Detached, purified nerve terminals from skate electric organ for biochemical and physiological studies.
The electromotor system of Torpedo is a well-established model for studies on the chemistry of cholinergic synaptic transmission (for review see Whittaker, 1992). Several other electric fish (e.g., Electrophorus, Malapterusus, and Raja) have comparable organs but, for biochemical studies, these have received little attention. The cholinergic nature of transmission in skate (Raja) electric organ was established by means of microelectrode techniques (Brock et al., 1958; Bennett, 1961) and enzyme histochemistry of AChE (Couteaux, 1963). More recently, quantal release of transmitter from these terminals was shown to be similar to that found at mouse and frog neu-romuscular junctions (Kriebel et al., 1987, 1988). In common with these neuromuscular synapses, but unlike the neuroelectrocyte synapses in Torpedo, the synaptic vesicles in skate are homogeneous in size (Fox et al., 1988). Innervated electrocytes that retain electrical responsiveness to presynaptic stimulation can be isolated from skate electric organ following collagenase treatment at room temperature (Fox et al., 1990). These authors also showed that extended exposure of the organ to collagenase results in denervation of electrocytes and formation of clumps of detached nerve terminals encapsulated by Schwann cells. We have used these observations as a starting point for the present study, in which we combined biochemical measurements with microscopy and electrophysiology to further investigate the innervation of skate electric organ and to monitor in greater detail the collagenase-induced detachment of nerve terminals. We report here the conditions that produce functional electrocytes and detached nerve terminals freed from adherent Schwann cells. A preliminary report of some of this work was presented at the General Scientific Meeting of the Marine Biological Laboratory, Woods Hole, Massachusetts (Dowdall et al., 1989).
Materials and Methods
Most experiments were done using adult specimens of Raja erinacea supplied by the Marine Resources Department of the Marine Biological Laboratory in Woods Hole. These varied in maximum width from 16.5-37 cm. Fish were killed with a blow to the head. Tails were amputated and electric organs removed by blunt dissection. In some experiments, specimens of R. naevus (cuckoo ray) were used. These were obtained from the Gatty Marine Station, St. Andrews, Scotland, and maintained in aquaria at 15 [degrees] C in Nottingham, England.
Treatment of electric organs
Dissected organs were placed in plastic petri dishes and immersed in skate saline of the following composition (mM): NaCl (280), KCl (12), Ca[Cl.sub.2] (10), Mg[Cl.sub.2] (2), urea (360), glucose (1), and HEPES buffer (10) pH 7.3. Measurements were made of the total length and wet weight of organs. Additionally, single transverse sections of 5 mm were removed from the middle of one of each pair of organs; after being weighed, these were used to determine the number of electrocytes in the organ. Counting of dissociated electrocytes was possible after they had been incubated overnight in 1% (w/v) collagenase at room temperature. Wells of a plastic macrophage migration plate covered with glass coverslips were used for incubation chambers. In some experiments the diameter of electrocytes was determined by aligning cells edge to edge over a 2-[mm.sup.2] reticle.
Total tissue homogenates for measuring enzyme activities were prepared from organs that were snap-frozen in liquid nitrogen, then reduced to powder. Tissue powders were extracted with 50 mM phosphate buffer (pH 7.4) containing 300 mM NaCl, 20 mM EDTA, and 0.5% (w/v) Triton X-100; a Teflon-glass homogenizer was used to produce 10% (w/v) homogenates.
Bulk electrocyte preparations were produced by incubating whole electric organs in an equal volume of skate saline containing 1% (w/v) collagenase (Worthington CLS I) for up to 7 days at 6 [degrees] C. For comparison, higher temperatures were also used in some experiments. In all cases the incubation vessels (normally 35-mm culture dishes) were left undisturbed throughout the incubation. Collagenase dissolved in skate saline contained insoluble material, and this was removed by centrifugation in a microfuge and gel-filtration through a Sephadex PD 10 column. The collagenase-containing fraction (brown) was recovered and adjusted where necessary to 1%. Collagenase-treated organs were dispersed by gentle aspiration of material and medium and expulsion with plastic transfer pipettes (10 cycles).
Dissociated electrocytes were washed free of smaller cellular material (fragments of electrocytes and nerve fibrils, glia and nerve terminals) by sequential washing and sedimentation at unit gravity in polypropylene centrifuge tubes at 0-4 [degrees] C. Tissue suspensions ([approximately]20% w/v) in skate saline formed loose pellets of electrocytes when left to settle for 3 min. The supernatant was gently removed, retained, and replaced by an equal volume of fresh saline. Following resuspension of the loose pellet, this washing procedure was repeated 3-4 times, with all washings being combined with the initial supernatant. Particulate material was concentrated from the supernatant fractions by centrifugation at 17,000 [g.sub.av] for 20 min with a Sorvall SS-34 rotor. The resultant pellet (fraction [P.sub.1,2]) was further fractionated into large ([P.sub.1]) and small ([P.sub.2]) particulate material by differential centrifugation in a microfuge (Sorvall Microspin 24S) for 1 min at 3000 rpm followed by 5 min at 13,500 rpm. Fraction [P.sub.1] contained fragments of electrocytes and nerve fibrils together with some smaller particles. None of this larger debris was seen in fraction [P.sub.2] which was therefore used as the crude nerve terminal preparation for subsequent biochemical analyses.
Unfixed samples of dissociated electrocytes in saline were viewed with standard light microscopy at low (binocular dissecting microscope) and high (Zeiss Axiophot) magnification. Nomarski and phase optics were used at the higher magnifications to identify detached nerve terminals. For electron microscopy, samples were fixed in 2.5% glutaraldehyde, 10% sucrose (0.292 M) in 0.2 M sodium cacodylate buffer pH 7.4 at 4 [degrees] C for 12-24 h. Primary fixation buffer was removed and samples were washed in fresh buffer prior to post-fixation in 1% osmium tetroxide in 0.4 M sodium cacodylate and en bloc staining with 1% uranyl acetate in 0.25 M sodium acetate buffer pH 5.5. Ethanol, propylene oxide, and Epon 812 were used throughout as dehydrating, transferral, and embedding agents.
Lactate dehydrogenase (LDH) was measured by following the oxidation of NADH spectrophotometrically (Johnson, 1960) in a final volume of 1 ml. Acetylcholin-esterase (AChE) was measured using a final incubation volume of 1 ml (Ellman et al., 1961). Choline acetyltransferase (ChAT) was used as a marker for nerve terminal cytoplasm and was measured by means of a radiometric assay described by Fonnum (1975), with the following modifications. The final incubation volume of 10-100 [[micro]liter] contained NaCl (300); EDTA (20); choline chloride (10); acetyl CoA (0.2); eserine sulfate (0.15); sodium phosphate buffer, pH 7.4 (50); 0.5% Triton X-100; and 3H- or 14C-labeled acetyl CoA (500 nCi/ml). Incubations were at 26 [degrees] C for time periods up to 2 h, and three time points were used for each determination. Reactions were terminated by transfer of aliquots (5-50 [[micro]liter]) to mini-scintillation vials containing 1.6 ml of 10 mM phosphate buffer and 0.67 ml acetonitrile containing sodium tetraphenyl-boron (10 mg/ml). Labeled ACh was measured by liquid scintillation after addition of 3.5 ml of water-immiscible scintillation fluid, gentle shaking, and phase separation. Labeled acetyl CoA remained in the lower (aqueous) layer and therefore made no contribution to the count rate. This procedure gave the same results as one in which the volumes of reagents after termination were 3 times greater (as originally described by Fonnum, 1975). Both collagenase and skate saline were found to inhibit ChAT activity when added to extracts of whole electric organ. This inhibitory effect was therefore minimized by resuspension of pelleted fractions in ice-cold 0.45 M NaCl followed by a brief centrifugation in a microfuge. This washing procedure was repeated prior to enzyme assay.
Dissociated electrocytes were bathed in saline and viewed with a dissection microscope. Excitatory miniature endplate potentials (MEPPs) and evoked potentials (EPPs) were recorded with standard microelectrode techniques. EPPs were produced with supramaximal stimulation with either a suction electrode or field stimulation. For suction electrode stimulation, the surface of electrocytes was drawn against the suction electrode tip. Field stimulation was achieved by placing Ag-AgCl wires along the sides of the bath.
Electrocytes show few, if any, spontaneous MEPPs, and intracellular MEPPs are very small (Kriebel et al., 1987). MEPP frequencies and amplitudes were increased by pushing the electrode against the cytoplasmic surface to record focally. The MEPPs studied here are "pressure evoked." Healthy nerve endings are indicated by low MEPP frequencies and sensitivity to electrode pressure such that frequencies are reversibly increased as the electrode is advanced. The condition and density of endings during collagenase treatment were evaluated from the response of the terminal to electrode pressure, which induced MEPPs, and by field stimulation, which generated EPPs.
Electrocyte numbers and size
Skates have spindle-shaped electric organs that are slightly flattened dorsoventrally. In the present study, individual organs of R. erinacea varied from 9-22 cm in length and from 2.5-5.0 cm in maximum diameter. Organ wet weight varied from 116-1246 mg (mean 553 [+ or -] SEM 47; n = 34 fish) and was positively correlated with fish size. Electrocyte numbers per organ varied between 1.1 and 6.2 x [10.sup.3], with a mean of 2.5 and an SEM o [+ or -] 0.19 x [10.sup.3], n = 28. The mean number for R. erinacea is therefore essentially the same as that of 2.4 x [10.sup.3] for R. clavata (Brock et al., 1953). The small coefficient of variance demonstrates that the number of electrocytes was independent of organ size. Thus growth of electric organs is due to the increase in size of individual electrocytes. This conclusion is borne out by the mean electrocyte diameters, which varied from 0.25 to 0.65 mm for organ weights of 116 to 800 mg. Dissociated electrocytes as seen at low magnification are illustrated in Figure 1.
Viability and temperature studies
Viability studies of electrocytes in dissected organs were carried out with R. naevus and R. erinacea. R. naevus: Organs were stored in saline at 6 [degrees] C for periods up to 18 days and small (1-2 cm) sections removed as required. Penetration of intracellular electrodes into electrocytes was greatly facilitated by prior treatment of the tissue with collagenase. This resulted in the partial dissociation of the loosely packed electrocyte columns into smaller pieces in which individual electrocytes were attached to the fine branches of common efferent nerves. The higher temperatures were detrimental to electrocyte resting potentials and also produced sticky preparations. Temperatures of 30 [degrees] C or lower produced electrocytes with healthy resting potentials ([approximately]-60 mV). The most stable electrocytes were produced by extended incubation periods (11-18 h) at 6 [degrees] C. When these conditions were used, the physiological state of electrocytes in vitro was remarkably stable. Resting and evoked potentials were essentially unchanged even after 11 days in vitro [ILLUSTRATION FOR FIGURE 2 OMITTED]. After 14 days electrocytes gave smaller responses, and at 20 days no viable cells could be found.
Nachmansohn and Meyerhof (1941) and, more recently, Fox et al. (1990) determined the ACh content in skate electric organ. We therefore determined the activity of two cholinergic enzymes (ChAT and AChE) in extracts of whole organ and in fractions prepared from collagenase-digested organ. The activity of a general cytoplasmic enzyme, lactate dehydrogenase (LDH), was also determined as a measure of overall cellular density.
Both AChE and ChAT activities were tested for their sensitivities to established enzyme inhibitors. BW 284 c51 (a selective inhibitor) inhibited AChE activity completely at 1 mM and by 50% at 2 nM. Therefore, any contribution from butyrylcholinesterase to the measured activity was negligible. Bromoacetylcholine, a specific inhibitor of ChAT (Tucek, 1982), was an effective inhibitor in the skate extracts (50% inhibition at 10 nM and 95% at 1 mM). A sensitivity comparable to that of this inhibitor has been reported for ChAT from Torpedo electric organ (Eder-Coli and Amato, 1985).
Enzyme activities are given in Table I together with the corresponding values for Torpedo, some unpublished; [TABULAR DATA FOR TABLE I OMITTED] values for ACh from both organs are shown for comparison. The activities of all three enzymes were much lower in skate than in Torpedo, and the results for ChAT, in particular, closely parallel the previously reported concentrations for ACh (Fox et al., 1990). AChE activity was comparable to that previously reported for R. undulata (Nachmansohn and Meyerhof, 1941). Taken together, these findings show that the terminal volume in skate electric organ would be 3%-5% of that in Torpedo. If allowance is made for the lower cellular density in skate and the larger volume of individual electrocytes (as indicated by LDH activity), then the terminal volume of skate electric organ is between 10% and 18% of that found in Torpedo.
Denervation by collagenase action
The action of collagenase on skate electric organ has two distinct, but partially overlapping, phases: first, the dissociation of innervated electrocytes; and second, the denervation of individual electrocytes and the concomitant release of nerve terminals into the surrounding fluid (Fox et al., 1990). Biochemical markers (ChAT and AChE) were used to follow the time course of this denervation. A simple fractionation scheme was devised to separate detached nerve terminals from the much larger electrocytes (see Materials and Methods).
The activities of ChAT and AChE in the small particulate fraction from collagenase-treated electric organ are shown as a function of incubation time and temperature in Figure 3. At 6 [degrees] C the dissociation of the electric organ was very much slower than at room temperature, requiring days rather than hours to run its full course. AChE activity was detectable after 1 day but required a further 3 days to reach a maximum, thereafter declining. The appearance of ChAT activity in the small particulate fraction was broadly similar to this pattern, sharing the same peak at 4 days but with a more marked lag period. The loss of both activities after 4 days suggests lower yields of nerve terminals after extended periods of collagenase digestion.
The difference in tissue dissociation rates at 6 [degrees] C (this study) and 26 [degrees] C (Fox et al., 1990) was surprisingly large ([approximately]40-fold) and much greater than the 6.3-fold expected for a single enzyme reaction with a [Q.sub.10] of 2.5. The observed temperature dependence is, however, consistent with cooperative enzyme action. Collagenase, by itself, is known to be ineffective for total tissue dissociation and is therefore used in conjunction with other proteases. The collagenase preparation from Clostridium is not highly purified and contains sufficient quantities of contaminating proteases to effect tissue dissociation. The time course of denervation at 16 [degrees] C [ILLUSTRATION FOR FIGURE 3B OMITTED] was 5 times faster than at 6 [degrees] C, as judged by the rate of accumulation of AChE and ChAT activities in the small particulate fraction (peaking for both at 20.5 h). In parallel with these changes, soluble AChE activity progressively increased in the incubation fluid up to 20.5 h, thereafter declining (see legend to Fig. 3). At its maximum this was some 32% of the AChE activity of whole tissue and presumably arises from the action of collagenase on collagen-tailed isoforms of this enzyme. In Torpedo these forms constitute 40% of total AChE; the hydrophobic (6S) form, localized in the presynaptic membrane, accounts for the other 60% (Li and Bon, 1983). It seems likely that the AChE activity of fraction [P.sub.2] from skate electric organ corresponds to the 6S form in Torpedo.
Comparison of enzyme activities in fraction [P.sub.2] with those measured for total tissue suggests a very poor recovery of nerve terminals in this crude fraction. ChAT activity was only 0.05%-0.15% of total tissue, whereas AChE was 10%-20%. The large discrepancy between these figures is almost certainly due to the problem of measuring ChAT in extracts prepared from collagenase-treated tissue (see Enzyme assay section in Materials and Methods). It is possible that the nerve terminals were seriously damaged by the washing technique used to remove traces of collagenase. AChE activity is therefore a more reliable indicator of nerve terminal recovery than is ChAT.
The morphology of electrocytes after treatment of organs for 4 days at 6 [degrees] C is shown in Figures 4 and 5. At this stage, the dissociation of electrocytes from innervating nerves is almost complete, with the latter having a "balled-up" appearance that we refer to as a nerve-ending cluster (NE) in Figures 2 and 4. With Nomarski optics [ILLUSTRATION FOR FIGURE 4 OMITTED], we see clusters of isolated nerve endings. Separation of nerves from electrocytes leaves some nerve terminals attached to the electrocyte surface, as revealed by the electron micrograph shown in Figure 5. The terminals are 1-3 [[micro]meter] in diameter and are filled with electron-lucent synaptic vesicles together with somewhat larger clear vacuoles and numerous intraterminal mitochondria.
The most striking feature of the detachment process at 6 [degrees] C was the dissociation of Schwann cells from the terminals. In contrast, at room temperature the Schwann cells creep over newly exposed surfaces of nerve terminals and eventually encapsulate detached terminals (see fig. 15 of Fox et al., 1990). Conceivably, the lower temperature inhibits the motility of the Schwann cells and thus prevents encapsulation. When salines from washed electrocytes are examined with Nomarski optics, free nerve terminals can be identified [ILLUSTRATION FOR FIGURE 6 OMITTED]. Detached Schwann cells have quite a different appearance because of their single large nuclei.
Previous physiological studies on transmission at the neuro-electrocyte junction of skate were carried out on freshly dissected tissue, either when it was in an intact, untreated state (Kriebel et al., 1987) or after it had been briefly exposed to collagenase at room temperature (Fox et al., 1990). In view of the greatly extended periods of exposure to collagenase used in the present study, it was important to establish the basic electrophysiological properties of electrocytes so treated. Accordingly, electrocytes dissociated by 6 [degrees] C incubations were checked by using extracellular and intracellular microelectrodes. Dissociated electrocytes produced endplate potentials of 50100 mV when stimulated repetitively at 2 to 10 Hz with suction electrodes applied to innervating nerves (up to 4 days) or the innervated external surface of electrocytes (4-7 days). For incubation periods up to and including 4 days, MEPPs at normal, albeit low, frequencies were obtained. As in previous studies with fresh tissue, MEPP amplitudes of two size classes were detected (Kriebel et al., 1987, 1988). Histograms of MEPP amplitude distributions show two distinct classes. The larger class has a bell-shaped distribution, so these are referred to as bell-MEPPs; the smaller class has a skewed distribution, so these are referred to as skew- or sub-MEPPs (Fig. 7, traces A and [C.sub.1]). The intracellularly recorded MEPPs in Figure 7 are focally recorded and are in the millivolt range. When the electrode tip was in the middle of the electrocyte, MEPPs were less than 100 [micro]V because of the low input impedance. Therefore, truly intracellular recordings may not show MEPPs, which could be lost into the noise. We were always careful to push the electrode against either the inside or outside surface of the electrocyte when evaluating the physiological condition of the terminals. We usually used the cytoplasmic surface because [K.sup.+][Cl.sup.-] leakage would not increase the MEPP frequency (Kriebel et al., 1988). In contrast, potassium ion leakage from an extracellularly placed electrode increased MEPP frequency. The K ion effect was independent of the pressure (deformation) effect that resulted from pushing the electrode against the cytoplasmic surface. MEPP frequencies were generally low but increased markedly in response to slight pressure from the recording electrode against the innervated surface of the electrocyte membrane (Fig. 7, trace [C.sub.2]). After 6-7 days of treatment, spontaneous MEPP frequencies were markedly lowered, but pressure-evoked MEPPs could be elicited. The fall in MEPP frequency between 4 and 6 days corresponded to the advanced state of denervation and agreed with biochemical observations.
In the present study we show that skate electric organs dissociate into their component cellular elements (electrocytes, Schwann cells, nerves, and nerve terminals) when incubated in saline solutions containing 1% collagenase at temperatures down to 6 [degrees] C. In contrast, we have not been able to dissociate Torpedo nerve endings from their electrocytes. The maintenance of skate tissues at these lower temperatures has several important advantages. It is more physiological, because skates prefer cold water; it results in very stable electrocyte preparations [ILLUSTRATION FOR FIGURE 2 OMITTED]; and it inhibits the movement of the Schwann cells and thus prevents encapsulation of detached nerve terminals during collagenase-induced denervation of dissociated electrocytes [ILLUSTRATION FOR FIGURE 5 AND 6 OMITTED]. Nerve terminals prepared using collagenase at 6 [degrees] C are therefore purer and probably in a better physiological state than corresponding preparations derived from tissue incubated at room temperature. The disadvantage of using lower temperatures is the relatively long digestion period. However, as judged by electrocyte viability, this does not compromise the physiological quality of subsequent preparations. Use of temperatures between 6 [degrees] and 16 [degrees] C might accelerate the digestion somewhat without compromising purity or physiological state, but this would require testing.
A notable feature of the detached nerve terminals described in this paper is that they are distinguishable from Schwann cells so that they can be patch clamped. Preliminary studies suggest the presence of the A current of K channels (D. R. Matteson, pers. com.) and a calcium channel similar to the Q sub-type (Richardson et al., 1995). With this preparation, presynaptic ion channel physiology can be investigated on isolated cholinergic nerve terminals without recourse to manipulations such as fusion (Umbach et al., 1984; Edry-Schiller et al., 1991).
The detached nerve terminals from skate electric organ deserve comparison with "synaptosomes," which are formed by homogenizing Torpedo electric organ. In both cases the tissue disruption techniques employed lead to the dissociation of presynaptic terminals from the innervated surface of the electrocytes. With Torpedo, these detached terminals can be bulk-isolated by conventional subcellular fractionation and are recoverable in synaptosome fractions: such preparations have been widely used for biochemical studies (see Dowdall and Zimmermann, 1977; Michaelson and Sokolovsky, 1978; Morel et al., 1977; and Zimmermann et al., 1979). The % yield of Torpedo synaptosomes is very low (reviewed by Whittaker, 1992) but reasonable quantities can be isolated because the organs are large and very richly innervated. With skate, the bulk purification of detached nerve terminals is likely to prove much more difficult to achieve because of the limited numbers of terminals available. Thus even if the % yield of detached nerve terminals is higher than in Torpedo, this is unlikely to compensate for the much smaller size and lower density of innervation of the organs.
Despite this limitation, the skate electric organ nerve terminals are still attractive subjects for physiological studies. In detached form, they have several features that are essential for the application of patch-electrode techniques: size; accessibility; and the absence of adherent, encapsulating Schwann cells. So far the use of patch electrodes on vertebrate nerve terminals has been possible only with isolated peptidergic endings of 1-10 [[micro]meter] diameter from rat neurohypophysis (Lemos and Nowycky, 1989) and with the giant, cholinergic, calyx synapse of the chick ciliary ganglion (Stanley, 1991). Nerve terminals with diameters up to 5 [[micro]meter] are present in the electric organs of Raja clavata and R. montagui (Richardson et al., 1995), and these might provide an opportunity to examine dynamic aspects of presynaptic channel activity directly in their normal environment, rather than in isolated synaptosome preparations (Umbach et al., 1984; Edry-Schiller et al., 1991). A "nerve plate" preparation consisting of a plexus of nerve fibers with associated Schwann cells and nerve fibrils with attached terminals (equivalent to the nerve-ending clusters described in this study) can be isolated from skate electric organ following collagenase treatment. It is encouraging for prospective studies that functional voltage-sensitive calcium channels have been detected in the nerve terminals of these preparations with the use of microfluorimetric imaging techniques (Richardson et al., 1995).
The electrophysiological finding that the evoked response is "normal" up to the dissociation condition at which the terminals detach strongly indicates that the process of transmitter release is physiologically normal in skate isolated terminals. This conclusion is further supported by the similarity in the ultrastructure of nerve endings before and after detachment.
We thank Erika Kriebel for valuable technical assistance, Virginia Kriho for help with electron microscopy, and Dr. Jean Vautrin for participation in some preliminary electrophysiological experiments. Supported by grants to MEK from NSF (BNS-8809803, 211 2117) and NIH (NS-25683).
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|Author:||Kriebel, Mahlon E.; Dowdall, Michael J.; Pappas, George D.; Downie, David L.|
|Publication:||The Biological Bulletin|
|Date:||Feb 1, 1996|
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