Molecular Architecture of a Sensory Ending Using Correlative Fluorescence and Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

The effects of microtubule dissociating agents on the physiology and cytology of the sensory neuron in the femoral tactile spine of the cockroach, Periplaneta americana L

Microtubules are prominent cellular components of the mechanosensory and chemosensory sensilla associated with the insect cuticle, and a range of hypotheses have been proposed to account for their role in sensory transduction. Chemical agents such as colchicine and vinblastine, which dissociate microtubules, also interfere with transduction in these sensilla, and this has been attributed to their anti-microtubule activity. We have now examined the dynamic properties of sensory transduction in the mechanosensitive neuron of the cockroach femoral tactile spine, after the application of colchicine, vinblastine and lumicolchicine. Concurrently we have examined the ultrastructure of the same sensory ending by transmission electron microscopy. All of the drugs reduced the mechanical sensitivity of the receptor. Colchicine and vinblastine achieved this reduction without altering the dynamic properties of the receptor but lumicolchicine changed the dynamic response, and increased the relative sensitivity to rapid move­ments. Conduction velocity, another measure of neuronal function, which relies upon ionic currents flowing through the membrane, was reduced by all three drugs. The effects of the drugs upon the ultrastructure of the sensory ending were also disparate. In the case of colchicine there was complete dissociation of microtubules in the tubular body and distal dendrite before a total loss of mechanical sensitivity. Vinblastine was less effective in dissociating microtubules, although more effective in the reduction of mechanical sensitivity. With lumicolchicine the dominant morphological effect was a severe disruption of the dendritic membrane. We conclude from these experiments that microtubules are not essential in the transduction of mechanical stimuli by cuticular receptors and that the effects of these drugs upon mechanosensitivity are not directly related to their dissociation of the microtubules in the tubular body, but are more likely to arise from actions upon the cell membrane. These actions could include effects upon tubulin in the membrane or upon other membrane components.

Further morphological and electrophysiological studies on snake muscle spindles

1. Electron microscope studies using ruthenium red, horseradish peroxidase, and colloidal lanthanum disclosed a feature characteristic of the snake spindle capsule: it consists of a single layer of flattened cells connected in series, each of which forms a cylinder enclosing the intrafusal fiber. 2. In intact spindles none of the markers penetrated the capsule. The diffusion barrier appeared to be located along the intercellular space near the inner surface of the capsule layer. Through the openings at the ends of the capsule the marker gained access to the periaxial space, but only for a limited distance. 3. Through a punctured hole in the capsule, ruthenium red had access to the periaxial space, disclosing a dense network consisting of fine filaments and granular structures. Since this space was also shown to contain alcian blue-positive substrate, the network may represent acid mucopolysaccharide (probably hyaluronic acid + protein molecules), which may account for the observed limited longitudinal diffusion of this dye in the periaxial space. The intercellular space between the intrafusal fiber, the sensory ending, and the satellite cell was filled with ruthenium red, and no special junction was found between these cells. 4. Using glass microelectrodes, potential profiles across the sensory region of muscle spindles of the snake were examined. 5. When the electrode was advanced through the capsular region, two steps of negative potential were usually observed. The first negative step of 5-10 mV was confirmed by fluorescent dye injection to be due to penetration of the capsule. The second larger step of 40-60 mV negative to the bathing solution was demonstrated by the dye injection to be due to penetration of the intrafusal fiber. Occasionally, a negative potential of 20-30 mV relative to the bathing solution was also recorded. This potential, characterized by small spikelike potentials occurring in synchrony with nerve impulses, was considered to be due to penetration of the sensory ending. 6. both the capsule and the intrafusal fiber showed rectification to injected currents. From input resistance, input capacitance and surface area specific resistance and capacitance were estimated with some assumptions to be 4.1-4.8 k omega . cm2 and 5.1-14.3 micro F/cm2 for the capsule, and 1.1-2.8 k omega . cm2 and 11-23 micro F/cm2 for the intrafusal fiber. The intrafusal fiber of short-capsule spindles lacked an action potential, whereas that of long-capsule spindles responded with an all-or-none action potential to depolarizing current. 7. In about half of the short-capsule spindles examined (8/15), current injected through an intracellular electrode in the intrafusal fiber neither initiated impulses in the afferent axon nor affected background nerve activity. The rest of the spindles responded to depolarizing current with initiation of nerve impulses and concomitant maintained contraction of the intrafusal fiber during applied current...

Structure and Abundance of Receptors of the Unspecialized External Epithelium of Nassarius Reticulatus [Gastropoda, Prosobranchia]

The epithelial receptors of Nassarius are primary sense cells. The sensory endings are abundant on the tentacle tips, anterior border of the foot and ventral external surface of the siphon tip. Densities are a small multiple of 1032 endings/mm2.Type 1 ciliated endings are of ubiquitous occurrence. The ultrastructure of type 1 endings is uniform, though the static and motile bundles of sensory cilia may be distinguished by the random or uniform orientation of the ciliary subfibrils and basal feet. Ciliated receptors probably include both chemo- and mechanoreceptors.A distinct ultrastructural type of sensory ending (type 2), confined to the siphon tip, consists of a small pit in the peripheral dendrite close to the epithelialsurface. The pit contains whorled microvilli and a few cilia. It may be photoreceptive.A small amount of convergence of sensory information may occur in peripheral nerve.

The anatomy and ultrastructure of the labyrinth of the lamprey ( Lampetra fluviatilis L.)

The anatomy of the labyrinth of the lamprey ( Lampetra fluviatilis ) is described. The am­pullae of the two semicircular canals are each equipped with a complex three-armed sensory crista. They may be considered homologues of the ampullae of the vertical canals of the gnathostomes. However, the complexity of their cristae encourages the assumption that they may cover a spatial range of responses to angular acceleration which includes responses to accelerations in a horizontal plane controlled in the gnathostomes by the horizontal semi-circular canal. The otolith-bearing end organs are found to be located on a common macular structure. This is subdivided into an anterior horizontal, a vertical, and a posterior horizontal macula, each of which portions carries a characteristic arrangement of sensory cells. On the basis of an electronmicroscopic analysis of the orientation of the hair cells in the three main portions of the macula a revision of the homologies found in the older literature appears to be called for. It is suggested to homologize the anterior horizontal macula with the macula utriculi, the vertical macula with the macula sacculi and the posterior horizontal macula with the macula lagenae of the labyrinth of gnathostome animals. A separate sensory ending in the dorsal part of the labyrinth, the dorsal macula, may be the homologue of the macula neglecta. Ultrastructurally the end organs of the lamprey labyrinth conform with those of the gnathostome labyrinth with the exception of the presence of a new type of sensory hair cell which is equipped with a stiff kinoeilium of extraordinary length accompanied by extremely short stereocilia. This cell is found preponderantly in the vertical macula (macula sacculi). A striated organelle in the cytoplasm of the hair cells appears to be uniquely confined to the labyrinth of the lamprey. Morphologically the lamprey labyrinth differs from all other chordate labyrinths including that of its fellow cyclostome Myxine by the presence of large ciliated chambers in its centre in which long and powerful cilia maintain a permanent pattern of four endolymph vortices. The ciliated chambers are in open communication with the ampullae and with the spaces con­taining the otolith-bearing maculae. The analysis of the functional significance of the anatomical and ultrastructural findings will be described in a separate paper.