electroreceptor functioning and morphology: functioning during histological fixation

9
Camp. B~oe~~m. P~i~sj~~. Vol. 75A, No. 4. pp, 569 to 577, 1983 03~-9629~x3 ~3.~+0.~ Printed in Great Britain Q 1983 Pergamon Press Ltd ELECTRORECEPTOR FUNCTIONING AND MORPHOLOGY: FUNCTIONING DURING HISTOLOGICAL FIXATION C. EIGENHUIS* and J. DONCKER Laboratory of Comparative Physiology, State University of Utrecht, Utrecht, The Netherlands. Telephone: 030-716221 (Received 26 November 1982) Abstract-i. The impact of chemical fixatives on electroreceptor functioning was studied electrophysio- logically. 2. The oxidative agents KMnO, and 0~0, eliminate the sensory response within a few seconds, leaving a disturbed spike train pattern. 3. Glutaraldehyde and formaldehyde eliminate the sensory response gradually in a period of several minutes; after that the afferent fibre shows repetitive activity. 4. It is concluded that the physiological state of the electroreceptor is not preserved during chemical fixation; the ultimate morphological picture will reflect an unphysiological state, i.e. the response to the fixative. INTRODUCTION Since Lissmann & ~achin (1958) introduced the “electric sense” in biology, many investigators have contributed to the description of the structure and function of electroreceptors which are considered to be primitive sense organs. Soon after Lissmann’s dis- covery attention was focused on the tuberous organs of the “active” electric fish, but also the ampullary organs of the less conspicuous “passive” electric fish were recognized by many authors as an attractive subject for study (Fessard, 1974). The subject has attracted investigators because the morphology seems rather simple and because the principles underlying information transfer by means of this frequency modulated system will probably be of general value in all acoustics-lateral sense organs, including the human ear. The present paper gives the results of our first attempt to elucidate the mechanism of information transfer in the ampullary electroreceptor of Icralurus nehulosus by making and comparing coincident snap- shots of its structure and physiological behaviour. Both morphologists and physiologists think that three cell strucures are involved in stimulus transduc- tion. Firstly, receptor cells are found to bear apical microoilli, which would lower the electrical resistance of the apical membrane. Secondly, zonulae occlu- dentes or tight &~ctions are found between receptor cells and accessory cells; these junctions are thought to prevent stimulus leakage through the intercellular compartment and thus they direct stimulus currents through the receptor cells. Last but not least, the receptor cells contact arborizations of the afferent nerve via conspicuous, morphologically differentiated synapses, which are thought to be the energy ampli- fiers of the transduction system. The morphological characteristics are depicted in Fig. 1. The ultrastruc- ture has been described by Mullinger (1964). * Send offprint requests to: C. Eigenhuis, Laboratory of Comparative Physiology, State University of Utrecht, Jan van Galenstraat 40, 3572 LA Utrecht, The Netherlands. An electrophysiological recording technique, which was introduced by Szabo in 1962 and has since been used successfully by several other authors (e.g. Roth, 1973; Bretschneider et al., 19801, permits the action potential pattern of the electroreceptor’s nerve fibre to be monitored in an intact animal. By applying a variety of electrical and chemical stimuli and using Szabo’s technique we hoped to be able to relate func- tional and ultrastru~tural aspects of the electrorecep- tor. It is obvious that in such a study the process of fixation is a crucial step. In this study we present the electrophysiological recordings of the electroreceptor activity during pen- etration by four histological fixatives, commonly used in ujtrastructure research. To our knowledge this is the first report comprising physiological data obtained from an in situ sense organ at the very moment this organ is fixed for morphological examin- ation. MATERIAL AND METHODS Animals The experiments were carried out on the brown bull- head, Zctalurus nebulosus (LeS). The fish came from fish ponds near Zonhoven, Belgium, and were kept in our faboratory for several months in Utrecht tap water (for ionic contents of Utrecht tap water, see Peters et al., 1975). The fish were fed on trout food (Trouvit, Trouw & Co., Putten, Netherlands). The results reported in this paper were obtained from animals with a body weight of between 50 and 100 g. Fish were immobilized with Flaxedil (Specia, Paris) in Ringer (Wolf. 1963) in doses of 2 pg/g fish, and placed in a plastic tray. Tap water at a temperature of 15-18°C was circulated through the mouth and gills of the fish to supply it with oxygen. Glass capillary micro-electrodes filled with 3 M KC1 were used to record spikes from the lumen of the electro- receptors. The impedance of the electrodes was between 10 and 20 Mega. The tip diameter was about i pm (see 569

Upload: j

Post on 05-Jan-2017

221 views

Category:

Documents


8 download

TRANSCRIPT

Page 1: Electroreceptor functioning and morphology: Functioning during histological fixation

Camp. B~oe~~m. P~i~sj~~. Vol. 75A, No. 4. pp, 569 to 577, 1983 03~-9629~x3 ~3.~+0.~ Printed in Great Britain Q 1983 Pergamon Press Ltd

ELECTRORECEPTOR FUNCTIONING AND MORPHOLOGY: FUNCTIONING DURING HISTOLOGICAL FIXATION

C. EIGENHUIS* and J. DONCKER

Laboratory of Comparative Physiology, State University of Utrecht, Utrecht, The Netherlands. Telephone: 030-716221

(Received 26 November 1982)

Abstract-i. The impact of chemical fixatives on electroreceptor functioning was studied electrophysio- logically.

2. The oxidative agents KMnO, and 0~0, eliminate the sensory response within a few seconds, leaving a disturbed spike train pattern.

3. Glutaraldehyde and formaldehyde eliminate the sensory response gradually in a period of several minutes; after that the afferent fibre shows repetitive activity.

4. It is concluded that the physiological state of the electroreceptor is not preserved during chemical fixation; the ultimate morphological picture will reflect an unphysiological state, i.e. the response to the fixative.

INTRODUCTION

Since Lissmann & ~achin (1958) introduced the “electric sense” in biology, many investigators have contributed to the description of the structure and function of electroreceptors which are considered to be primitive sense organs. Soon after Lissmann’s dis- covery attention was focused on the tuberous organs of the “active” electric fish, but also the ampullary organs of the less conspicuous “passive” electric fish were recognized by many authors as an attractive subject for study (Fessard, 1974). The subject has attracted investigators because the morphology seems rather simple and because the principles underlying information transfer by means of this frequency modulated system will probably be of general value in all acoustics-lateral sense organs, including the human ear.

The present paper gives the results of our first attempt to elucidate the mechanism of information transfer in the ampullary electroreceptor of Icralurus

nehulosus by making and comparing coincident snap- shots of its structure and physiological behaviour.

Both morphologists and physiologists think that three cell strucures are involved in stimulus transduc- tion. Firstly, receptor cells are found to bear apical microoilli, which would lower the electrical resistance of the apical membrane. Secondly, zonulae occlu- dentes or tight &~ctions are found between receptor cells and accessory cells; these junctions are thought to prevent stimulus leakage through the intercellular compartment and thus they direct stimulus currents through the receptor cells. Last but not least, the receptor cells contact arborizations of the afferent nerve via conspicuous, morphologically differentiated synapses, which are thought to be the energy ampli- fiers of the transduction system. The morphological characteristics are depicted in Fig. 1. The ultrastruc- ture has been described by Mullinger (1964).

* Send offprint requests to: C. Eigenhuis, Laboratory of Comparative Physiology, State University of Utrecht, Jan van Galenstraat 40, 3572 LA Utrecht, The Netherlands.

An electrophysiological recording technique, which was introduced by Szabo in 1962 and has since been used successfully by several other authors (e.g. Roth, 1973; Bretschneider et al., 19801, permits the action potential pattern of the electroreceptor’s nerve fibre to be monitored in an intact animal. By applying a variety of electrical and chemical stimuli and using Szabo’s technique we hoped to be able to relate func- tional and ultrastru~tural aspects of the electrorecep- tor. It is obvious that in such a study the process of fixation is a crucial step.

In this study we present the electrophysiological recordings of the electroreceptor activity during pen- etration by four histological fixatives, commonly used in ujtrastructure research. To our knowledge this is the first report comprising physiological data obtained from an in situ sense organ at the very moment this organ is fixed for morphological examin- ation.

MATERIAL AND METHODS

Animals

The experiments were carried out on the brown bull- head, Zctalurus nebulosus (LeS). The fish came from fish ponds near Zonhoven, Belgium, and were kept in our faboratory for several months in Utrecht tap water (for ionic contents of Utrecht tap water, see Peters et al., 1975). The fish were fed on trout food (Trouvit, Trouw & Co., Putten, Netherlands). The results reported in this paper were obtained from animals with a body weight of between 50 and 100 g. Fish were immobilized with Flaxedil (Specia, Paris) in Ringer (Wolf. 1963) in doses of 2 pg/g fish, and placed in a plastic tray. Tap water at a temperature of 15-18°C was circulated through the mouth and gills of the fish to supply it with oxygen.

Glass capillary micro-electrodes filled with 3 M KC1 were used to record spikes from the lumen of the electro- receptors. The impedance of the electrodes was between 10 and 20 Mega. The tip diameter was about i pm (see

569

Page 2: Electroreceptor functioning and morphology: Functioning during histological fixation

C. EIGENHUIS and J. DONCKER

Fig. 1. (a) Transverse section of a receptor cell (R) in the electroreceptor organ of ~~ru~~r~s. 1, iumen; pb. presynaptic body; t, afferent nerve terminal. Scale bar: 1 hrn. (b) Microvilli (m) at the apex of a receptor cell (R). The apex of the accessory cell (A) shows few villi. tj, tight junction; 1, lumen. Scale bar: I pm. (c) Higher magnification of a synapse. pb, presynaptic body; pv, presynaptic vesicle; sr. synaptic rodlet; R,

receptor cell: t, afferent nerve terminal. Scale bar: 0.5 Wm. Osmium fixed.

Geddes, 1372). The experimental set-up does not differ essentially from that described by Peters & Bretschneider (1980). The electroreceptors were stimulated electrically by sinusoidally alternating currents applied via the recording electrode.

Application of cherniculs

In the experiments local application of a fixative was made possible by placing a ring of a silicon rubber (STA-

SEAL, Detax-Dental, F.R.G.) on the skin of the fish. Such a ring isolates a compartment from the water surrounding the fish. The skin within the compartment, usually 10-20 mm’, can then be exposed to any desired solution.

Four different agents were tested: KMnO,, OsO,, glu- taraldehyde and formaldehyde. All fixatives were dissolved (KMnO,, 0~0,) or diluted (aldehyde fixatives) in Utrecht tap water. without any buffers being added. We followed this procedure because the fish had become adapted to this

Page 3: Electroreceptor functioning and morphology: Functioning during histological fixation

Electroreceptor functioning during fixation 571

b: to

Fig. 2. ExampIe of an a&rent fibre spike-train before and after the application of 20 mM KMnOo. (a,) Spontaneous activity in tap water becomes modulated activity as soon as a 3 Hz sinusoidal current srimulus (aJ is applied (arrow). (b) The arrow indicates the precise moment (t,) at which the fixative is applied to the modulating eiectroreceptor, (c) Activity 3 min after fixative application: modulation is much less obvious than in b. (df After 4 min the modulation has disappeared completely, the average frequency has increased considerably and the recorded spike amplitude has become smaller. (e) The spike pattern has become irregular after 9 min. ffi Spikes are no longer detected $2 min after application

of the fixative; (g) The 3 Hz stimulus, with which b, c, d, e and fare synchronized.

medium, and changes in the ionic constitution of the medium are known to influence electroreceptor functioning (Roth, 1971; Zhadan & Zhadan, 1975; Peters et nl., 1975; Bretschneider et al., 1980). In this way extra variables were minimized. We used the following concentrations of fixa- tive:

0~0~: 4, 20 and 80 mM (0.1, 0.5 and 2”/, resp.); KM&,: 20, 80 and 120mM (0.3, 1.2 and i.9%); glutara~dehyde: 100, 300 and SOOmM (i. 3 and S”/;); formaidehyd~: 300, 600 and I200 mM (0.9. I.8 and 3.6%).

Ali experiments began with the recording of the spon- taneous activity of the electroreceptor in tap water. Then a 3 Hz sinusoidal current was applied through the recording electrode. We deliberately applied rather strong stimuli, which elicited electroreceptor responses in the non-linear part of the input/output characteristic (see Bretschneider rt al., 1980). The strength of the stimulus is comparable to a stimulus of about 300 nA as used by Bretschneider rr al. In the recordings the resulting phase-locked spike pattern was easily recognized; this was a guarantee that we had an operative modulating mechanism at the start of the exper- ixents. The tap water in the compartment was then re- moved by blotting paper and a drop of the fixing agent (0.05-0.1 ml) was put into the com~rtment. The efectro- physiological recording and the spike train pattern are not necessarily disturbed by these manipulations. ff electron microscopical examination was to follow the electrophysio- logical experiment, the fixative and the silicon ring were removed from the fish and a small piece of skin (2 x 2 mm2) that had been in contact with the fixative was excised and prepared further. The fish was then put back into the aquarium, where it showed normal behaviour again within a few hours. The minor wound caused by the micro-biopsy healed quickly.

RESULTS

Sensitivity ta asmolarity and pH of fresh water sol- utions

A possible sensitivity to osmotic changes was inves- tigated by applying fresh water solutions of 400mM saccharose or 400 mM urea. The spontaneous activity and the response to a 3 Hz stimulus current did not change with these media. Tap water of pH I I.0 did not influence the electrttreceptor ~un~tion~n~, nor did tap water of pH 5.0. The pH was adjusted by simply adding small amounts of&O1 M NaUH or 0.01 NHCI, respectively. The solutions were not buffered. From these results we conclude that osmotic effects or pH effects are of minor importance during our experi- ments with the fixatives.

After application of KMnO,, the following sequence of events was seen (Fig. 2):

(1) disappearance of the response to the 3 Hz stimu- lus current ;

(2) increase in the average frequency to about twice the initial resting frequency, accompanied by a de- crease in spike amplitude;

(3) interruption of the high frequency activity by periods of non-activity. The interruptions lasted longer and occurred more frequently, as time passed;

(4) finally the activity was totally eliminated.

This is approximately the sequence seen with all concentrations used. However, the sequence was not always exactly as described. For instance. there was

Page 4: Electroreceptor functioning and morphology: Functioning during histological fixation

572 C. EIGENHUB and J. DONCKER

Fig. 3. Example of an afferent fibre spike-train before and after the application of 80 mM 0~0,. All traces are synchronized with the 3 Hz current stimulus (e). (a) The arrow indicates the precise moment (ta) at which the fixative is applied to a modulating electroreceptor. (b) Already 20 set after application the modulation is much less obvious than in (a). (c) One minute after application the modulation has disappeared completely, the average frequency has increased and the recorded spikes have become

smaller. (d) After 1 min and 20 see spikes can no longer be detected.

sometimes a short increase in the average frequency which was not accompanied by a decrease in spike amplitude; yet there was still good modulation.

The time-scale of the sequence depended on the concentration used. The modulation disappeared within 15 set after application of 120 mM KMnO,, whereas the modulation could be recognised for much longer (for up to 2 mm) after the application of 20 mM KMnO,. About 5 min after the application of 120 mM KMn04 spikes could no longer be detected, whereas after application of 20mM it could take 1 I min for the afferent nerve to be silenced. However, experiments in which the same concentration was used showed a considerable variability in the time- scale. After the application of 80 mM intermediate values were found.

In some experiments, after the aRerent nerve had been silenced, we quickly removed the fixative and washed the skin with tap water. On a number of occasions a low frequency activity, which did not show modulation, returned.

After the application of 0~0~ the following sequence of events was seen (Fig. 3):

(1) disappearance of the modulation; (2) increase in the average spike frequency; (3) decrease in the recorded spike amplitude; (4) finally spikes could no longer be detected: the

afferent fibre was considered to be silent.

This sequence was not seen within the first 30min after the application of 4mM 0~0~. Even after 30min there was still an excellent modulation to a 3 Hz stimulus.

After application of 20 mM 0~0~ the modulation gradually disappeared in a few minutes. Usually this was accompanied by a gradual decrease in the aver- age frequency. Then the average frequency started to

increase to about twice the resting frequency and any residual modulation was rapidly eliminated. The high frequency was maintained as long as the spikes were recognizable, and was not interrupted as it was after the application of KMnO,. It could take up to 12 min for the afferent fibre to be silenced.

After the application of 80mM 0~0~ the modu- lation disappeared within 5-10 sec. Then there was a short period of unmodulated activity before the aver- age frequency started to increase. After l-2min the spike amplitude became too small to be recognized and the afferent fibre was considered to be silent.

After application of formaldehyde the following sequence of events was observed (Fig. 4):

(1) a gradual, but considerable decrease in the aver- age frequency, while the modulation remained recog- nizable ;

(2) a period of low frequency activity, in which modulation is no longer recognizable;

(3) an increasing tendency to what should be called repetitive activity. accompanied by a gradual decrease in spike amplitude.

For different concentrations there were differences in the time-scale of the sequence. After application of 1200 mM there was a decrease in the average fre- quency from 40 Hz (resting frequency) to 15 Hz after about 2 min, whereas roughly the same decrease (35 Hz to about 10 I-Iz) was found 5-7 min after appli- cation of 300mM formaldehyde. The afferent fibre was considered to be silent about 8 min after the ap- plication of 1200 mM and 15-20 min after the appli- cation of 300 mM. After application of 600 mM inter- mediate values were found.

The phenomenon “repetitive activity” will be de- scribed in some detail under the heading “glutaralde- hyde” (see below).

Page 5: Electroreceptor functioning and morphology: Functioning during histological fixation

Electroreceptor functioning during fixation

g: 3Hz Fig. 4. Example of an afferent fibre spike-train at various set times after the application of 3OOmM. formaldehyde. (a) Just after appIication of the fixative the activity shows normal modulation. (b) After 4 min the modulation is still obvious, but the average frequency has decreased markedly. (c) The low frequency activity shows no modulation at 9min after the application of the fixative. (d) Incipient repetitive activity after f3 min: the spikes occur in pairs. fe) Repetitive activity after IS min: tripfe spikes are seen. (ff After 19 min only the first spike of each burst is recognizable. (g) The 3 Hz stimulus, with

which at1 traces are synchrn~i~ed.

When the fixative was quickly removed from the skin after a receptor had become silent, low frequency activity would sometimes return, but it never showed modulation.

After the application of glutaraldehyde the follow- ing sequence of events was observed (Fig. 5):

(1) gradual disappearance of the modutation; (2) decreasing average frequency; (3) increasing repetitive activity and a gradual de-

crease in spike amplitude.

The sequence of events is about the same as with formaldehyde. For different concentrations there were differences in the time-scale of the sequence. The fas- test sequence was seen after the application of 600 mM glutaraldehyde. Within 6-8 min the receptor was silent. Sometimes the gradual decrease in the modulation seemed to overlap with the increasing re- petitive activity. After the application of 300 mM glu- taraldehyde the modulation could be recognized for 2-6 min. Repetitive activity could be very explicit, but could also hardly be present at all. Usuatly the atfer- ent fibre was silent after about 10 min (but see Fig. 5!). The slowest time-scale was found with 100 mM glu- taraldehyde. It took about 30min for the afferent fibre to become silent. Figure 5 shows an example of an experiment in which explicit repetitive activity occurred: in the first phase of repetitive activity we see pairs of spikes (Figs 4d, 5~). There is still no differ-

ence in amplitude between the first spike and the second spike of one pair. The interval between the first and the second spike of each pair becomes pro- gressively smaller. When this interval is about 12 msec, a third spike appears after every pair: then the interval between the second and the third spike decreases and a fourth spike appears, etc. When com- plete bursts are seen, the first spike of such a burst is of near-normal ampIitude, whereas the successive spikes jn the burst become progressively smaller {Fig. 5d and 5e). Although all spikes finally become too small to be detected, the first spike of a burst can be recognized for a much longer time than the repetitive spikes (Fig. Sf).

DISCUSSION

The experimental procedure we followed allows the study of a completely intact in situ organ. This means that we don’t have to reckon with the impact of surgi- cal procedures or artificial media and we can study “just” the impact of fixatives on the system. A minor concession we have to make, however, lies in the im- mobiiization of the fish, which is carried out by the administration of Flaxcdit ~gallamine thr~ethiodide). Flaxedit paralyzes the voluntary cholinergic muscle activity. So, naturally occuring giil movements of the fish, which would have caused bioelectric phenomena that would have been reflected in the afferent output of the electroreceptor, are prevented by Flaxedil.

Although the ampullary electroreceptors are con- sidered to be relatively simple sense organs, it is

Page 6: Electroreceptor functioning and morphology: Functioning during histological fixation

C. EICEHHIJIS and J. DONCKER

a: t0

e: tlTp

f: t,,,

g: 3Hz nw

Fig. 5. Example of an afferent fibre spike-train at various set times after the application of 300mM glutaraldehyde. (a) Normal modulated activity during the first minute after application. (b) After 5 min the modulation has not yet disappeared completely; the average frequency has decreased. (c) Incipient repetitive activity shows no modulation; 10 min after application. (d) Increased repetitive activity after 13 min. (e) After 17 min the repetitive activity has further increased; the amplitude of the repetitive spikes has become smaller. (fJ After 20 min even the first spike of every burst has become small. (g) The 3 Hz

stimulus with which all traces are synchronized.

obvious that the study of a complete organ is still a very complicated matter. In addition, the electro- receptor cannot be seen apart from the skin in which it is located, and the skin on his own could be con- sidered an organ.

In our approach the available physiological infor- mation of this complicated system comes from only one of the constituent cells. i.e. the afferent nerve. It is to this rather limited information that we try to relate the impact of the penetrating fixing agents. It will be clear that just on this basis a full analysis of the fixing process is not possible.

Nevertheless, even considering the limitations of our experiments, we still think that the obtained in- formation is an important step towards a better understanding of the function and the structure of the electroreceptor, because new aspects are revealed of the process of fixing, which constitutes the essential link between physiology and morphology.

In this study we succeeded in monitoring the effects that penetrating fixatives have on the functioning of the electroreceptor organ. We found that ail four chemical fixatives applied drastically altered electro- receptor functioning before the afferent fibre was eventually silenced. This means that any ultrastruc- tural picture of an electroreceptor organ will reflect the physiological state of the electroreceptor reacting to the applied fixative.

One might question the specificity of the impact of the fixatives, in the sense that osmotic influences and pH influences are not compensated for in our experi- ments. The experiments with 400 mM solutions and with tap-water of pH 5.0 illustrato that osmolarity and pH arc of minor importance in electroreccptor

functioning in ~crul~rzis. The same holds true for elec- troreceptors in Kr~ptopt~~l~s, as reported by Roth (1982).

It is striking that the oxidative agents and the alde- hyde fixatives interfere with electroreceptor function- ing in their own characteristic way: after the modu- lation has disappeared the oxidative fixatives cause a marked increase in the average frequency, which can be considered as a lasting excitation, whereas the aldehyde fixatives gradually inactivate the organ. In view of these findings. one would expect to find some specific morphological differences between, for instance, electroreceptors primarily fixed in 0~0, and those primarily fixed in glutaraldch!~de. According to Gamier & Wachtel (1970). who compared these two fixation procedures for the electroreceptors of Nttpo- pormrs. the general ultrastructure after primary tixa- tion in 0~0, does not differ “significantly” from the ultrastructure after primary fixation in glutaralde- hyde. The study of Szamier and Wachtel indicates that {{ morphologic~~l differences do indeed exist in our system they will only be revealed after close scrutiny.

Interesting information about the functioning of electroreceptors could be derived from our experi- ments if the electrophysiologically monitored changes in functioning can be related to the penetration rate and chemical properties of the fixatives. This is useful if the penetrating agent sztc~rssiz~l?: reaches the rele- vant structures in a system with distinct polarity, as is the cast in the electroreceptor. Accurate information about the penetration rate would be obtained. if the diffusion front of the fixative in the tissue could be visualized at various set times after the application of

Page 7: Electroreceptor functioning and morphology: Functioning during histological fixation

Electroreceptor functioning during fixation 575

the fixative. Preliminary experiments indicate that this front can indeed be visualized for 0~0, and KMnO, by means of X-ray element analysis of sectioned material. Our future research will focus on this matter.

Although we are still short of information about penetration rates, the present results enable us to compare the kinetics of the two types of fixative. In this discussion we define the term “delay” as the period of time between the moment the fixative is dropped on the skin and the moment the first change in spike train pattern is recognized. The delay after application of high concentrations of the oxidative fixatives KMnO, and 0~0, is only 5-10sec. X-ray element analysis reveals that no Mn can be detected in the receptor cells such a short time after the appli- cation of KMnO,. This indicates that the rapid disap- pearance of the modulation is caused by the interac- tion of the fixative with the apical membranes. From the literature (e.g. Elbers, 1966) it is known that oxi- dative fixatives eliminate the semi-permeability of membranes; this will cause loss of the membrane potential and therefore also electrical disturbance of the basal, presynaptic membrane, where the hypo- thetical modulating mechanism (Bennett, 1965; Ben- nett & Clusin, 1979) would be inactivated. However. as long as the exact nature and location of the modu- lating mechanism are not known. one cannot rule out the possibility that the mechanism is located in the morphologically specialized apical membrane itself, where it could be inactivated directly by the fixative. Such a modulating mechanism could exist in the form of membrane proteins that have the properties of voltage sensitive Ca’+ channels (see Szabo & Fessard, 1974). Recent research (Pumplin et al., 1981) indicates that Ca’+ channels can be recognized in freeze-frac- tured material. Perhaps freeze-fracturing of the ampullary electroreceptor will tell us whether Ca’+ channels are present in the microvilli or in the presyn- aptic membrane.

In the foregoing it has been supposed that disap- pearance of the modulation is caused by inactivation of the modulating mechanism, no matter where it is located in the receptor cell. There is another possi- bility. One could imagine that changes in the electri- cal properties of the surrounding tissue after appli- cation of the fixative create a shunt pathway for the stimulus current. This would lead to an apparent loss of sensitivity, although the modulating mechanism is in fact not disturbed.

We found that the aldehyde fixatives needed one minute or more to abolish the modulation, whereas we found that the oxidative fixatives needed much less time. The differences in time-scale could be attributed not only to differences in penetration rates, but also to differences in the reaction rate or the nature of the interactions of the fixative with the tissue. It is well known that aldehyde fixatives primarily react with proteins, but do not completely abolish enzymatic ac- tivity; generally moderate activity remains. For glu- taraldehyde the numberous quantitative studies on this aspect have been reviewed by Hopwood (1972). Retention of enzymatic activity is considered to vary inversely with the crosslinking property of the fixa- tive. Therefore, formaldehyde is a still better preserver of enzymatic activity than glutaraldehyde (Sabatini et

al., 1963; Torack, 1965; Janigan, 1965). In contrast, KMn04 and 0~0, have an oxidative capacity to de- stroy enzymatic activity. Their fixing capacity pre- dominantly concerns the lipid component of mem- branes (e.g. Riemersma, 1968; Pelttari & Helminen, 1979). Not only do the fixatives show different time- scales, but they also have different impacts on the average frequency. After application of the aldehyde fixatives a decrease in the average frequency is seen. In terms of the current hypothesis on electroreceptor functioning this could mean a decrease in transmitter release. The oxidative fixatives cause an increase in the average frequency suggesting an increase in trans- mitter release. These findings are in contradiction with the few electrophysiological data on fixation of synapses available in the literature: De Robertis (1959) mentions the results of experiments carried out by Amassian on peripheral synapses of R~M.J cutcs- hiam. Here, 10sec after 0~0, is brought in contact with the cehac ganglion, synaptic transmission is completely abolished. For three different aldehyde fixatives Smith & Reese (1980) found with all concen- trations tested an increase in the frequency of quanta1 release in the neuromuscular junction of grass frogs. Our findings could be made consistent with those mentioned by De Robertis and Smith and Reese if we assumed that there is an inhibitory transmitter which modulates a high frequency pacemaker in the afferent neuron. Such a model system has been proposed by Roth (1973) for the ampullary receptor of Krrptop- rerus. However, further investigation (Roth, 1978) made him believe in the existence of an excitatory transmitter in both Kr~yfoptrrus and Ictuhrus. More- over, there are strong indications that L-glutamate acts as an excitatory transmitter in the ampullae of Lorenzini (Akoev, 1980) the medium receptors of Gnathonernus (Steinbach & Bennett, 1971) and the ampullary electroreceptor of Kryptopterus (Teeter & Bennett, 1981). The existence of an inhibitory trans- mitter in Ictdurus therefore seems unlikely. If the im- pact on the average frequency is not due to adaptive properties of, or to disturbing influence on, the spike generator in the afferent neuron, we can say that the impact of fixatives on the functioning of synapses is strongly dependent on the type of synapse.

Before we can decide whether the increase in aver- age frequency after application of the oxidative fixa- tives is due to an increased synaptic activity or to interaction with the spike generating membrane of the neuron, we must be able to visualize the state of ac- tivity of the synapse. The literature contains reports on some studies in which the state of activity of synapses is reflected in the morphology after chemical fixation (Bennett ef al., 1975; Atwood rt al., 1972; Schaeffer & Raviola, 1975). Differences in the vesicle content in the presynaptic neuroplasma and/or the presynaptic membrane surface are interpreted as being due to an imbalance between the endocytotic and exocytotic membrane processes necessary for the recycling of presynaptic vesicles. This imbalance is either introduced experimentally by a metabolic in- hibitor such as dinitrophenol (DNP) or by cooling the preparation, or it is naturally present in a very phasic system. If this imbalance could be introduced in the electroreceptor system, we would perhaps have an answer to our problem.

Page 8: Electroreceptor functioning and morphology: Functioning during histological fixation

576 C, EtG~t4HUis an d J. DON~KER

The repetitive activity seen in the late phase of the aldehyde fixation is possibly an effect of the fixative on the afferent neuron. In Xenoplfs luetiis repetitive activity of sensory fibres after insecticide poisoning has been shown to be related to an induced negative after”potent~a~ caused by interaction of the insecticide with Na’ thannets in the nerve membrane (Van den Bercken, 1972). Moreover, according to E‘ozzard & Dotninguez (1969) formaldehyde and glutaraldehyde induce negative after-potentials in Purkinje fibres of sheep. This could mean that. in our system too, re- petitive activity is caused by a negative after-potential induced by direct contact of the aldehyde fixative with the afferent fibre.

GEDDES L. A. (1972) Ekctrodes and rhe Meusurewmt of Bioelectric Ewnts. Wiley Interscience, New York.

WOPWOOO D. (1972) Theoretical and practical aspects of glutaraldehyde fixation. Histoclzen~ J. 4, X7-303.

JANIGAN D. F. (1965) The effects of aldehyde fixation on acid ph~~sphatase activity in tissue blocks. J. ~~sroc~~t~. Cyrochefn. fJ* 476-483.

LI~SMANN H. W. & MACHIN K. E. (1958) The mechanism of object location in Gy,nnurchus dock-us and similar fish. d. exp. Biol. 35, 451-486.

MULLI~GER A. M. (1964) The fine structure of ampullary electric receptors in Amiurus. Proc. R. SOC. Ser 5 160, 345-359.

ilckrro~fc~dgi,nierrts--The authors are indebted to Dr R. C. Peters and Professor Dr P. F. Elhers for their stimulat- ing support throughout this work; to Professor Dr F. J. Verheijen. Dr F. Bretschneider, Dr R. C. Peters and Pro- fessor Dr P. F. Elbers for critical coinmgnts on the manu- script; to Mr W. J. C. Loos for prepariq the figures: to Miss S. M. McNab for making linguistic improvements and to Miss F. E. M. van Vliet for typing the manuscript. This work was supported by a grant from the Foundation for Fundamental Biological Research (BION), which is subsidised hy the Netherlands Organization for the Ad- vancement of Pure Research IZWOt.

PELITARI A. & HELM~NEN H. J. (1979) The effects of various fxations on the relative thickness of cellular membranes in the ventral lobe of the rat prostate. Nistochevr. J. It, 599-611 t

PETERS R. C. & BRE”W!HNEIDER F. (1980) Electroreceptive microampultae in the African mudfish Cfnrius lazera (Cuv and Vat 1840). Xn Advances in Pb~~~io~ug~c~~~ S&m Vol. 3 I Smsor~ Ph~sj~)~~g~ oj Aytratic tower Verrehmrt~s (Edited bji SZARO T. & f&H G.). p. 13. Pergamon Press, Oxford.

PETERS R. C., BKETSCHNEIDER F. & SCHREUDER J. J. A. (1975) Influence of direct current stimulation on the ion- induced sensitivity changes of the electroreceptors (Small Pit Organs) of the bullhead. Ictnlurus neh~tIosrts (LeS). iVerA. J. Zoo!. 25, 359--397.

REFERENCES

Atxm G. N. & ANIIRIANOV G. N. (1981) The action of divaient ions and drugs on thermal and electric sensi- tivity of the ampullae of Lorenzini. In Ar~t~~nces in Plz~sioloyiccll Sciencr Vol. 3 I. SensOry PhySiOfOgp qf ,+ptic LOWW Vwrehrcrtrs (Edited by SZABC) T. & CZ&

G.). p. 57. Pergamon Press. Oxford. Arwt~t~r H. L., LANC? F. & MORTON W. A. (1972) Synaptic

vesicles: selective depletion in crayfish excitatory and in- hibitory axons. S&ncr 176, 1353-I 355.

BENNETT M. V. L. (1955) Electroreceptors in mormyrids. In Co/d Spring Htrrh. s3’1np. quuflf. Biai. xxx. Smsuq R~cq~tors (Edited by FKI~CH L.), p. 245. Cold Spring Ha&or, New York.

PUM~LIN D. W.. REESE T. S. & Lwns R. (1981) Are the presynaptic membrane particles the caicium channels? Proc. mf. Acari. Sci. USA 78, 7210-7213.

RIEMERSMA J. C. (1968) Osmium tetroxide fixation of lipids for electron microscopy a possible reaction mechanism. Bio&rtr. hioph~~ Actu 152. 718-727.

ROTH A. (197 1) Zur Funktionsweise der Elektrorezeptoren in der Haut von W&en (~~ta~~~us): Der EinBuss der Ionen im Siisswasser. Z. fieryi. Physsiol. 75. 3O3--322.

ROTH A. (19733 Ampuilary eiectroreceptors in catfish: a&r- ent fiber activity before and after removal of the sensory cells. J. conlp. Physiol. 87, 259-275.

ROTH A. (1978) Further indications of a chemical synapse in the electroreceptors of the cd&. J. camp. Phpsiol, 126, 147 150.

BFNNcrT M. V. L. & CLCSI~. W. T. (1979) Transduction at electroreceptors: origins at sensitivity. In Sociely qf Gm- crd Ph~sioloyists Skies 33. MrinhranP ~ru~~s~Ifcfi~~~ ~~~c~~~~ljs~~is (Edited by CANt R. A. & i&Wl.tNG J. E.1. p. 91. Raven Press. New York.

RUTH A. (1982) Sensitivity of catfish ~lectror~ceptors: dependence on freshwater ions and skin potential. J. cornp. Phgsioi. 147, 329- 337.

BENINETT M. V. L.. Monet P. G. & HIGHST~EIN S. M. (1976) Stim~i~ation induced depletion of vesicles, fatigue of transmission and recovery processes at a vertebrate cen- tral synapse. in Cofd Spritq Hnrh. S$q). qtinnr. Biol. XL. The Sq”apse (Edited by FOKD N.). p, 25. fold Spring Harhor, New York.

SAMTINI D. D.. BEN~CW K. & BARRNETT R. J. (1963) Cyto- chemistry and electron microscopy. The preservation of cellular ultrastructurc and enzymatic activity by alde- hyde fixation. J. Cell Bid. 17, 19-58.

SCHAEFFER S. F. & RAV~OLA E. (19753 Uttrastructural analysis of functional changes in the synaptic endings of turtle cone ceils. In Cotd SD&U Narh. Senln. quant. Biul. XL. The S?nupse (Edited’ by -FORD N:), b. i2l. Cold Spring Harbor. New York.

BKETSCWN~IUER F., PETERS R. C., PEELE P. M. & DOR- angrily A, W. C. (1980) Functioning of catfish electro- receptors: statistical distribution of sensitivity and ~uctuat~ons of spontaneous activity. f. ronlp. ~~~si~~. 137, 273-279.

SMITH J. E. & REESE T. S. (1980) Use of aldehyde fixatives to determine the role of synaptic transmitter release. f. eq B&f. 89, 19.-29.

DtRoar~~~s E. (19S9) Submicroscopic morphology of the synapse. In itzf~F}zlzti~~tal Review-of‘ Crtojogy vbl. VIII (Edited by BQ~RNE M. E. & DANIELLI J. F.). p. 61. Aca- demic Press, New York.

STEI~~~C~~ A. B. & BENNETT M. V. L. (1971) Pr~synaptic actions of Ca and Mg and post synaptic actions of giuta- mate at a sensory synapse. Biol. Bull. mar. hioi. tub. Woods Hole 141, 4O3.

SZABO T. (1962) Spontaneous electricai activity of cuta- neous receptors in Mormyrids. Nature. Lo&. 194, 6OCk-60 I. Etmas P. F. (1966) Ion permeability of the egg of L~mnara

st~fglzff~is L. on fixation for electron microscopy. B~oc~z~~~?. hiophIX. Acrct 112, 318-329.

F~~SARD A. (Editor) (1974) Etectroreceptors and 0ther specialized receptors in lower vertebrates. Handbook qf Semnr~~ Physiology 111/I Springer Verlag, Berlin.

FOZZAKI) W. A. & DOMINGUEZ C. (1969) Effect of formal- dehyde and gluteraldehyde on electrical properties of Cardiac Purkinje fibrrs. J. gen. Phrsinl. 53, 5X&540.

SZARO T. & FESSARD A. (L974) Physiology of electrorecep-

SZ~MIER R. ‘8. & WACWTE~ A. W.‘(19?O) Special cutaneous receptor organs of fish VI: Ampullary and tuberous organs of Hypopontus. J. ultrastr. Res. 30, 450-471.

TEETER J. H. & BENNETT M. V. L. (1981) Synaptic trans- mission in the ampul’tary electroreceptor of the trans-

Page 9: Electroreceptor functioning and morphology: Functioning during histological fixation

Electroreceptor functioning during fixation 577

parent catfish Kryptopterus. J. cornp. Physiol. 142, drin on myelinated nerve fibres. Ph.D. thesis, State Uni- 311-371. versity of Gtrecht.

TORACK R. M. (1965) Adenosine triphosphatase activity in WOLF K. (1963) Physiological salines for fresh-water the rat brain following differential fixation with formal- teleosts. Proaue. Fish Cdt. 25. 1355140. dehyde, glutaraldehyze and hydroxyadipaldehyde. 1. ZHADAN G. G: & ZHADAN P. fvl. (1975) Effect of sodium, Histochetn. Cytochem. 13, 191-205. potassium and calcium ions on electroreceptor function

VAN DFN BERCKEN J. (1972) The effect of DDT and Diel- in catfish. Neurophysiology (Kiev) 7, 403-410.