Fine structure of the ordinary lateral line organ

Summary The lateral line organ of the spotted shark is characterized by its semi-cylindrical shape. Each organ (neuromast) is so closely apposed to the next that the individual neuromasts are almost continuous. The neuromast is composed of receptor cells, supporting cells and mantle cells. The receptor cells bear one kinocilium and up to 40 stereocilia. Bi-directional arrangement of the receptor cells as occurs in teleosts was demonstrated. Afferent and efferent nerve endings were found at the base of the receptor cells. The supporting cells extend from the basal lamina to the free surface. Long microvilli and a cilium-like ciliary rod project from the top of each supporting cell. The cell contains relatively few elements of the Golgi apparatus and little rough endoplasmic reticulum, but mitochondria and filaments are abundant. The mantle cell limits the lateral margin of the neuromast. It is distinguished from the supporting cell because of its long crescent-shaped nucleus and scarce, short microvilli. Myelinated nerve fibres are found in the subepithelial connective tissue but not in the epithelium.The fine structure of the shark lateral line organ suggests that this organ is in an intermediated step of evolution between that of lamprey and teleost.     

Gustatory receptors and neighbouring cells in the surface layer of an amphibian taste disc: in situ relationships and response to cell isolation

Summary The outermost epithelial cell layer of frog taste buds consists of (1) mucus-secreting cells; (2) thin, sheet-like processes of wing cells, which enclose the basal and lateral aspects of the mucus cells; (3) slim apical processes (sensory dendrites) of taste receptor cells; the latter ascend within wing-cell processes and each process terminates apically in one villus-like rod, a dense, highly ordered bundle of 500–1500 actin filaments supporting the chemoreceptive membrane. Staining with fluorescent phalloidin indicates that there are about 760 receptor terminals in a taste disc of 200 m in diameter, or 200000 per tongue. Viable spindle-shaped taste receptor cells were isolated using a lowcalcium / collagenase / mechanical agitation protocol. Following fluorescent labelling of apical membranes prior to cell separation, the label remained at the dendritic tip of these cells during isolation. The tip was still stainable with phalloidin. Thus the sensory dendrite, including its apical pole, had been completely removed from the encasing wing-cell process. However, scanning electron microscopy and staining by lectin and phalloidin indicated that many taste receptor cells had undergone cytoskeletal reorganization in response to cell separation; they became flask-shaped following the shortening and thickening of their sensory dendrite. The cells also began to disassemble the actin filament bundle at the chemoreceptive apical cell pole.     

The time course of taste bud regeneration after glossopharyngeal or greater superficial petrosal nerve transection in rats

We previously have published data detailing the time course of taste bud regeneration in the anterior tongue following transection of the chorda tympani (CT) nerve in the rat. This study extends the prior work by determining the time course of taste bud regeneration in the vallate papilla, soft palate and nasoincisor ducts (NID) following transection of either the glossopharyngeal (GL) or greater superficial petrosal (GSP) nerve. Following GL transection in rats (n = 6 per time point), taste buds reappeared in the vallate papilla between 15 and 28 days after surgery, and returned to 80.3% of control levels (n = 12) of taste buds by 70 days postsurgery. The first appearance and the final percentage of the normal complement of regenerated vallate taste buds after GL transection resembled that seen previously in the anterior tongue after CT transection. However, in the latter case, regenerated taste buds reached asymptotic levels by 42 days after surgery, whereas within the time frame of the present study, a clear asymptotic return of vallate taste buds was not observed. In contrast to the posterior (and anterior) tongue, only 25% of the normal complement of palatal taste buds regenerated by 112 days and 224 days after GSP transection (n = 9). The difference in regenerative capacity might relate to the surgical approach used to transect the GSP. These experiments provide useful parametric data for investigators studying the functional consequences of gustatory nerve transection and regeneration.     

Chorda tympani nerve transection at different developmental ages produces differential effects on taste bud volume and papillae morphology in the rat

Chorda tympani nerve transection (CTX) results in morphological changes to fungiform papillae and associated taste buds. When transection occurs during neonatal development in the rat, the effects on fungiform taste bud and papillae structure are markedly more severe than observed following a comparable surgery in the adult rat. The present study examined the potential "sensitive period" for morphological modifications to tongue epithelium following CTX. Rats received unilateral transection at 65, 30, 25, 20, 15, 10, or 5 days of age. With each descending age at the time of transection, the effects on the structural integrity of fungiform papillae were more severe. Significant losses in total number of taste buds and filiform-like papillae were observed when transection occurred 5-30 days of age. Significant reduction in the number of taste pores was indicated at every age of transection. Another group of rats received chorda tympani transection at 10, 25, or 65 days of age to determine if the time course of taste bud degeneration differed depending on the age of the rat at the time of transection. Taste bud volumes differed significantly from intact sides of the tongue at 2, 8, and 50 days post-transection after CTX at 65 days of age. Volume measurements did not differ 2 days post-transection after CTX at 10 or 25 days of age, but were significantly reduced at the other time points. Findings demonstrate a transitional period throughout development wherein fungiform papillae are highly dependent upon the chorda tympani for maintenance of morphological integrity.     

The cellular organization and nervous supply of the basilar papilla in the lizard,Calotes versicolor

Summary The basilar papilla of the lizard Calotes versicolor contains about 225 sensory cells. These are of two types: the short-haired type A cells in the ventral (apical) part of the organ, and the type B cells with long hair bundles, in the dorsal (basal) part of the organ. The type A cells are unidirectionally oriented and are covered by a tectorial membrane while the type B cells lack a covering structure and their hair bundles are oriented bidirectionally. Apart from those differences, the type A and type B cells are similar. They are columnar, and display the features common to most sensory cells in inner ear epithelia. The sensory cells are separated by supporting cells, which have long slender processes that keep the sensory cells apart. Close to the surface of the basilar papilla a terminal bar of specialized junctions interlocks adjacent cells. Below this, adjacent supporting cells are linked by an occluding junction.The cochlear nerve enters from the medial (neural) aspect. The fibres of the nerve lose their myelin sheaths as they enter the basilar papilla. Each sensory cell is associated with several nerve endings. All the nerves identified were afferent. Marked variations were seen between nerve endings in the basilar papilla, but no morphological equivalents of any functional differences were observed.This work is supported by grant no. B76-12X-00720-11A from the Swedish Medical Research Council, and by funds from the Karolinska Institute, Stockholm, Sweden.     

Acetylcholinesterase-containing nerve cells in the pineal complex and subcommissural area of the frogs,Rana ridibunda and Rana esculenta

Summary In Rana esculenta and Rana ridibunda the frontal organ and the pineal organ (epiphysis cerebri) form a pineal complex. Approximately 60 nerve cells of the frontal organ and 220–320 nerve cells of the pineal organ display a positive acetylcholinesterase reaction (Karnovsky and Roots, 1964). The dorsal wall of the pineal organ is considerably richer in acetylcholinesterase-positive neurons than the ventral wall (ratio 31); a group of unusually large-sized nerve cells occurs in the rostral portion of the frog pineal. Two different types of nerve cells were observed in the pineal complex: multipolar and pseudounipolar elements. The former are embedded in the pineal parenchyma and their processes penetrate radially into the plexiform layer, whereas the latter are distributed along the roots of the pineal tract near the basal lamina. The ratio of the multipolar to pseudounipolar neurons is 14 for the frontal organ and 35 for the pineal organ. The multipolar elements may be interneurons; the pseudouni-polar cells send one of their processes into the pineal tract. At the caudal end of the pineal organ 30–50 unipolar nerve cells are clustered in juxtaposition with the pineal tract, and other 30–50 unipolar neurons are scattered along the basis of the subcommissural organ. Some of these nerve cells emit their processes toward the mesencephalon and others toward the pineal organ via the pineal tract. The results are discussed with respect to previous physiological and morphological findings on the pineal complex of Anura.Supported by a fellowship from the Alexander von Humboldt Foundation, Federal Republic of Germany, to K. Wake. Completed November 22, 1973.Supported by the Deutsche Forschungsgemeinschaft.     

Decline of IXth nerve taste responses following nerve transection

Summated impulse discharges to taste solutions were recordedfrom intact and transected IXth nerves in the Mongolian gerbil(Meriones unguiculatus). Five taste stimuli were used: 0.3 MNH₄Cl, 0.3 M NaCl, 0.01 M HCl, 0.01 M quinine hydrochloride,and 0.5 M sucrose. 0.3 M NH₄Cl was the most effective stimulus.Taste responses from intact nerves were stable for more than10 hours. Following IXth nerve transection, the peak summatedresponse to 0.3 M NH₄Cl declined by 50% in a mean of 119 min.(Some animals failed to show this taste response decline inthe winter months.) The transected IXth nerve's spontaneousactivity and responses to other taste solutions also typicallydeclined. The continued presence of normal compound action potentialsindicated that the transection-induced decline in taste responsesdid not result from a failure of impulse propagation mechanismsin the nerve trunk. The results are consistent with the propositionthat transection interferes with axonal transport of materialsvital to the short-term maintenance of taste responses.     

The ultrastructure of taste and touch receptors of the Frog's taste organ

Summary The taste buds from fungiform papillae and the hard palate of frogs were investigated with the scanning and transmission electron microscopes. An immature pre-taste cell and a mature taste cell can be differentiated. Only the mature taste cell exhibits synaptic contact with the afferent taste fibre. Glandular and satellite supporting cells envelop the thin apical processes of the sensory cells. At the base of the taste disc up to 10 Merkel cells form a complex with nerve endings. There are two types of myelinated fibres, large and small. The small fibre innervates the taste cells, the thicker nerve fibre the Merkel cells. The occurrence of two types of receptors explains physiological results.Supported by the Deutsche Forschungsgemeinschaft Rezeptorphysiologie.     

Innate immune responses of human tracheal epithelium to Pseudomonas aeruginosa flagellin, TNF-alpha, and IL-1beta

We measured innate immune responses by primary human tracheal epithelial (HTE) cells grown as confluent, pseudostratified layers during exposure to inflammatory activators on apical vs. basolateral surfaces. Apical Pseudomonas aeruginosa strain PAK (but not flagellin mutant PAK·fliC), flagellin, and flagellin + PAK·fliC activated NF-B and IL-8 expression and secretion. In contrast, HTE cells were insensitive to LPS compared to flagellin. Flagellin activated NF-B in columnar but not basal cells. IL-1 + TNF- elicited responses similar to those of flagellin. Basolateral flagellin or IL-1 + TNF- caused 1.5- to 4-fold larger responses, consistent with the fact that NF-B activation occurred in both columnar and basal cells. MyD88 (toll receptor-associated adapter), IL-1 receptor (IL1R)1, and TNF- receptor (TNFR)1 were expressed in columnar and basal cells. ZO-1 was localized to tight junctions of columnar cells but not to basal cells. We infer the following. 1) Flagellin is necessary and sufficient to trigger inflammatory responses in columnar cells during accumulation of P. aeruginosa in the airway surface liquid (ASL); columnar cells express toll-like receptor 5 and MyD88, often associated with flagellin-activated cell signaling. 2) IL-1 + TNF- in the ASL also activate columnar cells, and these cells also express IL1R1 and TNFR1. 3) Apical flagellin, IL-1, and TNF- do not activate basal cells because tight junctions between columnar cells prevent access from the apical surface to the basal cells. 4) Exposure of basolateral surfaces to inflammatory activators elicits larger responses because both columnar and basal cells are activated, likely because both cell types express receptors for flagellin, IL-1, and TNF-. toll-like receptor; nuclear factor-B; interleukin-8; tumor necrosis factor; interleukin-1     

Endothelial modulation of neural sympathetic vascular tone in canine skeletal muscle

The effect of nitric oxide synthase (NOS)inhibition and endothelin-A(ET_A)-receptor blockade onneural sympathetic control of vascular tone in the gastrocnemius musclewas examined in anesthetized dogs under conditions of constant flow.Muscle perfusion pressure (MPP) was measured before and after NOSinhibition(N-nitro-L-argininemethyl ester; L-NAME) andET_A-receptor blockade [cyclo-(D-Trp-d-Asp-Pro-D-Val-Leu);BQ-123]. Zero and maximum sympathetic nerve activities wereachieved by sciatic nerve cold block and stimulation, respectively. Ingroup 1 (n = 6), MPP was measured1) before nerve cold block,2) during nerve cold block, and3) during nerve stimulation.Measurements under these conditions were repeated afterL-NAME and then BQ-123. The sameprotocol was followed in group 2 (n = 6) except that the order ofL-NAME and BQ-123 was reversed.MPP and muscle vascular resistance (MVR) increased afterL-NAME and then decreased tocontrol values after BQ-123. MVR decreased after BQ-123 alone and, withthe addition of L-NAME,increased to a level not different from that observed during thecontrol period. MVR fell during nerve cold block. This response was notaffected by administration ofL-NAME followed by BQ-123, butit was attenuated by administration of BQ-123 before L-NAME. The constrictor responseduring sympathetic nerve stimulation was enhanced byL-NAME; no further effect wasobserved with BQ-123, nor was the response affected when BQ-123 wasgiven first. These findings indicate that endothelin contributes to1) basal vascular tone in skeletalmuscle and 2) the increase inskeletal muscle vascular resistance after NOS inhibition. Finally,nitric oxide "buffers" the degree of constriction in skeletalmuscle vasculature during maximal sympathetic stimulation.

     

Taste Bud Cell Generation in the Perihatching Chick

Chick taste bud primordia initially appear in late gestationon embryonic day 17 (E17), 4 days before hatching. To trackDNA synthesis and subsequent taste bud cell proliferation betweenE17 and the second day post-hatching (H2), single 25 µCiinjections of tritiated thymidine (specific activity = 72.5Ci/mmol) were administered in ovo during E15, E16, E17 or E18.Anterior mandibular oral epithelium was processed for lightmicroscopic autoradiography. Sections through each taste bud'scenter were analysed for label (6 silver grains/gemmal cellnucleus), and bud diameter. Results indicated a major part ofgemmal cell DNA synthesis does not occur until after E19 irrespectiveof the day of thymidine injection, suggesting postmitotic orquiescent (decycled) cells assemble to form the early bud primordium(E17–19) based on local tissue interactions. All budsexamined from E20–H2 contained labelled cells. The dayof injection was important since 5-day survival cases afterE16 injection yielded about 25% the number of labelled cells/budas compared with equivalent survival cases following E17–18injections. These results are discussed with respect to parallelchanges in bud shape and increasing bud diameter, and cell proliferationin possible extra- and intragemmal sources of bud cells.     

Stimulation of the gerbil's gustatory receptors by methyl glycopyranosides

The gustatory responses from the chorda tympani nerve of theMongolian gerbil Meriones unguiculatus were tested with somemethyl glycopyranosides and inhibitors. Except for one compound,methyl 4,6 dichloro-4,6-dideoxy -D-galactopyranoside, all sugarsstimulated the taste nerve. In our previous research, we foundone of the most effective methyl glycosides was methyl -D-glycopyranoside(MAD-Glu). We also found that methyl glycosides, which differedfrom MAD-Glu by the orientation of a substituent group at C-1,C-2 and C-4, were always less effective taste stimuli. In ourpresent study, we have extended this finding to include theC-3 epimer which was also found to be less effective than MAD-Glu.In a second set of experiments, we looked at deoxy sugars. Incontrast to orientation effects found in the epimers, substitutionof the hydroxyl groups of MAD-Glu by hydrogen atoms (deoxy-sugars)had variable effects. The 3-deoxy and 6-deoxy derivatives werefound to be as effective as MAD-Glu, while the 4-deoxy derivativewas less effective. In addition, substituting chlorine atomsfor the hydroxyl groups appears to have a deleterious effect.For example, the 6-chloro derivative was less effective thanMAD-Glu while the 4,6-dichloro derivative of methyl -D-galactopyranosidedid not stimulate the nerve at all. Finally, in addition tostructure-activity experiments mentioned above, we examinedthe possibility that the sugar taste response may involve otherphysiological factors. In this vein, we found that the sugartransport inhibitors phloretin and phlorhizin and the sodiumchannel blocker, amilonde had no effect on the gerbil's sucroseor glucose taste responses     

Basal chloride currents in murine airway epithelial cells: modulation by CFTR

We have isolatedciliated respiratory cells from the nasal epithelium of wild-type andcystic fibrosis (CF) null mice and used the patch-clamp technique toinvestigate their basal conductances. Current-clamp experiments onunstimulated cells indicated the presence ofK⁺ andCl conductances and, undercertain conditions, a small Na⁺conductance. Voltage-clamp experiments revealed three distinct Cl conductances.I_tv-indep wastime and voltage independent with a linear current-voltage(I-V)plot; I_v-actexhibited activation at potentials greater than ±50 mV, giving anS-shapedI-Vplot; andI_hyp-act wasactivated by hyperpolarizing potentials and had an inwardly rectifiedI-Vplot. The current density sequence was I_hyp-act = I_v-act  I_tv-indep. Theseconductances hadCl-to-N-methyl-D-glucaminecation permeability ratios of between 2.8 and 10.3 and were unaffectedby tamoxifen, flufenamate, glibenclamide, DIDS, and5-nitro-2-(3-phenylpropylamino) benzoic acid but were inhibited byZn²⁺ andGd³⁺.I_tv-indep andI_v-act werepresent in wild-type and CF cells at equal density and frequency.However, I_hyp-actwas detected in only 3% of CF cells compared with 26% of wild-typecells, suggesting that this conductance may be modulated by cysticfibrosis transmembrane conductance regulator (CFTR).

     

Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection

The intact sciatic nerve contains levels of nerve growth factor (NGF) that are comparable to those of densely innervated peripheral target tissues of NGF-responsive (sympathetic and sensory) neurons. There, the high NGF levels are reflected by correspondingly high mRNANGF levels. In the intact sciatic nerve, mRNANGF levels were very low, thus indicating that the contribution of locally synthesized NGF by nonneuronal cells is small. However, after transection an increase of up to 15-fold in mRNANGF was measured in 4-mm segments collected both proximally and distally to the transection site. Distally to the transection site, augmented mRNANGF levels occurred in all three 4-mm segments from 6 h to 2 wk after transection, the longest time period investigated. The augmented local NGF synthesis after transection was accompanied by a reexpression of NGF receptors by Schwann cells (NGF receptors normally disappear shortly after birth). Proximal to the transection site, the augmented NGF synthesis was restricted to the very end of the nerve stump that acts as a "substitute target organ" for the regenerating NGF-responsive nerve fibers. While the mRNANGF levels in the nerve stump correspond to those of a densely innervated peripheral organ, the volume is too small to fully replace the lacking supply from the periphery. This is reflected by the fact that in the more proximal part of the transected sciatic nerve, where mRNANGF remained unchanged, the NGF levels reached only 40% of control values. In situ hybridization experiments demonstrated that after transection all nonneuronal cells express mRNANGF and not only those ensheathing the nerve fibers of NGF-responsive neurons.     

Innervation und zentralnervöse Verbindungen des Frontalorgans von Rana temporaria und Rana esculenta

Zusammenfassung Mit neurohistologischen Techniken (Nauta-Verfahren) wurde der Anteil des Stirnorgans von Rana temporaria und Rana esculenta an der zentralnervösen Projektion des lichtempfindlichen Pinealkomplexes geprüft. Nach operativer Unterbrechung des Nervus pinealis im dorsalen Lymphsack lassen sich degenerierende Nervenfasern sowohl im Tractus pinealis als auch im stirnorgannahen Stumpf des Nervus pinealis nachweisen. Die ersteren werden als cerebropetale (afferente), die letzteren als zum Stirnorgan ziehende (efferente) Faserelemente gedeutet. Es ist gelungen, die hirnwärts gerichteten Nervenfasern des Stirnorgans bis in die unmittelbare Umgebung des sekretorischen Subcommissuralorgans zu verfolgen; zerfallende Faserfragmente liegen dicht der Basis des Subcommissuralorgans an. Anders als nach Durchtrennung des Tractus pinealis (vgl. Paul, Hartwig und Oksche, 1971) ließen sich nach Unterbrechung des Nervus pinealis keine Degenerationszeichen im mesencephalen Zentralen Grau darstellen.

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Innervation and central nervous connexions of the frontal organ in Rana temporaria and Rana esculenta Fiber degeneration after surgical interruption of the pineal nerve

Summary The contribution of the frontal organ of Rana temporaria and Rana esculenta to the central nervous projections of the light-sensitive pineal complex has been investigated with neurohistological techniques (Nauta-method). After surgical transection of the pineal nerve within the dorsal lymph sac, degenerating nerve fibers have been observed within the pineal tract and also in the proximal stump of the pineal nerve. Those in the pineal tract have been interpreted as cerebropetal (afferent) connexions of the frontal organ, and those in the pineal nerve as fibers directed towards the frontal organ (efferent elements). The cerebropetal fibers of the frontal organ have been traced to the subcommissural region where they degenerate in close juxtaposition with the secretory subcommissural organ. In contrast to the findings obtained after transection of the pineal tract (see Paul, Hartwig and Oksche, 1971), no degenerating fibers have been observed in the mesencephalic central grey after surgical interruption of the pineal nerve.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS ^1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells ^5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site ^{6, 7, 13-15}. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve ^16-19 or injecting vectors into their target the superior colliculus ¹⁰. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.Download video file.^{(69M, mov)}     

Hearing and glycoconjugates: localization of Ley, Lex and sialosyl-Lex in guinea pig cochlea, particularly at the tectorial membrane and sensory epithelia of the organ of Corti

Immunohistological examination of guinea pig cochleas was performedusing a panel of 25 monoclonal antibodies directed to variouslacto-, ganglio- and globo-series carbohydrate epitopes as wellas mucin-type epitopes. Lacto-series structures were found tobe localized at specific sites of the tectorial membrane (TM)and Corti's organ, i.e. 13 fucosyl type 2 chain (Le^x) at Kimura'smembrane, marginal band and covering net of TM; 12, 13 difucosyltype 2 chain (Le^y) at covering net; and sialosyl-Le^x and sialosyl-iat Kimura's membrane and sensory epithelia, particularly sensorytips of hair cells of Corti's organ. In striking contrast, ganglio-seriesstructures (GM3, GD3, GD2, 9-O-Ac-GD3) were detected at spiralganglion cells, neuronal fibres and stria vascularis, but werecompletely absent from Corti's organ and most of the TM. Otherepitope structures defined by various antibodies were not detectableat any location. The functional roles of lacto-series carbohydrateepitopes expressed at TM and Corti's organ remain unknown. However,the expression of Le^y (but not other structures) in associationwith developmental deficiency of TM induced by 6-N-propyI-2-thio-uracilin rats suggests that Le^y plays some role in normal TM development.The presence of Le^x at Kimura's membrane and sialosyl-Le^x athair cell sensory tips of Corti's organ suggests the intriguingpossibility that these fucosylated/sialosylated carbohydratestructures play some role in interactions (either attractiveor repulsive) of these inner ear components, which have beenimplicated in the physiology of hearing, i.e. the conversionof sound waves to nerve impulses. cochlea Corti's organ glycoconjugate sialosyl fucosyl type 2 chain tectorial membrane     

Guard Cells Extrude Protons Prior to Stomatal Opening--A Study using Fluorescence Microscopy and pH Micro-electrodes

A method for the demonstration of pH changes in the apoplastis described. The fluorescent pH indicator pnmulin was usedto follow pH changes in the epidermis of leaves of Commelinacommunis during stomatal movements. Previously darkened leavesexposed to light showed quenching of fluorescence in the apoplastsurrounding theuard cells up to 20 min before the stomata opened.This indicated that proton efflux by the guard cells precededstomatal opening. This result was substantiated by apoplasticpH measurements using pH micro-electrodes. Acidification ofthe apoplast spread outwards from the guard cells to the surroundingsubsidiary and epidermal cells. This phenomenon persisted forsome time after subsequent stomatal closure, supporting thehypothesis that closure is brought about by a process otherthan the cessation of proton pumping. Key words: Commelina communis, stomata, proton pumping     

Dye-coupling among frog (Rana catesbeiana) taste disk cells

• 1.1. Dye-coupling among taste disk cells in the bullfrog fungiform papillae was examined histologically by injecting a fluorescent dye (Lucifer yellow) into the cell, and the effects of the dye-coupling on depolarizing responses induced by taste stimuli were studied electrophysiologically.
• 2.2. With dye injection into a taste cell, dye-coupling was found between taste cells (23%) or between taste cell and supporting cell (28%). With dye injection into a supporting cell, dye-coupling was found between supporting cells (34%) or between supporting cell and taste cell (27%).
• 3.3. Depolarizing responses recorded from either a taste cell or a supporting cell to stimulation with 0.5 M NaCl or 10 mM quinine-HCl were the same in amplitude whether the dye-coupling to another cell was present or not. On the other hand, depolarizing responses recorded from a taste cell for 0.5 mM acetic acid became significantly larger when dye-coupled to a supporting cell.
• 4.4. It is concluded that gustatory transduction for acid stimuli is influenced by supporting cells coupled to taste cells.

     

Cell Potentials and Turgor Pressures in Epidermal Cells of Tradescantia and Commelina

Cell membrane potentials have been measured both in epidermalstrips and intact leaf sections of Tradescantia virginiana andCommelina communis, and in epidermal cells over green and overalbino mesophyll cells of T. albiflora var. albovittata. Membranepotentials (_cell) in strips were considerably lower than thosein intact sections and were insensitive to light and to theabsence or presence of calcium. Their response to external cationlevels was indifferent to ionic species. However, in intactleaf sections incubated with calcium present, membrane potentialsresponded to K⁺ levels but not to Na⁺. were more negative thancells in epidermal strips, and responded to changes in illumination. Long-term recordings of _cell and vacuolar K⁺ levels in T. virginianaduring stomatal closure suggest that the fluctuations of _cellwere unrelated to K⁺ movement (which we could not detect) andthus probably to stomatal movement as well. Turgor pressures measured in epidermal cells of intact leafsections of T. virginiana were found to be of the same magnitudeas those previously reported for epidermal strips. It is concludedthat epidermal cells maintain their solute contents during strippingwithout the involvement of an electrophysiological transportsystem. With the possible exception of lateral subsidiary cells,there was no evidence suggesting that ordinary epidermal cellsare capable of osmotic adjustment even when additional KCI wassupplied in the osmoticum. Absolute turgor levels in intactleaf sections kept at constant external KCI were unrelated tosteady state _cell.