Geoffrey Q. Fox

Immunohistochemical localization of cholinergic nerve terminals

SummaryMost of the published light-microscopic methods for the localization of cholinergic nerve pathways present various difficulties of interpretation. The production and characterization of an antiserum that binds specifically to cholinergic terminals is described. The antiserum was raised to small synaptosomes prepared from the purely cholinergic electric organ of Torpedo marmorata. It was shown to lyse cholinergic synaptosomes in a mixed population derived from guinea-pig cortex. After partial purification by adsorption onto nonspecific antigens, it was used to label nerve endings in several tissues of Torpedo, rats and guinea pigs using indirect immunofluorescence histochemistry. The antiserum appears to provide a highly specific means of localizing cholinergic nerve endings in these tissues.

A morphometric analysis of exocytosis in KCl-stimulated bovine chromaffin cells

Abstract.Transmission electron microscopy has been used to morphometrically evaluate exocytosis in bovine adrenal medulla chromaffin cells as the mechanism of catecholamine release. Purified cell suspensions were stimulated with KCl at varying strengths and durations and then conventionally processed for ultrastructural analysis. Quantitation of exocytotic images of dense cored chromaffin granules was a major objective and such images were found in all preparations, attesting to the efficacy of chemical fixation to preserve this event. However, because hundreds of cell profiles had to be screened to find a single granule in the process of release this low frequency precluded any meaningful correlations with estimates of granular involvement based on catecholamine release. Neither KCl molarity nor duration altered this finding nor did these variables significantly affect other parameters linked to exocytotic activity. For example, cell size and numbers of ’empty’ granules and vesicles remained constant and attempts to label ’any’ organelle with 30-nm colloidal gold or lanthanum precipitate proved unsuccessful. In short, if exocytosis is responsible for release, it would appear to function without leaving a morphological trace. An alternative hypothesis, therefore, is outlined which better accommodates existing data.

Organotypic culture of embryonic electromotor system tissues from Torpedo marmorata

Abstract An explant culture system has been used to study the electric organ and electric lobe tissues of Torpedo marmorata at different stages during the development of the electromotor system. The myotubes in tissue expiants, taken from the electric organ primordia of 33–38 mm body-length embryos prior to electrocyte differentiation, contract spontaneously on explantation and have electrogenic membranes. The myotubes subsequently lose these properties in vitro and can differentiate in the absence of neural tissue into immature electrocytes which have morphologically characteristic postsynaptic membranes. Isolated expiants of differentiated electric organ tissue from 60–100 mm body-length embryos can be maintained for 3 to 4 weeks in vitro but cellular outgrowth is minimal. In contrast, a rapid, dense outgrowth of cells and a subsequent regeneration of myotubes occurs when differentiated electric organ explants are co-cultured with electric lobe tissue from embryos of the same stage. Cellular outgrowth from differentiated electric-organ tissue expiants can be stimulated by spinal cord, medulla, cerebellum and heart tissues but a subsequent regeneration of myotubes has not been observed. Myotube regeneration in the presence of electric lobe tissue is maximal with tissue from 60–80 mm body-length embryos. The myotubes that regenerate from differentiated electric organ expiants have not been observed to differentiate into electrocytes. Neuritic outgrowth in vitro occurs with electric lobe tissue taken at two different embryonic stages. The first stage corresponds to a period when most of the neuroepithelial cells in the lobe anlagen are withdrawing from the mitotic cycle and projecting axons into the branchial arches. The second, later stage is when the electromotorneurones are normally generating axon collaterals that are invading the interelectrocyte space of electrocyte columns. Maximum neuritic outgrowth at this second, later stage is obtained with tissue from 60–80 mm body-length embryos. Although neuritic invasion of electrocyte column expiants can be obtained in electric organ—electric lobe co-cultures at this later stage, synapses similar to those observed during the early stages of synaptogenesis in the electric organs in vivo have not been observed in vitro .

The morphology of the oval nuclei of neonatal Torpedo marmorata

SummaryThe morphology of the oval nucleus of neonatal Torpedo marmorata is described at the light and electron microscopic level of examination. The nucleus is unique relative to other central electromotor centers of electric fish so far described being bilaterally symmetrical, composed of two nerve cell types, and possessing no gap junctions between neurons and their processes. This particular structural plan presents difficulties in accounting for presumed synchronous discharge since it has been strongly argued that electrotonic coupling by means of gap junctions is the primary process by which synchronization is accomplished. Close membrane apposition and dendritic bundling, common features within the nucleus, are discussed as possible alternative structural correlates.

A morphometric analysis ofTorpedo synaptic vesicles isolated by iso-osmotic sucrose gradient separation

The presynaptic terminal vesicle population of Torpedo electric organ is heterogeneous in size, consisting of two prominent subpopulations that comprise 80% of the total. The use of standard iso-osmotic sucrose gradients with zonal centrifugation to isolate vesicle fractions that co-localize with the acetylcholine (ACh) peak results in the recovery of: (1) 10% of the total estimated vesicle population; and (2) a single 68-nm diameter vesicle size class. The whereabouts of the major 90-nm subclass, which accounts for 60% of the total terminal population and which has long been considered to represent the resident ACh population, has been investigated. Assuming this subclass to have undergone severe osmotic stress, the effects of hypo- and hyper-osmotic salines, buffers and fixatives were examined and found to produce only negligible changes on vesicle size. Isolation of vesicles by hypo-osmotic shocking of synaptosomes purified on a Ficoll gradient, however, resulted in a reasonable approximation of the in situ distribution. As the iso-osmotic sucrose gradient procedure utilizes frozen blocks of electric tissue, this step is suspected of being involved in the loss, perhaps because of the slow freezing rates employed. These findings indicate that the 90 nm subclass is lost rather than transformed during isolation by sucrose gradient separation and that dimensionally, the cholinergic vesicle is a constant-sized and relatively stable structure.

Morphological, physiological and biochemical observations on skate electric organ

SummaryThe electric organs of two species of skate have been examined morphologically, physiologically and biochemically. They can be easily dissociated into innervated or denervated component electrocytes by a Torpedo Ringers solution containing 1% collagenase. Collagenase treatment did not, however, separate the Schwann cell cover capping the synaptosomes. Isolated electrocytes generate normal MEPP frequencies and show evoked responses for two days in Torpedo Ringers. The nerve terminals retain excitability and transmitter release properties up to the time of separation. Since isolated terminals and denervated electrocytes show normal ultrastructural characteristics for up to 12 h, the skate electric organ provides several preparations which are not attainable with Torpedo tissue. Acetylcholine (ACh) content of supernatant fractions containing the synaptosomes was comparable to that found in Torpedo (sps.). Collagenase specifically eliminates the basal lamina associated with the synaptic junctional region. Neuronal cell death and synaptic terminal degeneration were also noted in the adult organs of both species. The skate electric organ is ideally suited for the study of cholinergic development and transmission.

Dynamic responses of presynaptic terminal membrane pools following KCl and sucrose stimulation

The cholinergic presynaptic terminals of Torpedo electric organ have been examined morphometrically following stimulation by KCI and sucrose. The objective was to confirm correlations predicted by the vesicle hypothesis between miniature end-plate potentials (MEPPs) and morphometric changes in terminal ultrastructure. Both secretegogues generated high frequencies of MEPPs and also distinctive though differing ultrastructural changes. The synaptic vesicles show classes of 68 and 90 nm diameters and both store acetylcholine (ACh). KCl stimulation depleted the 90 nm class first whereas sucrose reversed the order of depletion. Very few instances of actual vesicle fusion were seen. Dose-response correlations between vesicle density and secretegogue strength (mM) and duration were higher with sucrose. Both secretegogues produced declines in vesicle numbers and densities and yielded multimodal distributions of large vesicles with an average 160 nm mean diameter. No meaningful correlations were detected between numbers of MEPPs and vesicles and little evidence was found to indicate that vesicles were fusing to terminal plasma membrane in numbers approximating MEPP release. Linear regression analysis was used to quantitatively examine relationships between the vesicle membrane pool and other pools of the putative exo/endocytotic pathway. Correlation coefficients between vesicle and terminal plasma membrane pools were non-significant and of positive sign, indicating independent, similar responses. Non-significant, negative coefficients were obtained when vacuole and 160 nm vesicle membrane values were included. These tests further argue against claims that vesicles are actively fusing with the plasma membrane. These conflicting findings for both secretegogues preclude meaningful correlations between vesicle changes and numbers of MEPPs generated and again emphasize the difficulty of validating the vesicle hypothesis by ultrastructural means. On the other hand, the study shows that vesicular, vacuolar and terminal membrane pools are dynamically changing during transmitter release, presumably interacting with cytosolic membrane constituents. A dynamical release process therefore has been proposed to account for the two classes of MEPPs, the rapid changes in class ratio and the mutable characteristics of the bell-MEPP that presently challenge the quantal-vesicular claims of prepackaged, immutable, exocytotically released packets of transmitter. This model features a state for each MEPP class with class and size determined at moment of release. For example, a single flicker of a channel would generate the sub-MEPP (defined subunit of an MEPP) and 7-20 flickering channels would generate the bell-MEPP.

Dynamic responses of presynaptic terminal membrane pools to electrical stimulation

The anatomical tenets of the quantal-vesicular hypothesis of neurotransmission are a 1:1 ratio between numbers of releasable quanta and vesicles, a reciprocal response between vesicle and terminal membrane pools and constancy of the total membrane pool. We have used electrical stimulation and morphometry to study these relationships in the cholinergic presynaptic terminals of Torpedo electric organ. Our results show that during neurotransmission changes in vesicle numbers do not correlate with quantal release, vesicle and terminal membranes do not change in reciprocal fashion and total nerve terminal membrane does not remain constant. We conclude that these vesicular tenets of quantal release are not verifiable at the Torpedo electric organ junction.

Effect of Nerve Stimulation, K+ Saline and Hypertonic Saline on Classes of Quanta, Quantal Content and Synaptic Vesicle Size Distribution of Torpedo Electric Organ

The Torpedo electric organ is an extremely useful preparation for the study of cholinergic mechanisms because of its easy accessibility to biochemical, morphological and physiological methods. A kilogram of electric organ can be obtained from one animal of which 10% are synaptic terminals. At least 3 classes of synaptic vesicles have been described based on density (Stadler and Kiene, 1987) and 2 classes based on diameter (Zimmermann and Whittaker, 1974, 1977). Its unique structure of vertical columns of stacked electrocytes enables one to uniformly field stimulate the tissue and obtain reliable quantitative end-plate potential.

Development of the electromotor system of Torpedo marmorata: Distribution of extracellular matrix and cytoskeletal components during acetylcholine receptor focalization

SummaryA combination of direct fluorescence and indirect immunofluorescence microscopy has been used to compare the distribution of the acetylcholine receptor with the distribution of major cytoskeletal and extracellular matrix components during electrocyte differentiation in the electric organs of Torpedo marmorata. Laminin, fibronectin and extracellular matrix proteoglycan are always more extensively distributed around the differentiating cell than the acetylcholine receptor-rich patch that forms on the ventral surface of the cell. The distribution of acetylcholinesterase within the ventral surface of the differentiating electrocyte closely resembles the distribution of the acetylcholine receptor. Areas of apparently high acetylcholine receptor density within the ventrally forming acetylcholine receptor-rich patch are always areas of apparently high extracellular matrix proteoglycan density but are not always areas of high laminin or fibronectin density. Desmin levels appear to increase at the onset of differentiation and desmin initially accumulates in the ventral pole of each myotube as it begins to form an electrocyte. During differentiation F-actin-positive filament bundles are observed that extend from the nuclei down to the ventrally forming acetylcholine receptorrich patch. Most filament bundles terminate in the acetylcholine receptor-rich region of the cell membrane. Electronmicroscopic autoradiography suggests that the filament bundles attach to the membrane at sites where small acetylcholine receptor clusters are found. The results of this study suggest that, out of the four extracellular matrix components studied, only the distribution of acetylcholinesterase (which may be both matrix- and membrane-bound at this stage) closely parallels that of the acetylcholine receptor, and that F-actin filament bundles terminate in a region of the cell that is becoming an area of high acetylcholine receptor density.