapeptide (PMBN) on the transformation of E. coli HB101 with pBR322 plasmid DNA was investigated. Pretreatment of cells with PMBN (followed by suspending the cells in PMBN-free medium) did not stimulate the development of competence induced by the calcium heat pulse. In the absence of calcium-ions, a high PMBN concentration (1 mM) was able to induce a low transformation frequency provided that PMBN was not removed before the addition of DNA.     

Fast neurogenesis from carotid body quiescent neuroblasts accelerates adaptation to hypoxia

Unlike other neural peripheral organs, the adult carotid body (CB) has a remarkable structural plasticity, as it grows during acclimatization to hypoxia. The CB contains neural stem cells that can differentiate into oxygen‐sensitive glomus cells. However, an extended view is that, unlike other catecholaminergic cells of the same lineage (sympathetic neurons or chromaffin cells), glomus cells can divide and thus contribute to CB hypertrophy. Here, we show that O₂‐sensitive mature glomus cells are post‐mitotic. However, we describe an unexpected population of pre‐differentiated, immature neuroblasts that express catecholaminergic markers and contain voltage‐dependent ion channels, but are unresponsive to hypoxia. Neuroblasts are quiescent in normoxic conditions, but rapidly proliferate and differentiate into mature glomus cells during hypoxia. This unprecedented “fast neurogenesis” is stimulated by ATP and acetylcholine released from mature glomus cells. CB neuroblasts, which may have evolved to facilitate acclimatization to hypoxia, could contribute to the CB oversensitivity observed in highly prevalent human diseases.     

Substance P-like immunoreactivity in rat and cat carotid bodies: light and electron microscopic studies

Substance P-immunoreactive (SP-1) structures in the carotid bodies of rats and cats were examined with the light and electron microscopes. In both species SP-I varicose nerve fibers were located singly in the interstitial connective tissue in close association with blood vessels. They were small unmyelinated fibers enveloped in a common Schwann cell sheath with other SP-negative fibers. Some of SP-I fibers contained large dense-cored granules and small clear vesicles in addition to microtubules and mitochondria and probably represented nerve fiber varicosities. The latter often were found incompletely invested by Schwann cell sheaths. SP-fibers were found occasionally in the envelopes of supporting cells at the periphery of parenchymal cell groups. However, none of the nerve terminals making synaptic contacts with glomus cells exhibited SP-like immunoreactivity. In cat carotid bodies some glomus cells showed moderate to intense SP-like immunoreactivity. The intense SP-I glomus cells displayed numerous dense-cored vesicles of 85 to 140 nm in diameter and frequently showed synaptic contacts with SP-negative nerve terminals. In rat carotid bodies we were unable to detect consistent SP-immunoreactivity in glomus cells. Our results do not favor the hypothesis that SP is a neurotransmitter/modulator in the chemoreceptor afferents synapsing on glomus cells in either the cat or rat carotid body. However our results support the hypothesis that SP in cat glomus cells may play a role in the modulation of chemoreceptor activity.     

Improved demonstration of exocytotic profiles in glomus cells of rat carotid body after perfusion with glutaraldehyde fixative containing a high concentration of potassium

Extensive secretion by exocytosis was demonstrated in the glomus (type I) cells of the adult rat after perfusion of carotid bodies with a potassium-rich (high K) glutaraldehyde fixative. Similar secretory profiles were very rare with a glutaraldehyde fixative containing a low concentration of potassium (low K). The increase in the incidence of exocytotic profiles in glomus cells with the high K fixative was highly significant, whereas no statistical difference could be observed in the incidence of coated pits with the different fixatives. Exocytotic profiles were characterized by the following features: (1) they predominated in non-synaptic regions, but were occasionally observed near synapses between two glomus cells; they were not observed near synapses between glomus cells and nerve terminals; (2) extruded electron-dense material associated with coating of the cell membrane was frequent; (3) different stages of dissolution of the extruded granule material was evident. The possible role of exocytosis as a mode of secretion in the glomus cells and the characteristics of the new high K-glutaraldehyde fixative are discussed.     

Autocrine and paracrine actions of ATP in rat carotid body

Carotid bodies are peripheral chemoreceptors that detect lowering of arterial blood O(2) level. The carotid body comprises clusters of glomus (type I) cells surrounded by glial-like sustentacular (type II) cells. Hypoxia triggers depolarization and cytosolic [Ca(2+)] ([Ca(2+)](i)) elevation in glomus cells, resulting in the release of multiple transmitters, including ATP. While ATP has been shown to be an important excitatory transmitter in the stimulation of carotid sinus nerve, there is considerable evidence that ATP exerts autocrine and paracrine actions in carotid body. ATP acting via P2Y(1) receptors, causes hyperpolarization in glomus cells and inhibits the hypoxia-mediated [Ca(2+)](i) rise. In contrast, adenosine (an ATP metabolite) triggers depolarization and [Ca(2+)](i) rise in glomus cells via A(2A) receptors. We suggest that during prolonged hypoxia, the negative and positive feedback actions of ATP and adenosine may result in an oscillatory Ca(2+) signal in glomus cells. Such mechanisms may allow cyclic release of transmitters from glomus cells during prolonged hypoxia without causing cellular damage from a persistent [Ca(2+)](i) rise. ATP also stimulates intracellular Ca(2+) release in sustentacular cells via P2Y(2) receptors. The autocine and paracrine actions of ATP suggest that ATP has important roles in coordinating chemosensory transmission in the carotid body.     

长时间低氧对大鼠颈动脉体培养细胞的细胞内pH和膜电位的影响

本文的目的是研究长时间低氧对离体培养的大鼠颈动脉体球细胞（ｇｌｏｍｕｓｃｅｌｌ）的影响。对实验组Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ（ＳＤ）大鼠,首先将其置于模拟５０００ｍ高度低氧环境的低压舱中饲养７—１０ｄ,然后麻醉动物,取出颈动脉体,将其分离成单个细胞和细胞群体（ｃｌｕｓｔｅｒｓ）。这些细胞在低氧条件（１１％Ｏ２,５％ＣＯ２,８４％Ｎ２）下培养２—３ｄ。取自正常ＳＤ大鼠的颈动脉体细胞被分为两组,分别将其培养在常氧（２１％Ｏ２,５％ＣＯ２,７４％Ｎ２）或低氧环境中。球细胞的细胞内ｐＨ（ｐＨｉ）和膜电位（ＭＰ）分别用Ｈ＋选择性微电极和常规微电极同时测量。结果表明：长时间低氧降低球细胞的ｐＨｉ,增加ＭＰ,其变化程度远远大于急性低氧的影响,而且当将细胞置于常氧中测量时其值不恢复。     

The effects of 6-hydroxydopamine on the appearance of granulated vesicles in glomus cells of the rat carotid body

The glomus cells of the rat carotid body reveal an intense fluorescence after exposure to paraformaldehyde vapor and contain catecholamines. After initial fixation in glutaraldehyde, many granulated vesicles are seen in the glomus cells. After initial fixation in osmium tetroxide, most of the vesicles are depleted of their dense interiors and granulated vesicles occur infrequently. Administration of 6-hydroxydopamine followed by initial fixation in osmium tetroxide leads to the reappearance of dense interiors in virtually all vesicles. 6-Hydroxydopamine apparently is taken up by the membrane pump of the glomus cell and is incorporated into the amine storage granules, thereby displacing the endogenous monoamines. Osmium tetroxide does not dissolve the 6-hydroxydopamine from the vesicles, as it apparently does for the normal vesicular contents. The 6-hydroxydopamine does not fluoresce, hence 6-hydroxydopamine administration results in a decreased intensity of formaldehyde induced fluorescence in the glomus cells. Administration of reserpine after 6-hydroxydopamine treatment (and subsequent initial fixation in osmium tetroxide) depletes the previously restored dense material from the vesicles of the glomus cells. 6-Hydroxydopamine acts like a monoamine in that it is taken up by the glomus cell, incorporated into the vesicles, and can be depleted from the vesicles by reserpine.     

Effect of acute hypoxia on glomus cell Em and psi m as measured by fluorescence imaging.

We have reinvestigated the hypothesis of the relative importance of glomus cell plasma and mitochondrial membrane potentials (E(m) and psi(m), respectively) in acute hypoxia by a noninvasive fluorescence microimaging technique using the voltage-sensitive dyes bis-oxonol and JC-1, respectively. Short-term (24 h)-cultured rat glomus cells and cultured PC-12 cells were used for the study. Glomus cell E(m) depolarization was indirectly confirmed by an increase in bis-oxonol (an anionic probe) fluorescence due to a graded increase in extracellular K(+). Fluorescence responses of glomus cell E(m) to acute hypoxia (approximately 10 Torr Po(2)) indicated depolarization in 20%, no response in 45%, and hyperpolarization in 35% of the cells tested, whereas all PC-12 cells consistently depolarized in response to hypoxia. Furthermore, glomus cell E(m) hyperpolarization was confirmed with high CO (approximately 500 Torr). Glomus cell psi(m) depolarization was indirectly assessed by a decrease in JC-1 (a cationic probe) fluorescence. Accordingly, 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an uncoupler of oxidative phosphorylation), high CO (a metabolic inhibitor), and acute hypoxia (approximately 10 Torr Po(2)) consistently depolarized the mitochondria in all glomus cells tested. Likewise, all PC-12 cell mitochondria depolarized in response to FCCP and hypoxia. Thus, although bis-oxonol could not show glomus cell depolarization consistently, JC-1 monitored glomus cell mitochondrial depolarization as an inevitable phenomenon in hypoxia. Overall, these responses supported our "metabomembrane hypothesis" of chemoreception.     

Granule matrix property and rapid "kiss-and-run" exocytosis contribute to the different kinetics of catecholamine release from carotid glomus and adrenal chromaffin cells at matched quantal size

Catecholamine-containing small dense core granules (SDCGs, vesicular diameter of ~100 nm) are prominent in carotid glomus (chemosensory) cells and some neurons, but the release kinetics from individual SDCGs has not been studied in detail. In this study, we compared the amperometric signals from glomus cells with those from adrenal chromaffin cells, which also secrete catecholamine but via large dense core granules (LDCGs, vesicular diameter of ~200-250 nm). When exocytosis was triggered by whole-cell dialysis (which raised the concentration of intracellular Ca(2+) ([Ca(2+)](i)) to ~0.5 μmol/L), the proportion of the type of signal that represents a flickering fusion pore was 9-fold higher for glomus cells. Yet, at the same range of quantal size (Q, the total amount of catecholamine that can be released from a granule), the kinetics of every phase of the amperometric spike signals from glomus cells was faster. Our data indicate that the last phenomenon involved at least 2 mechanisms: (i) the granule matrix of glomus cells can supply a higher concentration of free catecholamine during exocytosis; (ii) a modest elevation of [Ca(2+)](i) triggers a form of rapid "kiss-and-run" exocytosis, which is very prevalent among glomus SDCGs and leads to incomplete release of their catecholamine content (and underestimation of their Q value).     

Chemical and electric transmission in the carotid body chemoreceptor complex

Carotid body chemoreceptors are complex secondary receptors. There are chemical and electric connections between glomus cells (GC/GC) and between glomus cells and carotid nerve endings (GC/NE). Chemical secretion of glomus cells is accompanied by GC/GC uncoupling. Chemical GC/NE transmission is facilitated by concomitant electric coupling. Chronic hypoxia reduces GC/GC coupling but increases G/NE coupling. Therefore, carotid body chemoreceptors use chemical and electric transmission mechanisms to trigger and change the sensory discharge in the carotid nerve.     

The oscillatory responses of skate electroreceptors to small voltage stimuli

Tonic nerve activity in skate electroreceptors is thought to result from spontaneous activity of the lumenal membranes of the receptor cells which is modulated by applied stimuli. When physiological conditions are simulated in vitro, the receptor epithelium produces a current which flows inward across the lumenal surface. This epithelial current exhibits small spontaneous sinusoidal fluctuations about the mean that are associated with corresponding but delayed fluctuations in postsynaptic response. Small voltage stimuli produce damped oscillations in the epithelial current similar in time-course to the spontaneous fluctuations. For lumen-negative, excitatory stimuli, these responses are predominantly an increase over the mean inward current. For inhibitory stimuli they are predominantly a decrease. Increased inward current across the lumenal membranes of the receptor cells increases depolarization of the presynaptic membranes in the basal faces leading to increased release of transmitter and an excitatory postsynaptic response. Decreased inward current decreases depolarization of the presynaptic membranes leading to a reduction in transmitter release and an inhibitory postsynaptic response. Clear changes in postsynaptic response are detectable during stimuli as small as 5 microV with saturation occurring at +/- 400 microV. The evoked oscillations in epithelial current are damped and the postsynaptic responses decline during maintained stimuli with large off-responses occurring at stimulus termination. The initial peak of the off-response is similar to the response produced by onset of an oppositely directed stimulus. These observations substantiate the role of receptor cell excitability in the detection of small voltage changes.