Motile statocyst cilia transmit rather than directly transduce mechanical stimuli

We have investigated the role of motile cilia in mechanotransduction by statocysts of the nudibranch mollusk Hermissenda crassicornis. Movement of the cilia that experience the weight of statoconia causes increased variance of voltage noise and membrane depolarization of the statocyst hair cell. Two complementary approaches were used to immobilize the cilia. Vanadate anion was iontophoretically injected into hair cells. This reversible inhibitor of vibratile form and to assume a more classic, pliable beat pattern. Voltage noise decreased as the cilia slowed and bent more extremely, nearly disappearing as motility was lost. When the intracellular vanadate concentration approached 10(-5) M, the cilia were arrested in an effective stroke against the cell membrane. The cell no longer depolarized upon gravitational or local mechanical stimulation. Rapid reversal of ciliary inhibition by norepinephrine or slow reversal with time restored both the voltage noise and depolarization response. Cilia were rendered rigid and upright by covalent cross-linkage of their membrane "sleeve" to the 9 + 2 axoneme, using the photoactivated, lipophilic, bifunctional agent 4,4''-dithiobisphenyl azide. In the initial stages of cross-linkage, the cilia remained vibratile but slowed and moved through wider excursions. Voltage noise decreased in frequency but increased in amplitude. When the cilia were fully arrested, voltage noise was minimized while the resting potential and membrane resistance remained essentially constant. Mechanical stimulation of the rigid cilia, normal to the cell membrane, elicited a generator potential of the same amplitude but of greater duration than before treatment. Because cilia that are partially arrested by vanadate undergo increased bending, although the hair cell shows decreased noise, neither the axoneme nor the ciliary membrane proper would appear to be sites of direct transduction. In cells with beating but stiffened cilia, however, the voltage noise becomes amplified, implying an increased efficiency of transduction. We suggest that active but rigid flexure of the axoneme is involved in amplification and continuous signal detection. The basal insertion area is the most likely transduction site, being the terminal leverage point through which force is applied to the plasma membrane via the flexing ciliary shaft.     

Voltage noise accompanying chemically-induced depolarization of insect photoreceptors

Intracellular potentials are recorded from photoreceptors in a superfused preparation of the retina of a locust compound eye. Chloral hydrate and alkyl alcohols induce a rapid, superfusing reversible depolarization of these photoreceptors when dissolved in the saline. Analysis of voltage noise accompanying depolarization by chloral hydrate suggests that depolarizing ionic pathways are opened briefly and randomly in time in the photoreceptor membranes. This conclusion is supported by measurements of the cell resistance and of voltage noise amplitude as a function of membrane potential. Replacement of superfusate sodium by choline reversibly reduces the effects of chloral hydrate, suggesting that the ionic pathways opened are permeable by sodium. The voltage noise induced by chloral hydrate is compared to that during depolarization by steady illumination of the same cell. As the illumination intensity is increased, the amplitude and the shape of the power spectrum of light-induced voltage noise approach those of drug-induced noise at the same depolarization level. The possibility that these phenomena represent alterations in the mechanism of phototransduction is discussed.     

Spontaneous activity of hair cells of the snail statocyst and its changes during intracellular stimulation

The following conclusions were drawn from an electrophysiological study of statocyst hair cell activity inHelix lucorum using intracellular recording. The maximal input resistance of the receptors is observed with hyperpolarizing currents of not more than 0.1 nA, close in magnitude to that arising during inhibitory synaptic transmission. Background noise, a special type of activity of statocyst hair cells, is neither synaptic nor pacemaker in nature, but depends entirely on the degree of contact between the cilia and statoconia. The hair cells possess pacemaker properties which are manifested on depolarization. The zone of action potential generation of the receptors lies in the axon. Inhibitory interactions take place between hair cells, leading to the generation of IPSPs in their spontaneous activity, which do not disappear after division of the vestibular nerve.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 2, pp. 222–229, March–April, 1985.     

Structure and function of the Nautilus statocyst.

The two equilibrium receptor organs (statocysts) of Nautilus are avoid sacks, half-filled with numerous small, free-moving statoconia and half with endolymph. The inner surface of each statocyst is lined with 130,000-150,000 primary sensory hair cells. The hair cells are of two morphological types. Type A hair cells carry 10-15 kinocilia arranged in a single ciliary row; they are present in the ventral half of the statocyst. Type B hair cells carry 8-10 irregularly arranged kinocilia; they are present in the dorsal half of the statocyst. Both type of hair cells are morphologically polarized. To test whether these features allow the Nautilus statocyst to sense angular accelerations, behavioural experiments were performed to measure statocyst-dependent funnel movements during sinusoidal oscillations of restrained Nautilus around a vertical body axis. Such dynamic rotatory stimulation caused horizontal phase-locked movements of the funnel. The funnel movements were either in the same direction (compensatory funnel response), or in the opposite direction (funnel follow response) to that of the applied rotation. Compensatory funnel movements were also seen during optokinetic stimulation (with a black and white stripe pattern) and during stimulations in which optokinetic and statocyst stimulations were combined. These morphological and behavioural findings show that the statocysts of Nautilus, in addition to their function as gravity receptor organs, are able to detect rotatory movements (angular accelerations) without the specialized receptor systems (crista/cupula systems) that are found in the statocysts of coleoid cephalopods. The findings further indicate that both statocyst and visual inputs control compensatory funnel movements.     

The statocyst of Vampyroteuthis infernalis (Mollusca: Cephalopoda)

The statocyst shows a remarkable combination of features of decapods and octopods confirming that Vampyroteuthis is a relic somewhere near the ancestor of both groups. The lining of the statocyst separates from the outer wall, forming an inner sac, filled with endolymph, surrounded by perilymph. This is the condition found in octopods, never in decapods. The macula is partly divided into a macula princeps and macula neglecta, as in decapods but never in octopods. There are numerous statoconia, but no large statolith has been seen. The crista has four parts as in decapods, but they are not sharply separated. There are numerous small anticristae, with the general form found in decapods, differentiated into pegs and hooks.
The wall of the inner sac contains numerous hair cells. These hairs protrude between the epithelial cells. The bases of the cells are drawn out into fine processes, presumably some dendritic and some axonal. There is thus a plexus of nerve fibres all over the wall, communicating with the crista nerve.
There is a very large posterior sac of unknown function, lying behind the crista. It contains only one large anticrista and the opening of Kölliker's canal, which is very large.     

Carbonic anhydrase is required for statoconia homeostasis in organ cultures of statocysts from Aplysia californica

A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (L15) medium supplemented with salts and Aplysia haemolymph for four days at 17°C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 × 10^–4 M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.     

A crustacean statocyst with only three hairs: Light and scanning electron microscopy

Standard histological and SEM techniques have been used to examine the pair of statocyst organs located in the telson of the isopod, Cyathara polita. Each organ is formed as an invagination of the dorsal cuticle of the telson. The invagination narrows to form a stalk between the statocyst and dorsal surface. A canal courses longitudinally through this stalk and forms a continuous channel between the lumen of the cyst and the external environment. On the luminal floor of each statocyst, there are three pits; each correlates with a nodule protruding from the ventro-medial wall. From each pit, a single, bifurcating hair projects dorsally to contact the single concretion within the statocyst lumen. No other static organs have been found in this animal. Thus, maintenance of equilibrium in this species appears to be under the control of but six hairs, three in each statocyst. Innervation of each statocyst is provided by a branch of a nerve which connects anteriorly with the last abdominal ganglion.     

Responses of hair cells to statocyst rotation

A new technique is described for stimulating hair cells of the Hermissenda statocyst. The preparation and recording apparatus can be rotated at up to 78 rpm while recording intracellular potentials. Hair cells in front of the centrifugal force vector depolarize in response to rotation. Hair cells in back of the centrifugal force vector hypoerpolarize in response to rotation. Mechanisms by which the hair cell generator potential might arise are examined.     

Otoliths developed in microgravity

Little is known about mechanisms that regulate the development of the otoliths in the gravity-sensing organs. Several reported experiments suggest that the growth of the otoliths is adjusted to produce a test mass of the appropriate weight. If this is the case, larger than normal otoliths would be expected in animals reared in reduced gravity and a reduced mass, relative to 1-g controls, would be expected in animals reared at elevated g. In gastropod mollusks, the gravity-sensing organ is the statocyst, a spherical organ whose wall is made largely of sensory receptor cells with motile cilia facing the lumen. Dense statoconia in the cyst lumen interact with cilia of receptor cells at the bottom of the cyst and action potentials in their axons carry information on direction and magnitude of gravity and linear acceleration. In the marine mollusk, Aplysia californica, larvae reared at 2 to 5-g, the volume of statoconia was reduced in a graded manner, compared to 1-g control animals. In the statocyst of the fresh-water pond snail, Biomphalaria glabrata, reared in space in the Closed Equilibrated Biological Aquatic System (CEBAS), the number and total volume of statoconia was increased approximately 50%, relative to ground-reared controls. Lychakov found the utricular otolith to be 30% larger in space-reared Xenopus, whereas we found the saccular otolith to be significantly larger in newt larvae reared in space. In cichlid fish reared on a centrifuge, the saccular otolith was smaller than in 1-g controls. Here, we demonstrate that the otoliths of late-stage embryos of the swordtail fish, Xiphophorus helleri, reared in space on STS-89 and STS-90 (Neurolab) were significantly larger than those of ground-controls reared in functionally identical hardware.     

Model of statoconia accumulation in gravireceptors of mollusks

The kinetics of formation and accumulation of statoconia are different for Aplysia californica and Biomphalaria glabrata. In Aplysia californica, the fast growth of statoconia number occurs after the critical size (approximately 45 micrometers) of statocyst is reached; then the increase of statoconia number is proceeding with the nonmonotonic rate during the life of an animal. In Biomphalaria the growth of statoconia number occurs only in the initial phase. Then long-term evolution of statoconia in the absence of their generation is the result of their growth in the cyst lumen. In the case of Aplysia californica it is not clear whether a temporal change of the statoconia size distribution (SSD) is caused by statoconia growth in the cyst lumen similar to that in Biomphalaria (Model 1) or statoconia growth takes place in supporting cells until their release into the cyst lumen occurs. (Model 2). This problem is of practical importance because the majority of experiments related to the development of molluscan gravireceptors in altered gravity dealt with an initial phase of statoconia evolution in Aplysia californica and Biomphalaria glabrata. The purpose of the present work is the application of mathematical modeling to the analysis of mechanisms of statoconia formation by supporting cells.     

Development of gravity-sensing organs in altered gravity conditions: opposite conclusions from an amphibian and a molluscan preparation

Researchers examined early otolith development in microgravity using fertilized eggs of the Japanese newt, Cynops pyrrhogaster in space flight. Ground experiments examined statocyst, embryonic statolith volume, and statoconia in the post-metamorphic marine mollusk Aplysia californica reared at 1-g and 2-5.7-g. Results indicate that exposure to hypergravity decreased the otolith mass to compensate for increased weight in Aplysia. In the Cynops, there was no compensatory difference in otolith mass, though otoconia production in the endolymphatic system was enhanced.     

Electrical Activity in the Chemoreceptors of the Blowfly : III. Dendritic action potentials

All sizes of the chemosensory hairs on the labellum of the blowfly, Phormia regina, contain neurons which respond to chemical stimulation with diphasic impulses if the recording electrode is at the tip of the hair. The impulses are initially positive, then become negative and return to the base line. The negative phase can be abolished by the action of anesthetics (xylocaine, cocaine, procaine, chloral hydrate) and tetrodotoxin, an extract from the puffer fish. The negative phase is increased by strong salts, mechanical injury, and thermal injury, which also act to reduce or abolish the positive phase. Simultaneous recordings from the side and the tip of the hair show that both positive and negative phases appear first at the side. These results indicate that the impulse is initiated in or near the cell body at the base of the hair and under certain conditions is propagated antidromically up the dendrite to the tip.     

Mechanosensory afferents innervating the swimmerets of the lobster

Feathered hair sensilla fringe both rami of the lobster (Homarus americanus) swimmeret. The sensory response to hair displacement was characterized by recording afferent impulses extracellularly from the swimmeret sensory nerve while deflecting sensilla with a rigidly-coupled probe or controlled water movements. Two populations of hairs were observed: "distal" hairs localized to the distal 1/3 of each ramus and "proximal" hairs near its base. Distal hairs are not innervated by a mechanosensory neuron but instead act as levers producing strain within adjacent cuticle capable of activating a nearby hypodermal mechanoreceptor. Hair deflections of 25 degrees or more are required to evoke an afferent response and this response is dependent on hair deflection direction. The frequency and duration of the afferent discharge evoked are determined by the velocity of hair displacement. Each proximal hair is innervated by a single mechanosensory neuron responding phasically to hair deflections as small as 0.2 degrees in amplitude. Deflection at frequencies up to 5 Hz elicits a single action potential for each hair movement; at higher frequencies many deflections fail to evoke an afferent response. These sensilla, which are mechanically coupled, may be activated by the turbulent flow of water produced by the swimmerets during their characteristic beating movements.     

Hair Cell Interactions in the Statocyst of Hermissenda

Hair cells in the statocyst of Hermissenda crassicornis respond to mechanical stimulation with a short latency (<2 ms) depolarizing generator potential that is followed by hyperpolarization and inhibition of spike activity. Mechanically evoked hyperpolarization and spike inhibition were abolished by cutting the static nerve, repetitive mechanical stimulation, tetrodotoxin (TTX), and Co⁺⁺. Since none of these procedures markedly altered the generator potential it was concluded that the hyperpolarization is an inhibitory synaptic potential and not a component of the mechanotransduction process. Intracellular recordings from pairs of hair cells in the same statocyst and in statocysts on opposite sides of the brain revealed that hair cells are connected by chemical and/or electrical synapses. All chemical interactions were inhibitory. Hyperpolarization and spike inhibition result from inhibitory interactions between hair cells in the same and in opposite statocysts.     

Hair Cell Generator Potentials

A technique is introduced using a piezoelectric device to stimulate hair cells of a molluscan statocyst while recording their responses intracellularly. Statocyst displacements produced with the technique are calibrated with stroboscopic photography. Properties of the hair cells' response to currents and mechanical stimulation are studied. The hair cell generator potential arises from a conductance increase and, for a certain range, is a logarithmic function of the amplitude of the displacement stimulus.     

不同品种来源广藿香叶表皮毛特征比较分析

叶表皮毛特征作为药用植物的"微性状"特征,被越来越多地应用于植物学分类研究中,表皮毛的生长发育状态可以直接影响药材的整体品质。广藿香主要含有挥发油成分,叶表皮毛中的腺毛是其挥发油的主要合成分泌场所,而非腺毛可以起到保护作用,影响其长势和产量。为了探讨叶表皮毛特征在广藿香品种鉴别方面的意义,该研究以8个不同品种来源以及2个采收期(6月末和10月末)的广藿香的顶叶、第4对生叶及底叶为材料,采用水合氯醛透化法制片,于光学显微镜下观察拍照,使用Microsoft Excel 2019统计数据,采用SPSS 23.0统计学软件对叶表皮毛密度、腺毛直径进行方差分析、主成分分析和聚类分析。结果表明:同一品种来源,不同采收期的广藿香叶表皮毛密度有显著性差异,6月末采收的广藿香叶表皮毛密度显著低于10月末采收的;根据叶表皮毛(非腺毛、头状腺毛、盾状腺毛)密度及腺毛直径,可将相同采收期的8个不同品种来源广藿香分为4类,且可以将已知品种来源的南香、肇香和牌香分开,与传统分类结果有着很好的一致性。叶表皮毛密度和腺毛直径在广藿香的分类中具有一定的参考意义,并为广藿香药材品质形成机制的研究提供参考。     

Differential Effects of Chloral Hydrate and Pentobarbital Sodium on a Cocaine Level and Its Catecholamine Response in the Medial Prefrontal Cortex: A Comparison with Conscious Rats

Abstract: Adult male Sprague-Dawley rats anesthetized with chloral hydrate and pentobarbital sodium were used as two different treatment groups. Conscious rats were used as a control group. By using baseline (precocaine) concentration as 100%, after cocaine administration (3.0 mg/kg i.v.), the maximal dopamine (DA) increase occurring at the first microdialysis collection period (20 min) in the medial prefrontal cortex was 299 ± 46% for the chloral hydrate group, 168 ± 12% for the pentobarbital sodium group, and 325 ± 23% for the conscious group. At the same time, norepinephrine (NA) increases reached a maximum and were 162 ± 20%, 100 ± 5%, and 141 ± 17%, respectively. The maximal changes of DA and NA in the chloral hydrate group and in the control group were both significantly higher than that in the pentobarbital sodium group. Meanwhile, the cocaine concentration was higher over a 100-min period of time in the chloral hydrate group when compared with the pentobarbital group and the control group. The peak cocaine concentration in dialysate occurred in the same time slot of maximal DA and NA responses, which were 0.65 ± 0.08, 0.30 ± 0.02, and 0.41 ± 0.05 µ M , respectively. Anesthetics suppress the pharmacologic response of neurons, which may explain the difference in catecholamine response between the pentobarbital sodium and the conscious groups. Conversely, because there was no significant difference in DA and NA response between the chloral hydrate group and the conscious group, it may possibly be due to the balancing effect between the higher existing cocaine concentration and the anesthetic suppression on pharmacological response of neurons in the chloral hydrate group. The effect of guide cannula implantation on the cocaine-induced catecholamine response was also evaluated.     

一种简易小鼠尾部皮肤移植教学实验改良方法的建立

目的 为本校生物工程系实验动物学课程小鼠尾部皮肤移植实验课提供一种合适的操作方法.方法 分别用0.7%、1%、1.5%戊巴比妥钠和10%水合氯醛对小鼠进行麻醉试验,选出合适的麻醉方法;再进行小鼠尾部皮肤移植,对比玻璃套管法和创可贴法2种包扎方法的效果,以选出合适的手术方法.结果 0.7%和1%戊巴比妥钠麻醉持续时间过短,1.5%戊巴比妥钠和10%水合氯醛能维持足够手术操作所需的时间,但1.5%戊巴比妥钠易导致动物死亡,故采用10%水合氯醛麻醉小鼠较合适;玻璃套管法包扎的效果较创可贴法为好且更简便.结论 10%水合氯醛麻醉结合玻璃套管法包扎进行小鼠尾部皮肤移植是一种有效、简便、经济的小鼠尾部皮肤移植手术实验方法;此方法技术失败率低,适合学生实验课采用.     

Notes on Technic

Fixation in 20 to 40% pyridin or in 10% chloral hydrate followed by 10 to 40% pyridin gave the most consistent staining of pericellular structures in the spinal cord of cat. Chloral hydrate perfusion and soaking followed by ammoniated alcohol (Hoff's application of Cajal's method) was uniformly successful only when pyrogallol instead of hydroquinone was used as a reducing agent. Perfusion of the animal with chloral hydrate gave a rather questionable degree of improvement over fixation by simple soaking. The difficulty in selecting a routine procedure as the “best” became apparent when no single experimental variation was outstandingly superior in all animals.