J. Phyeiol. (1976), 260, pp. 143-158 With 1 plate and 4 text-ftguree Printed in Great Britain

143

PHYSIOLOGICAL AND MORPHOLOGICAL EFFECTS OF POST-GANGLIONIC AXOTOMY ON PRESYNAPTIC NERVE TERMINALS

BY H. R. BRENNER AND E. W. JOHNSON From the Department of Physiology, University of Colorado Medical Center, Denver, Colorado 80220, U.S.A. (Received 30 December 1975) SUMMARY

1. Electrophysiological and electron microscope studies were done on cells in the ciliary ganglion of chickens which had been axotomized on the day of hatching. 2. By the third day after post-ganglionic axotomy both electrical and chemical transmission through the ganglion were severely depressed; by the fifth day ganglionic transmission had disappeared. 3. Action potential initiation and conduction in axotomized cells and in their associated presynaptic nerve terminals were unimpaired 3-4 days after axotomy. 4. Depression of ganglionic transmission in 3-4 day axotomized preparations was due to a reduction in amplitude of both the excitatory post-synaptic potential (e.p.s.p.) and the electrical coupling potential in individual ganglion cells. 5. In addition to being reduced in amplitude, e.p.s.p.s in axotomized cells were more subject to fatigue during low frequency (1/sec) stimulation. 6. The reduction in e.p.s.p. amplitude was due to a reduction in both the mean quantal content of the e.p.s.p.s and the calculated depolarization produced by an individual quantum of transmitter. On the average the e.p.s.p. was reduced by a factor of about 4, the mean quantum content to about two thirds normal and the quantal size to about a third normal, compared with responses in unaxotomized cells of the same age. 7. Ultrastructural studies revealed a progressive maturation of presynaptic terminals in normal ganglia between 0 and 9 days after hatching. Over this period the content of synaptic vesicles and mitochonaria in the terminals increased and the background matrix became more dense. 8. After axotomy these signs of maturation were abolished or reversed, particularly from the third day onward. In addition there was an increase in the number of cell sections in which no synaptic terminals were observed.

144 H. R. BRENNER AND E. W. JOHNSON 9. It was concluded that loss of synaptic transmission was due to at least three factors: a reduction in release of transmitter from presynaptic terminals, a reduction in quantal size, probably due to a loss of postsynaptic sensitivity, and a partial loss of presynaptic contact. INTRODUCTION

The most conspicuous histological consequences of injuring the axon of a peripheral neurone are changes in its soma commonly referred to as chromatolysis (Liebermann, 1971; Matthews & Raisman, 1972), and proliferation of adjacent glial cells (Matthews & Nelson, 1975). These changes are accompanied by a depression of synaptic transmission between the presynaptic nerve terminals and the affected neurones (Brown & Pascoe, 1954; Acheson & Remolina, 1955; Hunt & Riker, 1966; Kuno & Llinas, 1970; Purves, 1975). Brown & Pascoe (1954) found acetylcholine (ACh) concentrations in venous perfusates from stimulated normal and axotomized superior cervical ganglia of cats to be similar and suggested that failure of synaptic transmission after axotomy was due primarily to a decreased effectiveness of ACh at the post-synaptic membrane rather than to a decrease in ACh release from the presynaptic terminals. Acheson & Remolina (1955) reported similar results from experiments on axotomized inferior mesenteric ganglia. They also found, a few days after axotomy, that the post-ganglionic compound action potential produced by repetitive preganglionic stimulation fatigued rapidly, due to a loss of synaptic transmission within the ganglion. Intracellular recording from axotomized frog sympathetic ganglion cells (Hunt & Riker, 1966) revealed a decrease in amplitude of excitatory post-synaptic potentials (e.p.s.p.s) and in resting potential and input resistances of the cells. These combined to lower the safety factor for synaptic transmission which, together with the normal fatigue of transmitter release during repetitive stimulation, could account for the rapid depression of the post-ganglionic compound action potential observed by Acheson and Remolina (1955). More recently, Purves (1975) has reported a reduction in e.p.s.p. amplitude in the axotomized superior cervical ganglion of the guinea-pig, accompanied by a decrease in the number of synapses on the affected cells. The electrical properties were unchanged. Detachment of presynaptic terminals from neurones undergoing chromatolysis, accompanied by invasion of glial processes into the synaptic clefts, has also been suggested as a reason for decline of synaptic efficacy by Matthews & Nelson (1975). Such detachment is not confined to autonomic ganglia. Several morphological investigations on chromatolysed motoneurones have shown the number of synaptic contacts to be

PRESYNAPTIC EFFECTS OF AXOTOMY 145 decreased (e.g. Blinzinger & Kreutzberg, 1968; Hamberger, Hansson & Sj6strand, 1970). Kuno & Llina's (1970) obtained electrophysiological evidence that axotomy of cat's spinal motoneurones was followed by loss of synaptic input to the soma and proximal dendrites of the affected cells. None of the studies summarized above have given any indication of an effect of post-synaptic axotomy on the physiological function of the presynaptic terminals. Kuno & Llina's (1970) found the quantal contents of e.p.s.p.s originating from the distal portions of dendrites of axotomized motoneurones to be essentially normal, suggesting that the presynaptic terminals remaining after axotomy were unaltered in function. On the other hand, various other studies have indicated that presynaptic terminals are dynamic structures capable of responding to post-synaptic changes with structural and functional adaptations. Thus, Black, Hendry & Iversen (1972) have shown that destruction of post-synaptic adrenergic neurones in developing mouse superior cervical ganglia inhibited normal maturation of presynaptic cholinergic nerve terminals. Ceccarelli, Clementi & Mantegazza (1972) have reported that preganglionic cholinergic fibres may be made to innervate the nictitating membrane in cats and that, upon such innervation, the fibres are modified to release catecholamines in addition to, or instead of, ACh. Such observations suggest that presynaptic neurones may respond to alterations in the function of their post-synaptic targets. The present investigation was aimed at establishing whether the loss of synaptic transmission after axotomy is accompanied by functional changes in the presynaptic terminals. The avian ciliary ganglion was chosen because of its relatively simple organization. The ganglion consists of two populations of cells, ciliary and choroid (Martin & Pilar, 1963a; Hess, 1965; Marwitt, Pilar & Weakly, 1971). The larger ciliary cells each receive synaptic input from only one preganglionic oculomotor nerve fibre and send their axons via the ciliary nerves to innervate the ciliary muscles and iris. In addition, the preganglionic fibres connected to this group of cells form large caliciform terminations which envelop some 25 % of the ciliary cell surface. This permits intracellular recording from the presynaptic terminals as well as from the post-synaptic cells (Martin & Pilar, 1963b). A brief report of the present results has appeared elsewhere (Johnson & Brenner, 1975). METHODS Axotomy. Fertilized chicken eggs were obtained from a local hatchery and incubated in the laboratory. Shortly after hatching, chickens were anaesthetized with

ether. The ventrocaudal part of the eyeball was exposed by cutting and reflecting the lower eyelid and attached skin. The ciliary nerves could then be seen through the sclera and cut with iridectomy scissors after which the eyelids were sutured together and the chicken allowed to recover from the anaesthesia.

H. R. BRENNER AND E. TV. JOHNSON

146

Electrophysiology. The procedure for dissection of the ciliary ganglion from 3 to 4-day-old chickens and the general electrophysiological techniques for intracellular and extracellular recording were as described by Martin & Pilar (1963a, b). Electrodes for extracellular stimulating and recording consisted of silver wires imbedded in the preparation chamber, one pair in contact with the oculomotor (pre-ganglionic) nerve, and the other contacting the ciliary (postganglionic) nerves. Glass pipettes for intracellular recording were filled with 3 m-KCl or 4 M-K-acetate and had resistances in the range of 70-100 Mo. They were connected to the input of a high impedance, negative-capacity pre-amplifier with a feed-back circuit for passing polarizing currents into the cell while at the same time recording the membrane potential (Fein, 1966). Signals from the pre-amplifier were displayed on a cathode ray oscilloscope and photographed. Electron microscopy. Ciliary ganglia from 1-, 3- and 9-day-old chickens were fixed for 4 hr in cold 2-5% glutaraldehyde in 0 I M Soerensens phosphate buffer (pH 7-3) containing 2 % sucrose. The ganglia were then washed three times in buffer solution containing 3 % sucrose, each wash being 20 min in duration, then postfixed in 1 % OS04, dehydrated in an ethanol series and embedded in Epon epoxy resin. Thin sections were post-stained with uranyl acetate and lead citrate and viewed in a Zeiss EM9S-2 electron microscope. A

p 10 msec

B

1

5

10

15 Stimulus number

20

70

10 msec

Text-fig. 1. Extracellular records of compound action potential from ciliary nerves produced by preganglionic stimulation at 1/sec. A, axotomized preparation; B, normal preparation. Stimulus number applies to both sets of records. Two peaks in compound action potentials related to electrical and chemical transmission at the ganglionic synapses. The chemical (second) component fatigues more rapidly in the axotconized preparation than in the normal. RESULTS

It was found by Martin & Pilar (1963a, b) that synaptic transmission in the avian ciliary ganglion can occur in two ways: electrically, by direct spread of the action currents from the presynaptic caliciform terminals into the ciliary cells, and chemically, through the release of ACh from the presynaptic terminals. These two modes of transmission, because of their different synaptic delays, are reflected as two components in the compound action potential recorded from the ciliary nerve in response to preganglionic stimulation. The first component represents the response of cells excited electrically, the second that of cells excited chemically. When the

147 PRESYNAPTIC EFFECTS OF AXOTOMY compound action potential was recorded from the ciliary nerves 3-4 days after axotomy, both components appeared smaller than normal; 6-7 days post-operatively both components had disappeared entirely. Text-fig. 1 shows records of compound action potentials from ciliary nerves of a normal and a 4-day axotomized ganglion, produced in response to preganglionic stimulation at a rate of 1/sec. The major point of interest is that the second component, representing the chemically activated cells, declined faster in the axotomized preparation than in the normal one. This is similar to observations reported previously by Acheson & Remolina (1955) on axotomized inferior mesenteric ganglia of cats. The decline in amplitude of the second component of the compound action potential could have been associated either with progressive failure of chemical synaptic transmission or with conduction failure in the axotomized cells themselves. The latter possibility seems unlikely, as no similar decline was observed in the electrical component (see also Pilar & Landmesser, 1972). More direct evidence on this point was obtained by recording intracellularly from axotomized cells. Text-fig. 2 shows action potentials produced in one such cell by injection of depolarizing current (A) and by antidromic stimulation of the ciliary nerves (B). Action potentials so produced were indistinguishable from those recorded from normal cells with respect to excitation threshold, amplitude and duration, and the ability of the cell to fire repetitively in response to maintained depolarization of the soma (cf. Martin & Pilar, 1963a). In contrast, the amplitude of the e.p.s.p. produced by preganglionic stimulation was invariably smaller than normal in the axotomized cells, as illustrated in Fig. 2C. It seemed clear from the evidence presented so far that the decline of the post-ganglionic compound action potential with repetitive stimulation was due to a reduced safety factor for synaptic transmission after axotomy. Given that many of the e.p.s.p.s in the ganglion cells were reduced to just above threshold for excitation, normal fatigue of transmitter release during repetitive stimulation would be expected to result in progressive loss of transmission in the ganglion cell population and a consequent reduction in the number of active fibres in the ciliary nerve. This idea was tested further by examining the amplitude changes of the e.p.s.p. itself during repetitive stimulation. These experiments revealed an additional factor contributing to the loss of transmission, namely an increased rate of decline of the e.p.s.p. amplitude in the axotomized cells. This is illustrated in Text-fig. 3. The lower series of records are e.p.s.p.s recorded from a normal cell at a stimulus rate of 1/sec. These are superimposed on hyperpolarizing pulses, applied to the cell to prevent initiation of action potentials during the responses. The first response just reached

H. R. BRENNER AND E. W. JOHNSON threshold, producing an action potential in the cell. By the tenth response, the e.p.s.p. had fallen to about 80 % of its initial amplitude and remained at this size for the remainder of the train. In the axotomized cell (B), the response was much smaller and the amount of depression during the train somewhat greater. In both cells the amplitude of the coupling potentials preceding the e.p.s.p.s remained constant during the train, 148

I

A

B

C

Text-fig. 2. Intracellular recordings from ganglion cell in 4-day axotomized preparation. A, action potentials initiated by injection of positive current and, B, by antidromic stimulation of ciliary nerve are indistinguishable from normal. C, synaptic potential (e.p.s.p.) produced by preganglionic stimulation is markedly depressed. Calibrations: 40 mV (A, B); four mV (C); 10 msec. I (in A) 2-5 x 10-10A.

indicating that the reduction in e.p.s.p. amplitude was not related to alterations in the presynaptic action potential. In the upper sequence of records in Text-fig. 3 this is confirmed directly. The presynaptic action potential, recorded from the calyx of an axotomized cell, remained unchanged during the 1/sec stimulus train.

PRESYNAPTIC EFFECTS OF AXOTOMY

149 The difference in the rate of depression of the e.p.s.p. in normal and axotomized cells is summarized in Text-fig. 4, in which the amplitudes of successive e.p.s.p.s in the response train are plotted as a fraction of the amplitude of the first response with all e.p.s.p.s corrected for non-linear summation (Martin, 1955), assuming a reversal potential of -15 mV (Martin & Pilar, 1963a). Each point represents the pooled results from A

LPw

0

Lo"

L..

L

oe

NO

40< 10 msec

Xaft_

C

4

A

t

1 4mV

10 msec

JJC-D~~~9~ Us--2-1 ~mv

10 15 20 70 msec 10 Stimulus number Text-fig. 3. Intracellularly recorded action potential from (A) a presynaptic terminal and (B) a ganglion cell in axotomized ganglion. Stimulation of preganglionic nerve at 1/sec. Presynaptic action potential is normal and does not fatigue during stimulus train. Post-synaptic response consists of coupling potential followed by e.p.s.p. Note fatigue of e.p.s.p. during train. C, similar sequence from cell in normal ganglion. E.p.s.p.s superimposed on hyperpolarizing pulses to prevent action potential initiation. Fatigue is less than in axotomized cell. Stimulus number applies to all three series.

1

5

6 normal (filled circles) and 6 axotomized (open circles) cells. In both populations the responses fell rapidly in amplitude during the first 10 sec and remained relatively constant thereafter. However, the depression was more severe in the axotomized preparations, with the responses falling to about 40 % of their initial amplitude as compared with a reduction to about 60 % of initial amplitude in normal cells. The synaptic changes produced by axotomy are summarized in Table 1. In normal cells, the average e.p.s.p. amplitude (iv) was about 19 mV. In cells in which the e.p.s.p. was superimposed on hyperpolarizing pulses the amplitude of the response was normalized to the resting potential, i.e. the observed amplitude was multiplied by a factor (Em - 15)/(Eh - 15) to

H. B. BRENNER AND E. W. JOHNSON obtain the amplitude at the resting potential, where Em and Eh are the resting and hyperpolarized membrane potentials respectively. In the axotomized cells the mean amplitude of the e.p.s.p.s was about 10 mV. These amplitude measurements do not represent the true effect of axotomy on the synaptic response for several reasons. First, in about 20 % of the axotomized cells no response at all was observed, and such cells are not included in the table. Consequently the mean amplitude after axotomy is over-estimated. Second, three of the six normal cells were curarized 150

1.0

08

046 -'m 0.4

02

0

10

20

40 30 Stimulus number

50

60

70

Text-fig. 4. Depression of e.p.s.p.s with repetitive stimulation at 1/sec. Ordinate: e.p.s.p. amplitude as fraction of amplitude of first response: abscissa: stimulus number. Pooled results from six control cells (0) and six axotomized cells (0). Points were obtained by measuring three to ten successive responses in each cell. Depression is more severe in axotomized preparations. Bars represent + s.E. of mean.

to reduce the e.p.s.p. below threshold. While the amplitude reduction produced by addition of tubocurarine (10 #g/ml.) was not determined, the response amplitudes were probably attenuated by a factor of at least 2 (see Martin & Pilar, 1964b). Finally, the amplitudes were not corrected for non-linear summation. The amplitude estimates given by iv' in the table provide a more accurate representation of the effects of axotomy. To obtain iv', e.p.s.p.s in the axotomized cells were corrected for non-linear summation, using the correction factor proposed by Martin (1955). The

PRBSTYAPTIC EFFECTS OF AXOTOI Y 151 resulting mean value of 16 mV represents the amplitude the e.p.s.p.s would have reached if the unit potentials which make up the responses had summed linearly. Expressed another way, the figure is proportional to the underlying synaptic conductance increase. E.p.s.p.s in the normal cells were corrected in the same way. In addition, the corrected amplitudes of the responses in control cells which had been curarized were multiplied by a factor of 2. The resulting corrected mean e.p.s.p. amplitude (66 mV) is again roughly proportional to the average synaptic conductance increase. While these corrections are admittedly limited in accuracy, they serve to provide a rough estimate of the effect of axotomy, namely a reduction in the synaptic response by a factor of at least 4. The correction factor for non-linear summation probably over-corrects (Martin, 1976), so that the values of v' for both populations are likely to be too large, the over-estimate being greater for the normal cells. On the other hand, no account was taken of the axotomized cells in which responses were absent, so the corrected response amplitude for the axotomized group is probably over-estimated by at least a similar factor. The mean quantum contents (m) of the e.p.s.p.s in normal and axotomized cells were also calculated. There are several ways of obtaining values for m (see Martin, 1966). The most accurate is to divide the mean amplitude of the evoked e.p.s.p.s by the mean amplitude of the spontaneously occurring miniature excitatory post-synaptic potentials (min. e.p.s.p.s), with appropriate correction for non-linear summation. This method could not be used in the present experiments because miniature potentials were extremely rare in normal cells and virtually absent in axotomized preparations. Attempts to increase the min. e.p.s.p. frequency in axotomized cells by applying brief tetani to the oculomotor nerve or by perfusing the preparation with hypertonic or high [K+] solutions were unsuccessful. Possibly, miniature potentials remained undetected because of the reduction in acetylcholine sensitivity of axotomized cells. Because of these difficulties, a more indirect measure of m was made for each of the ten cells in Table 1, using the coefficient of variation of the e.p.s.p. amplitude distribution and assuming that quantal release from the presynaptic terminals was a Poisson process. With this assumption, the mean quantum content of a series of responses is given by the relation m

=

(I+CV2)ICV2,

where CV is the coefficient of variation of the e.p.s.p. amplitudes in the series and cv the coefficient of variation of the min. e.p.s.p.s. A value of 1 1 was taken arbitrarily for the factor (1 + cv2) (Martin & Pilar, 1964b). Before calculating CV, the amplitude for each e.p.s.p. in the series was corrected for non-linear summation. Each calculation was from a series

152 H. R. BRENNER AND E. W. JOHNSON of 80-140 e.p.s.p.s evoked at a rate of 1/sec. The mean quantum contents m in six normal and four axotomized cells averaged 38-1 ± 8&9 (mean + s.E. of mean) and 15-6 + 5 9, respectively. In two additional axotomized cells the amplitudes of the responses did not reach a steady state but, instead, gradually fell to zero during the train. The average quantum contents for the ten cells analysed were, of course, obtained under conditions of a steady-state depression in which e.p.s.p. amplitudes in normal cells were reduced to about 61 % and those in axotomized cells to about 40 % of their resting values (Text-fig. 4). If it is assumed that the mean quantum contents were equally depressed, then the resting quantum contents in normal and axotomized cells may be estimated as about 62 and 39 respectively. Values corrected for depression in this way are given in Table 1 (m). Finally, knowing the corrected e.p.s.p. amplitudes and mean quantum contents, average amplitudes (i3,) of the min. e.p.s.p.s in normal and axotomized cells were calculated from the relation i3, = V'/m. The mean miniature potential amplitude was reduced from 1*07 mV before axotomy to 0-41 mV after axotomy. TABiE 1. Electrophysiological parameters of cells and presynaptic calyces in normal and axotomized ganglia. Em, resting membrane potential; E., action potential amplitude; 13, mean e.p.s.p. amplitude; v', mean e.p.s.p. amplitude corrected for non-linear summation and curarization (see text); m, mean quantum content of etp.s.p., calculated from coefficient of variation of amplitude distribution; v, = V'Im, calculated miniature potential amplitude; v3, mean amplitude of electrical coupling potential. Figures give mean values + S.E. of mean for numbers of observations in parentheses. Mean quantum content and miniature potential amplitude are less than normal in axotomized preparations

Axotomized

Normal

Cell Em (mV) ;v (mV)

60.3+193 (19)

59.3+16 (18) 10.0± 2*4 (6) 16 ± 6 (6) 38.9+ 9*2 (4) 0-41

m

18-8 ± 3-6 (6) 66 ± 22 (6) 61-5 ±14-4 (6)

v13 (mV) VC (mV)

15-4± 2.5 (15)

Em (mV) E, (mV)

78.6 ± 17 (16) 102-6 ± 3-8 (16)

v' (mV)

1P07

3.9+1P2 (18) Calyx 74-6 ± 3.5 (5) 99-8± 886 (5)

The above calculations are subject to numerous errors, the most serious being related to corrections for non-linear summation and the assumption that the quantal release followed Poisson statistics (Johnson & Wernig, 1971). Consequently, the absolute values given in Table 1 for the various parameters cannot be taken too seriously. However, the relative values

153 PRESYNAPTIC EFFECTS OF AXOTOMY can be used to provide a general picture of the consequence of axotomy. The reduction of the synaptic response by a factor of about 4 in the 3- to 4-day post-operative period appeared to be due to both a reduction in mean quantum content of the synaptic potential and a reduction in the size of the unit synaptic potential. The quantum content was reduced to about two thirds and the unit potential to about a third their normal values. There was considerable scatter about these mean values, largely because the effects of axotomy do not appear to proceed at a uniform pace for all cells in the ganglion. At 3 days cells could be found which differed little from normal; at 4 days many cells were devoid of synaptic responses. The major point to be made is that the loss of transmission cannot be ascribed to reduction of a single quantal parameter. Also shown in Table 1 are the mean amplitudes (vc) of the electrical coupling potentials in fifteen normal and eighteen axotomized cells. The normal amplitude of about 15 mV was reduced to less than 4 mV 3-4 days post-operatively. This reduction was not due to any change in the presynaptic action potential. The mean action potential amplitude (calyx, Ea) in five calyces in axotomized preparations was 100 mV, not significantly different from that in sixteen calyces in normal ganglia (103 mV).

Electron microscopy It has been shown previously by Pilar & Landmesser (1972) that axotomy is followed by the usual chromatolytic reaction in ciliary cells 3 days post-operatively. In an attempt to correlate physiological alterations in synaptic transmission with changes in synaptic morphology, electron microscopic studies were done on normal ganglia of newly hatched chickens and on normal and axotomized ganglia of 3- and 9-day-old chickens. A total of about 1100 thin sections of ganglion cells were examined. Each cell section was classified into one of four groups. Those containing ganglion cell soma with no apposed presynaptic terminals were classified as Group 0. Group I contained presynaptic terminals which were characterized by low vesicle density, a dearth of mitochondria and a lightly staining background matrix. An example of this, taken from a newly hatched chicken, is shown in P1. 1 A. Group III terminals are illustrated in P1. 1 B, taken from a normal 9-day ganglion. The presynaptic terminals had a high vesicle density, numerous mitochondria and a darkly staining background matrix. Group II terminals were intermediate in characteristics and could not be classified clearly into either of Groups I or III. The distributions of the cell sections into the four groups are shown in Table 2 for normal and axotomized ganglia. In the normal cells there appears to be a progressive differentiation of the presynaptic terminals with age from predominantly Group I terminals in newly hatched

H. R. BRENNER AND E. W. JOHNSON 154 chickens, through Group II to predominantly Group III at 9 days. This pattern is altered by axotomy. For example, in control ganglia from 9-dayold chickens, 84 % of the terminals were classified as Group III whereas in the axotomized ganglia of the same age only 28 % of the terminals fell into this category and 34 % were classified as Group I (PI. 1C). The distribution of terminals between Group I and Group III was essentially unaltered by axotomy in the three day ganglia. Thus it appears that axotomy arrests the progression from Group I to Group III between the third and ninth day of development. In addition there was a marked increase in Group 0 profiles after axotomy; i.e. the probability of finding a cell section devoid of presynaptic terminals was increased so that at 9 days such sections constituted 30 % of the total in axotomized cells as compared with 3-4 % in normal ganglia. At 3 days, the increase in Group 0 profiles was the major difference between the normal and axotomized cells. TABLE 2. Characteristics of presynaptic terminals in normal and axotomized ganglia. Approximately 1100 thin sections, each containing a complete ganglion cell profile, were examined. Sections were classified as follows (see P1. 1 for examples): Group 0, no presynaptic terminals in section; Group I, few synaptic vesicles and mitochondria in terminals with lightly staining background matrix; Group II, intermediate between Groups I and III; Group III, dense vesicles and mitochondria in terminals with darkly staining background matrix. Axotomies performed on day 0 (newly hatched). Numbers give percentages of total sections falling into each group

Group 0 Control Axotomized Group I Control Axotomized Group II Control Axotomized Group III Control Axotomized

Newly hatched

3 day

9 day

6-5 -

7-1 22-8

30-0

74.2

35'6 30-6

40 33-7

9.7 -

20-6 13.0

8-7 8-4

9.7 -

32-7 33.7

83-9 27.9

3.4

DISCUSSION

The results presented here strongly suggest that axotomy of the ciliary cells results in a change in the transmitter release properties of the presynaptic nerve terminals. Both the mean quantum content of the e.p.s.p. and the susceptibility to fatigue of the e.p.s.p. during relatively low frequency stimulation were altered in the axotomized cells 3-4 days post-

155 PRESYNAPTIC EFFECTS OF AXOTOMY operatively. The reduced quantum content may be related to the apparent

reduction in extent of synaptic contact after axotomy. Thus in the 3-day axotomized ganglion the mean quantum content was reduced by about a third and the number of cell profiles in which synaptic contacts were present was reduced from about 93 % in normal cells to about 77 % after axotomy, a reduction of about 17 %. As there is no obvious way to relate the observed morphological change quantitatively to an expected physiological change, we cannot say whether the reduction in synaptic contact could account for the reduction in quantum content. In any case, it would be difficult to imagine that the loss of synaptic contact could account for the increased susceptibility of transmitter release to fatigue. We have no evidence at this point whether the latter is due to impairment of ACh metabolism or to a defect in the release mechanism itself involving, for example, impaired vesicle re-cycling (Heuser & Reese, 1973). Experiments by Black et al. (1972) have shown that destruction of developing neurones in the superior cervical ganglion of young mice prevents the normal maturation of choline acetyltransferase activity in the presynaptic nerve endings in the ganglion. The more subtle damage produced by post-ganglionic axotomy in the present experiments may have a similar effect. Apart from the loss of synaptic contact already discussed, no other morphological changes were obvious at the time of loss of chemical transmission. The later morphological changes, observed 9 days post-operatively, provide further evidence for the idea that there is a retrograde interaction between the cells and their presynaptic terminals but, because of their delayed nature, shed little light on the reasons for failure of synaptic transmission. It is apparent from Table 1 that the dominant factor in the reduction of e.p.s.p. amplitude after axotomy was an apparent reduction in amplitude of the min. e.p.s.p. Although this information was obtained indirectly from statistical calculations of quantum content, it will be shown in the following paper (Brenner & Martin, 1976) that the sensitivity of the ganglion cells to iontophoretically applied ACh is markedly reduced after axotomy, and consequently, that a reduction in size of the unit potential at least of the magnitude calculated here would be expected. Purves (1975) has reported a somewhat smaller reduction in miniature e.p.s.p. amplitude in axotomized mammalian sympathetic ganglion cells. The reason for the loss of electrical transmission after axotomy remains obscure at this point. Again, one can postulate that a 17 % reduction in the observed number of synaptic contacts could lead to a 25 % reduction in amplitude of the electrical coupling potential. No other relevant morphological changes were obvious, such as a breaking up of the calciform

H. R. BRENNER AND E. W. JOHNSON 156 terminals into smaller bouton-like structures, which would decrease the effective cleft resistance (Martin & Pilar, 1964a), or a loss of the myelin sheath surrounding the synaptic apparatus (Hess, Pilar & Weakly, 1969). We have found no evidence for synaptic detachment, accompanied by invagination of glial processes into the synaptic cleft, as reported in axotomized motoneurones (e.g. Kuno & Llinas, 1970) and in axotomized rat superior cervical ganglia (Matthews & Nelson, 1975). In the axotomized ciliary ganglion detached synaptic terminals containing membrane densities and vesicles typical of transmitter release sites were never observed. The fact that the percentage of cell sections without terminals increased after axotomy does suggest, however, some form of shrinkage of the terminal apparatus. One experimental problem which is difficult to resolve is whether the presynaptic changes observed here are, in fact, due to axotomy or, instead due to disuse as a consequence of interruption of the normal pathway associated with accommodation and pupil constriction. Because the surgical procedure interrupts normal vision, one would not expect presynaptic activity in the ciliary ganglion to be normal. On the other hand, it has been shown by L0mo & Rosenthal (1972) that blocking of impulse traffic in motor nerves of the rat has no detectable effect on mean quantum content of the end plate potential or on the amplitude of miniature endplate potentials, at least for periods of blockade lasting up to 8 days. We wish to thank Dr A. R. Martin for helpful discussion and advice. This work was supported by Research Grants NS 10832 and NS 09660 from the U.S.P.H.S. and by a grant for travel to H. R. B. from the Holderbank-Stiftung (Switzerland). REFERENCES J. (1955). The temporal course of the effects of postACHESON, G. & REMOLINA, ganglionic axotomy on the inferior mesenteric ganglion of the cat. J. Physiol. 127, 603-616. BLACK, I. B., HENDRY, I. A. & IVERSEN, L. L. (1972). The role of post-synaptic neurones in the biochemical maturation-of presynaptic cholinergic nerve terminals in a mouse sympathetic ganglion. J. Physiol. 221, 149-159. BLINZINGER, K. & KREUTZBERG, G. (1968). Displacement of synaptic terminals from regenerating motoneurons by microglial cells. Z. Zellforsch. mikrosk. Anat. 85, 145-157. BRENNER, H. R. & MARTIN, A. R. (1976). Reduction in acetylcholine sensitivity of axotomized ciliary ganglion cells. J. Physiol. 260, 159-175. BROWN, G. L. & PASCOE, J. E. (1954). The effect of degenerative section of ganglionic axons on transmission through the ganglion. J. Physiol. 123, 565-573. CECCARELLI, B., CLEMENTI, F. & MANTEGAZZA, P. (1972). Adrenergic re-innervation of smooth muscle of nictating membrane by preganglionic sympathetic fibres. J. Physiol. 220, 211-227. FEIN, H. (1966). Passing current through recording glass micropipette electrodes. IEEE Trans. bio-med. Engng 13, 211-212.

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HAMBERGER, A., HANSSON, H.-A. & SJOSTRAND, J. (1970). Surface structure of isolated neurons. Detachment of nerve terminals during axon regeneration. J. cell Biol. 47, 319-331. HESS, A. (1965). Developmental changes in the structure of the synapse on the myelinated cell bodies of the chicken ciliary ganglion. J. cell Biol. 25, 1-19. HESS, A., PILAR, G. & WEAKLY, J. N. (1969). Correlation between transmission and structure in avian ciliary synapses. J. Physiol. 202, 339-354. HEUSER, J. E. & REESE, T. S. (1973). Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction. J. cell Biol. 57, 315-344. HuNT, C. C. & RIKER, W. K. (1966). Properties of frog sympathetic neurons in normal ganglia and after axon section. J. Neurophysiol. 29, 1096-1114. JOHNSON, E. W. & BRENNER, H. R. (1975). Preganglionic effects of postganglionic nerve section. Physiologit, Wash. 18, 265. JOHNSON, E. W. & WERNIG, A. (1971). The binomial nature of transmitter release at the crayfish neuromuscular junction. J. Physiol. 218, 757-767. KUNO, M. & LLINAS, R. (1970). Alterations of synaptic action in chromatolysed motorneurones of the cat. J. Physiol. 210, 823-838. LIEBERMAN, A. R. (1971). The axon reaction: a review of the principal features of perikaryal responses to axon injury. Int. Rev. Neurobiol. 14, 49-124. L0Mo, T. & ROSENTHAL, J. (1972). Control of ACh sensitivity by muscle activity in the rat. J. Physiol. 221, 493-513. MARTIN, A. R. (1955). A further study of the statistical composition of the endplate potential. J. Physiol. 130, 114-122. MARTIN, A. R. (1966). Quanta] nature of synaptic transmission. Physiol. Rev. 46, 51-66. MARTIN, A. R. (1976). The effect of membrane capacitance on non-linear summation of synaptic potentials. J. theor. Biol. (in the Press). MARTIN, A. R. & PILAR, G. (1963a). Dual mode of synaptic transmission in the avian ciliary ganglion. J. Physiol. 168, 443-463. MARTIN, A. R. & PILAR, G. (1963b). Transmission through the ciliary ganglion of the chick. J. Physiol. 168, 464-475. MARTIN, A.R.& PILAR, G. (1964a). An analysis of electrical coupling at synapses in the avian ciliary ganglion. J. Physiol. 171, 454-475. MARTIN, A. R. & PILAR, G. (1964b). Quantal components of the synaptic potential in the ciliary ganglion of the chick. J. Physiol. 175, 1-16. MARWITT, R., PILAR, G. & WEAKLY, J. N. (1971). Characterization of two ganglion cell populations in avian ciliary ganglia. Brain Res. 25, 317-334. MATTHEWS, R. M. & RAISMAN, G. (1972). A light and electron microscopic study of the cellular response to axonal injury in the superior cervical ganglion of the rat. Proc. R. Soc. B 181, 43-79. MATTHEWS, M. R. & NELSON, V. H. (1975). Detachment of structurally intact nerve endings from chromatolytic neurones of rat superior cervical ganglion during the depression of synaptic transmission induced by post-ganglionic axotomy. J. Physiol. 245, 91-135. PILAR, G. & LANDMESSER, L. (1972). Axotomy mimicked by localized colchicine application. Science, N.Y. 177, 1116-1118. PuRvEs, D. (1975). Functional and structural changes in mammalian sympathetic neurones following interruption of their axons. J. Physiol. 252, 429-463.

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H. R. BRENNER AND E. W. JOHNSON EXPLANATION OF PLATE

Sections through ciliary ganglion cells (go) showing synaptic contacts (arrows) and characteristics of presynaptic caliciform terminals (cx). Scale, 1 jam. A, terminal classified as Type I (Table 2), having low vesicle density, few mitochondria and lightly staining background matrix. From 1-day-old chicken. B, Type III terminal with dense vesicle population and darker background matrix. From 9-day-old chicken. C, section from 9-day axotomized ganglion classified as Type I.

The Journal of Physiology, Vol. 260, No. 1

H. R. BRENNER AND:E._W. JOHNSON

H. R. BRENNER AND E. W. JOHNSON

Plate 1

(Facingp. :158)

(Facing p. 158)

Physiological and morphological effects of post-ganglionic axotomy on presynaptic nerve terminals.

J. Phyeiol. (1976), 260, pp. 143-158 With 1 plate and 4 text-ftguree Printed in Great Britain 143 PHYSIOLOGICAL AND MORPHOLOGICAL EFFECTS OF POST-GA...
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