upper cervical ganglion. The characteristics of the spontaneous and evoked spike discharges of these preganglionio units upon somatic nerve stimulation were ...
Pfliigers Arch. 314, 199--216 (1970)
Single Unit Responses in the Cervical Sympathetic Trunk upon Somatic Nerve Stimulation* W]~F~I]) JXNIG a n d ROBERT F. S C ~ X D ~ II. Pllysiologisches Institut der Universit~it, Heidelberg Received September 8, 1969
Summary. 1. ]11 cats anesthetized with chloralose single unit activity was recorded from filaments of the cervical sympathetic trunk dissected caudal to the upper cervical ganglion. The characteristics of the spontaneous and evoked spike discharges of these preganglionio units upon somatic nerve stimulation were studied. The vagus and carotid sinus nerves were cut. 2. More than 500 units were identified. Their conduction velocities ranges from 20 m/s to less than 0.5 m/s. The units with conduction velocities below 2 m/s (28~ of our sample) were considered to be unmyelinated fibers. The peak of the conduction velocity histogram of the myelinated fibers was at 4 - - 6 m/s. 3. Both in the myelinated and in the unmyelinated fiber range mainly two types of sympathetic units were found: about 700/0 were not spontaneously active and did not exhibit evoked discharges, whereas 250/0 had both properties. The other 50/0 had either one or the other property. 4. As a rule the evoked response of a unit consisted of one spike only. More rarely units with 2 - - 4 evoked discharges per stimulus were seen. I n any given unit the evoked discharges occurred with a certain propability, which, for the majority of units, was between 40--600/0 in a series of 20 trials. 5. The sympathetic units responded either to cutaneous volleys, or to cutaneous and muscle volleys. ~ o units Were seen which responded to a muscle afferent volley but not to a cutaneous one. 6. The spontaneous activity was of low frequency. In the myelinated fiber range the average was 1.7 Hz. In the unmyelinated fiber range an average of 2.9 Hz was found. Following somatic nerve stimulation the spontaneous discharge was reduced or abolished for :periods up to I s independent of the occurrence of an evoked response. The maximum depression appeared immediately after the onset of the inhibition. Key-Words: Sympathetic Fibers -- Preganglionic ~ibers -- Cervical Sympathetic Trunk -- Afferent Fibers -- Cats. Schli)zselwSrter: Sympathische Fasern -- Pri~ganglion~re Fasern -- ttalssympathicns -- Afferente Fasern -- Katzen. Somatic afferent stimulation evokes a mass discharge in the cervical sympathetic trunk having a characteristic configuration (Schmidt and S e h S n f u ~ , 1968, 1970). T y p i c a l l y , t h e r e s p o n s e c o n s i s t s o f t h r e e c o m p o n e n t s w i t h a v e r a g e l a t e n c i e s o f 40, 70, a n d 110 ms. T h e r e a f t e r t h e * This work was supported by the Deutsche Forsehungsgemeinschaft.
200
W. JEnig and R. F. Schmidt:
s p o n t a n e o u s a c t i v i t y is suppressed for periods of u p to 1 s. I n preceding studies from this l a b o r a t o r y t h e properties of the mass reflex were analyzed, p a r t i c u l a r l y w i t h regard to t h e q u e s t i o n t h r o u g h which t y p e s of muscle a n d c u t a n e o u s afferents these discharges were evoked (Sate a n d Schmidt, 1966; S c h m i d t a n d Sch6nful~, 1970). However, t h e mass recordings could n o t give a n y clues a b o u t the n u m b e r a n d t y p e s of efferent fibers p a r t i c i p a t i n g i n the reflex. To get this i n f o r m a t i o n a n analysis of the properties of i n d i v i d u a l s y m p a t h e t i c fibers i n the cervical s y m p a t h e t i c t r u n k was started, a n d the results o b t a i n e d so far will be r e p o r t e d i n this paper. Our a t t e n t i o n was m a i n l y focussed on t h e following p r o b l e m s : 1. the discharge characteristics of the i n d i v i d u a l fibers; 2. the b e h a v i o u r of t h e i r s p o n t a n e o u s a c t i v i t y after somatic n e r v e s t i m u l a t i o n ; 3. the p r o p o r t i o n of fibers p a r t i c i p a t i n g i n t h e reflexes; a n d 4. the r e l a t i o n s h i p b e t w e e n t h e c o n d u c t i o n v e l o c i t y a n d t h e s p o n t a n e o u s a n d reflex a c t i v i t y . A p r e l i m i n a r y c o m m u n i c a t i o n has a p p e a r e d (J~nig a n d Schmidt, 1969).
Methods The experiments were performed on 15 adult cats (weight 2.2 to 4.2 kg) anesthetized with chloralose (50--70 mg/kg i.p., 11 animals) or decerebrated under ether anesthesia by occluding the arteria basilaris and the carotid arteries using a ventral approach (4 animals). All animals were immobilized by the i.v. injection of gallamine tricthiodide (Flaxedil). The artificial respiration was usually adjusted to an end-expiratory C02 of 2.5--3~ The mean blood pressure of the animals was continuously recorded and kept above 90 mm Hg, if necessary by infusion of ~acrodex or Haemaccel. The rectal temperature was kept between 37 and 38~ C. In order to eliminate effects from the peripheral baroreceptors the vagal and depressor nerves as well as the carotid sinus nerves were cut bilaterally and the ganglion nodosum was extirpated on both sides. The peripheral nerves of the left hind limb which were dissected and mounted for stimulation on platin electrodes were the following: the flexor muscle nerves peroneous longus, brevis and tertins plus the part of the deep peroneal nerve supplying tibialis anticus and extensor digitorum longus (PDP), the extensor muscle nerves gastrocnemius and soleus (GS), and the cutaneous nerves superficial peroneal (SP) and sural (SU) which included both branches that arise from the sciatic nerve in the middle of the ~high. All nerves were kept in a pool of warmed paraffin oil. The thresholds for electrical stimulation of the peripheral nerves were determined by recording from pairs of electrodes mounted on the peripheral nerves and/or the l~arent nerve trunk proximal to the stimulating electrodes. The sh-ength of stimulation of nerves will be given relative to threshold, which was expressed as 1.0 T. Usually, with 0.2 ms square pulses 1.0 T was in the range from 150 to 300 inV. To dissect single unit filaments 10 mm of the cervical sympathetic trunk immediately caudal to the upper cervical ganglion were put on a black perspex plate. The epinenral sheath was slit open, and, of the protruding filaments, a small bundle was cut at the ganglion and put on the perspex plate at a right angle to the nerve trunk. Using 25 to 50 times magnification of the dissection microscope this bundle was split up in the finest fibers strands possible. The strands were put on Ag/AgC1
Sympathetic Fiber Activity
201
electrodes, and by direct stimulation of the sympathetic trunk 40--50 mm caudal to the recording electrodes it was determined whether the bundle contained one or more intact fibers.
Results
I. Types o/Sympathetic Units in the Cervical Sympathetic Trunk Identification of Single Units A sympathetic microbundle (dissection see methods) was used for investigation as soon as direct stimulation of the cervical sympathetic t r u n k caudal to the recording electrode revealed t h a t it contained one to three intact fibers which were easily disce~ible from each other (Fig. 1 B). F r o m these records the conduction velocity of the fiber under investigation was determined. Next it was seen whether the unit showed a n y spontaneous activity, and whether it was able to follow tetanic stimulation. Thereafter, in 11 out of 15 experiments a testing progTam of about 8 min duration was used to characterize the behaviour of the fiber upon somatic nerve stimulation. This procedure will be described in connection with Fig.4. The identity of a peripherally and directly evoked spike discharge was confirmed b y their collision (Fig.lB). The total time of i 0 - - 1 2 min devoted to one unit allowed the classification of altogether 534 units in the eleven experiments. I n the other 4 experiments more detailed investigations were carried out on 21 units. I n these experiments all records were taken on tape and analyzed after the experiment using a CAT computer as a multi-channel counting device. Recording from a single unit took 1 to 3 hours. I t should be made clear at this point t h a t not all fibers in the cervical sympathetic trunk are of preganglionic origin. Detailed histological investigations have shown t h a t the cervical sympathetic trunk contains some 8000 fibers. About 3000 (37~ of these fibers are unmyelinated (Foley, 1943, 1945). Judging from the results of various types of degeneration experiments the myelinated fiber group near the upper cervical ganglion contains about 900/0 preganglionic, 1~ postganglionic, and 90/0 aberant vagal fibers. The corresponding percentages for the unmyelinated group are 51~ , 320/0 , and 17~ respectively (Foley and DuBois, 1940; Foley, 1943, 1945). The same authors and Ranson and Billingsley (1918) claim t h a t there are no dorsal root afferents in the trunk. These results justify the assumption t h a t most of the myelinated fibers (conduction velocity > 2 m/s) of our sample were ofpreganglionie origin. There is much less certainty as to the origin of the unmyelinated fibers, except in those eases (10 fibers) where direct tetanic stimulation revealed t h a t the postganglionic neuron was lying somewhere in the sympathetic Crank between the stimulating ad recording electrodes
W. Janig and R. F. Schmidt:
202
SU
A
S~
sym.p. e
"sm~ number
D
C
15
lO 5 IIIIIIII
"--"-' 0
0
number
10
m/s
20
b
E
;I I ItI It t t tI I ItI It l ~ I I '
1o
.~o
m/,
F
40'.
spent, activo
lO
Z-t
--
[]
+
-- -
I~
-
+
II
+
+
vq
o
Io
m/s
2'o
'o
io
m/~
2'0
l~ig. 1 A - - F . Spontaneous and reflex activity o/sympathetic fibers in the cervical trunk. The specimen in A and B are records from a sympathetic trunk filament which contained a unit which could be activated by direct stimulation of the trunk (B) or by somatic nerve stimulation (A). I n B the SU nerve was Stimulated before the sympathetic trunk b u t tile somatically evoked spike collided with the direct antidromic spike and did not appear on the screen. The underlined portion of the upper sweep is shown expanded in the lower sweep. The histograms in C - - F show the number of fibers (ordinates) identified in 4 different experiments plotted against their conduction velocity (abscissae). The fibers were identified as described in the t e x t and classified according to their spontaneous and reflex activity (see inset). Chloralose anesthesia except in F were an anemic deeerebration was performed. The ordinates in C and E also apply to D and F. A fiber was classified as being activated from somatic affercnts ff any single electrical stimulus to a hind limb nerve evoked a discharge if t h a t fiber in more than 30~ of all trials
Sympathetic Fiber Activity
203
(accessory ganglionic neurons, Foley, 1945). The implications of the remaining ambiguity will be dealt with in the discussion. Relationship between Conduction Velocity and Spontaneous and Reflex Activity With the screening program outlined above the isolated units were grouped according to their conduction velocities and were classified into four types depending on their spontaneous activity and their responsiveness to somatic nerve stimulation. In Fig. I C - - F the results of 4 experiments on the relationship between conduction velocity and spontaneous and reflex activity are shown. I t is seen that the majority of units were neither spontaneously active nor did they exhibit evoked discharges (open squares in Fig. 1). The second largest group were those units having both properties (filled squares). There were only a few units which had only one or the other property (key see inset). I t should further be noted from Fig. 1 that in the individual experiment there was a considerable variation of not only the absolute number of fibers which we were able to dissect, but also of their conduction velocity distribution (compare C, E, F with D), and the percentages of units showing a somatically evoked reflex discharge. For instance, in Fig.1E only 16~ of the units showed such a discharge, whereas in F 43~ were activated after a peripheral stimulus. (As indicated in Fig. 1A and described in more detail in connection with Fig.3 sympathetic units mostly responded with one discharge upon a single peripheral stimulus). In Fig.2 the results of 11 experiments of this type are pooled. The histogram contains 534 units of which 147 (27.50/0) had conduction velocities ~ 2 m/s with an average of 1.1 m/s; most probably these fibers were umnyelinated. Both in the unmyelinated and in the myelinated fiber range the units formed two major groups in regard to their spontaneous activity and their reaction upon somatic stimulation: Most of them (369 units, 690/0) were neither spontaneously active nor did they show any reflex discharge, whereas the second largest group (126 units, 23.6~ had both properties. There were 23 units (4.30/0) which showed reflex discharges but had no spontaneous activity and, finally, only 16 units (3.10/0) discharged spontaneously but were not excited by somatic stimulation. Thus the first major conclusion to be drawn from Figs. 1 and 2 is that a sympathetic unit showing reflex discharges usually is spontaneously active and vice versa. There is a remarkably uneven distribution of units of these two types in our population: The percentage of units exhibiting both spontaneous and evoked activity is less than 5 in fibers with conduction velocities above 10 m/s but it is about 30 in fibers with slower conduction velocities.
204
W. J~nig and R. F. Schmidt: nut ~ber 106 c~ [] 9
8G 6G
spont,
activ.
+ +
+ +
30 26 IG
0 0
lO
mls
20
Fig. 2. Spontaneous and reflex activity el sympathetic fibers in the cervical trunk. The histogram shows the number of fibers (ordinate) obtained in 11 experiments plotted against their conduction velocity (abscissa). The graph contains 534 fibers. They were identified as described in methods and ~heir classification is given in the inset. A fiber was classified as being activated from somatic affereuts if any single electrical stimulus to a hind limb nerve evoked a discharge of that fiber in more than 30~ of all trials
II. Reflex Responses and Modi/ication o/ the Spontaneous Activity of Sympathetic Units upon A//erent Nerve Stimulation The Variability of the Reflex Latency and the Discharge Probability of Single Units I n a series of somatically evoked sympathetic mass reflexes there was a considerable fluctuation both of the latencies and amplitudes of the various reflex components (Schmidt and Sch6nfug, 1969; ~igs. 1, 3). I n order to obtain more insight into these phenomena a detailed analysis of the reflex properties of sympathetic single units was made. The specimen records of ~ i g . 3 A are from a microbundle which contained three intact units (conduction velocities see Legend). The fibers b and c were spontaneously active. Following a single SU nerve stimulation (4.7 T applied at the start of the sweep) all three fibers showed reflex discharges, fibers a and b usually responding with one, fiber c with two discharges. A similar result was obtained when the flexor muscle nerve P D P was stimulated at high stimulus strength (Yig.3C). The records demonstrate several important properties of the reflex discharges: not every stimulus is followed b y a discharge (fibers a, b) or b y the same number of discharges (fiber c); each fiber discharges with another latency; and this individual latency varies by m a n y ms around an average value.
Sympathetic Fiber Activity
A
a
b
C
c
. _A ..........
J_
a
.... . . . .
........
!
. . . . . . . . . . . . .
_L_L
__.
. . . .
~
.
L L _
_
-
--
--I
.
. .
.
.
.
.
.
.
.
.
.
.
.
+r
-,
9
1
205
. . . . .
b
c
- ........... I l
. . . . . . . .
L ,.L
. . . . . . . .
-~ . . . . . . . . . . . . . . . 1 ~l ..... - .... ir. . . . .
. . . . .
J .
.
.
.
.
.
IL... ! . .r . .
L~.
t
,L rs
- - -
....
t _~
. . . . . . .
I
. . . .
9
.
.
.
.
.
.
.
Y._
.
.
1-
.....
I-7----
] . . . . . .
~
T
I
---
I--1 Y__-2__-_ _ Y ._-'T _~ F -~ .
.
.
.
.
.
.
.
9
9
9
9
,[ +
.
,
9
9
9
_
9
__ +
L
.
.
.
.
L
--
_
,
+
20m~
B
.
20rn~
D
p/Srns
p/5ms 0,~
SU ,~.TT
PDP 75 T
0.~ sffm.
st/m.
0
TO0
200
mr
3dO
0
100
200
ms
300
Fig.3A--D. The variability o/ the reflex discharge o/ individual sympathetic fibers. The filament under observation contained three fibers having conduction velocities of 9.1 (fiber a), 1.i (b) and 0.7 (c) m/s. The fibers b and e were spotaneously active (1--2 imp/s). The time histograms in B and D show the discharge probability (ordinates) and latency distributions (abscissae) of the spike discharges evoked after SU (]3) and PDP (D) nerve stimulation. In each case 50 successive trials at a repetition rate of 2.5 per s were recorded on tape and later processed in a CAT computer, using it with an address advance every 5 ms. The specimen in A and C are photographs of a tape play-back on a storage oscilloscope screen. The stimuli were applied at the sweep onset. Time scale 20 ms, same scale as abscissae in B and D
T h e v a r i a b i l i t y o f t h e i n d i v i d u a l latencies is m o r e c l e a r l y d i s p l a y e d in t h e h i s t o g r a m s of F i g . 3 B a n d D which were o b t a i n e d from 50 consecutive trials a t a r e p e t i t i o n r a t e of 1 p e r 2.5 s. A s is a l r e a d y e v i d e n t f r o m t h e specimen records, b o t h t y p e s of s t i m u l i e v o k e d discharges in all t h r e e fibers, b u t i t is n o w seen t h a t fiber a r e s p o n d e d m o r e a n d fibers b a n d c less f r e q u e n t l y to t h e S U t h a n to t h e P D P stimuli. Changing from S U t o P D P s t i m u l a t i o n p r o l o n g e d t h e a v e r a g e latencies o n l y slightly, t h e p e a k s of t h e h i s t o g r a m s shifting b y n o t m o r e t h a n 5 t o l 0 ms. The latencies o f t h e fiber discharges in ~ i g . 3 f l u c t u a t e d b y 10 (fiber a) to • 25 m s (fiber b) a r o u n d t h e i r r e s p e c t i v e a v a r a g e value. This v a r i a b i l i t y r a n g e was also f o u n d in all o t h e r fibers (8 units) which were a n a l y z e d as t h o s e i n ~ i g . 3 a n d w h i c h h a d c o n d u c t i o n velocities
206
W. J~nig and R. F. Schmidt:
5 m/s. There was a definite tendency to a wider variability range with fibers having slower conduction velocities. Here values up to =k 50 ms were found. As seen in Fig.3 sympathetic fibers usually responded with one (fibers a, b) or two (fiber c) discharges upon a single peripheral stimulus. I n our sample 80--900/0 or the myelinated fibers (conduction velocity > 2m/s) responded with one discharge, whereas 10--20~ showed 2--3 discharges. We never saw more than 3 discharges in this fiber group. In the C-fiber range (conduction velocity ~ 2 m/s) about 50~ of the fibers responded with 1--2 discharges and 50~ with 2--5 discharges. More than 3 discharges were rarely seen. I t has to be appreciated, however, that these slowly conducting fibers often showed a considerable spontaneous activity from which the evoked discharges were sometimes difficult to discern. The units shown in Fig.3 responded rather regularly to all kinds of somatic nerve stimulation. Other units had lower discharge probabilities or their discharge probability varied with the stimulus strength and/or the afferent nerve stimulated. To obtain a quantitative measure of the discharge probability the following procedure was adopted: the 4 peripheral nerves SU, SP, GS, and P D P were stimulated at two stimulus strengths, namely at 4 and 40 times threshold of the least sensitive nerve. (This resulted in not more than 8 to 80 times threshold stimuli to the most sensitive nerve since peripheral nerve thresholds ranged from 150 to 300 mV). The stimulus strengths were chosen because cutaneous nerve stimuli of 4 times threshold and muscle nerve stimuli of 40 times threshold strength evoke nearly maximal mass reflex discharges in the cervical sympathetic trunk (Schmidt and Sch6nfuB, 1969 ; Figs. 5, 6). At each stimulus strength 20 stimuli at a repetition rate of 1 per 2.5 s were given. The trials showing a reflex discharge (Figs. 1A, 3A, C) were counted ,~nd the probability of discharge was expressed as the ratio successful/unsuccessful trials • 100 (~ Fig. 4 shows histograms of the probability of discharge of altogether 118 units tested in this way. I n A the results obtained with low stimulus strength are assembled. 108 out of the 118 units (dotted contour line) responded to low strength stimulation of cutaneous nerves and the histogram shows that the majority of units had probabilities of discharge above 50~ . Stimulation of the P D P nerve evoked reflex discharges in 60~ of these units (thin contour line) whereas GS stimulation was even less successful, only 26 units (heavy contour line) displaying discharges with 200/0 probability or more (nearly all of these GS sensitive units were also responding to P D P stimuli (ef. Fig.3). As seen from the histogram the probability of discharge upon muscle nerve stimulation was below 50~ in most units. With high stimulus strength all l i 8 units were activated b y
Sympathetic Fiber Activity number of units A
L,-ST
..... SU, S P __ PDP
B
207
0 - 80T
GS
2( F"*~
'2o'
' " '6o' 'x' '~o6 discharge
20 probability
60
%
100
Fig.4Aand B. The variability of the reflex excitability of individual symlgathetie neurons. In this histogram the units are grouped according to their discharge probabilit~y (abscissae) determined by applying 20 stimuli to a peripheral nerve at a repetition rate of 1 per 2.5 s and counting the number of trials during which an evoked discharge occurred. As indicated by the inset, in A the results obtained on low threshold stimulation (4--8 T) of cutaneous (SU, SP 108 units), flexor (PDP, 60 units), and extensor muscle (GS, 26 units) nerves are shown. The respective numbers of units responding to high threshold stimulation in B are 116, 95, and 78 units cutaneous stimuli (Fig. 2B, same symbols as in A). The number of units responding to P D P and GS stimulation increased to 95 and 78 respectively and there was also a considerable increase of the average probability of discharge upon muscle nerve stimulation, both factors contributing to the increased mass reflex discharge obtained under these conditions. Cutaneous and Muscle Afferents Converging on Single Units I n the histograms of Figs. 5A, B it is shown to what extent cutaneous and muscle afferent volleys activated the same or different populations of sympathetic fibers. I n A the results obtained with 4 - - 8 times stimulus strength were accumulated. About 450/0 of the units discharged to cutaneous volleys only, the others either to cutaneous plus flexor (320/0), or to cutaneous plus extensor (30/0) or to all 3 types (200/0) of afferent volleys. I n B the results with high stimulus strength are given. The proportion of fibers activated exclusively b y cutaneous volleys is reduced to 24~ indicating t h a t m a n y of the cutaneous units in A are also activated b y high strength muscle afferents. Similarly, the propol~ion of fibers activated b y cutaneous and flexor muscle afferents is reduced to 210/0 and there are no longer fibers activated b y cutaneous and extensor muscle volleys only. Thus the number of fibers responding to all 3 types of afferent stimulation is increased to 55~ of our sample. 14
Pfliigers Arch. Bd. 314
W. Jgrdg and R. F. Schmidt:
208
[] SU,SP [] SU,SP.PDP
I~ISU.SRGS 9 SU,SP,PDP,GS
A number ,( - 8 T
B
_
~.O-80T
I0
0
0
I0
m/s 20
0
10
m/s 20
Fig.5A and B. Al/erenf /iber types evoking sympathetic reflex discharges. The fibers were arranged according to their conduction velocity (abscissae) and, as shown in the inset, were classified as being activated either by cutaneous (El), or by cutaneous plus flexor (E), or by cutaneous plus extensor ([~), or by cutaneous plus flexor and extensor (B) muscle afferents. In A sympathetic fibers from 11 experiments were pooled which were excited by stimuli of 4--8 T. B corresponds to A but all fibers ~re added which were activated by high threshold afferents
We never saw a sympathetic unit which was activated b y muscle afferent volleys only. Furthermore, neither in A nor in B a correlation between the fiber conduction velocity and the response pattern of the unit was seen. All units in A are also contained in B, i.e. inclusion of the high threshold afferents in the afferent volley never silenced a unit discharging to low threshold afferents. There are, however, 13 units in B which responded to high threshold afferents only. I n Fig.2 it was shown t h a t a b o u t 25~ of the units in our sample exhibited evoked discharges. I t has now been demonstrated tha~ these units do not form a homogeneous group in regard to the reflex latencies (Fig.3), their discharge probabilities (Fig.4) and their afferent connections (Fig. 5). F r o m the results of Fig. 4 it would appear t h a t the individual mass discharges following identical peripheral stimuli would exhibit v e r y similar amplitudes, which in their size depended on the overall discharge probability of the sympathetic units. However, as mentioned in the beginning of this section, the individual mass discharges fluctuated considerably in size, indicating t h a t the probability of discharge of the sympathetic units varied in par~21el to each other due to
Sympathetic Fiber Activity
209
mechanisms hitherto unknown (cf. Sehmidt and SchSnfuB, 1970). A similar coupling of the individual latency fluctuations has also been indicated by the changes in latency of the mass reflex components. The Changes of the Spontaneous Activity I t is well known that sympathetic nerves show a considerable spontaneous activity which is temporarily reduced or abolished after a somatically evoked discharge, a phenomenon described as "postexcitatory-depression" or "silent period" (for literature see Schaefer, 1960; Schmidt and SchSnfui~, 1970). Recording from single units offered the possibility to study the behaviour of spontaneously discharging units before and during this silent period. The spontaneous activity was analyzed in a total of 86 units. Of these 45 had conduction velocities above 2 m/s and 31 below 2 m/s. The other 10 fibers came from postganglionie neurons lying between the stimulating and recording electrodes on the sympathetic trunk (see Methods). The three fiber groups had the following average spontaneous discharge frequencies ( • standard deviation): 1.7 ~= 1.6 Hz (range 0.7--5 Hz), 2.9 • 2.0 Hz (range 0.5--7.5 Hz) and 4.0 • 1.7 Hz (range 1.5--7.5 ttz) respectively. With two exceptions no spontaneously discharging fibers with conduction velocities above 12 m/s were found although we recorded from 72 units in the 12 to 22 m/s range. I f a tmit discharged only occasionally a spontaneous impulse (at intervals ~ 2 s) no average discharge frequency was calculated; altogether 22 units of this type were noted which are not included in the 86 units characterized in this paragraph. Following somatic nerve stimulation the spontaneous discharge was reduced or abolished in nearly all fibers under observation. Typical examples from 3 different experiments are shown in Fig. 6A--C. A and B are records from single unit filaments, filament C contained two spontaneously discharging fibers. The nerves and stimulus strengths indicated were those which gave the maximum inhibitory effects. Here, as in m a n y other examples which we recorded, the amount and time course of the inhibition appeared to be independent of the reflex activation, which varied from nearly zero in unit B to a very frequent activation of the two units in C. The dissociation between reflex activation and inhibition was even more pronounced in the histograms of Fig. 6 D - - F which show the depression of the spontaneous activity in a single unit following stimulation of three different peripheral nerves. This unit never could be activated by the somatic stimuli used, but its rather frequent spontaneous discharges were fully inhibited after each of the stimuli shown.With weaker stimuli the inhibition was less complete but had the same time course. 14"
W. Janig and R. F. Schmidt:
210
A ~5~p~ ~',,
B
D
se3eT
E
iiI ~ P~P
l
loot
C
s~'rn
F
GS 83T
SP 28 T
PDP g3T
GS 27T
15
0L
b
1000 ms
2(~00
b
1000 ms
2000
Fig.6A--F. Modification o/the spontaneous activity of individual sympathetic fibers following somatic nerve stimulation. The results in A and B are from 2 single fiber filaments (conduction velocity 6.2 and 1.6 m/s respectively). C is the same filament as in Fig.3 and D--F are from a further single fiber filament (conduction velocity 5.7 m/s). Each filament is from a different experiment. The time histograms show the changes in spontaneous activity following stimulation (arrows) with the nerves indicated. Same recording and processing techniques as in Fig. 3B, D, but with an address advance every 40 ms
Delay, amount and duration of the inhibition varied from unit to unit, with average values which were in the same order of magnitude in all experiments. The delay took 100--350 ms, the amount ranged from very weak to complete inhibition, and the duration was 500 to 1200 ms. Usually the maximum depression appeared early after the onset of the inhibition (eft Fig.6). Occasionally the inhibition was followed b y a period of slightly enhanced spontaneous activity (Fig.6D, E). 57o relation between the delay, amount and time course of the inhibition could be detected, suggesting t h a t the factors determining these parameters operate relatively independent from each other. I n multi-fiber strand of the cervical sympathetic trunk Iggo and Vogt (1960) also recorded spontaneous activities which in most cases were in the order of 1 Hz for individual units. On the other hand our observations are in some respects slightly different from those of Beacham
Sympathetic Fiber Activity
211
and Perl (1964) on thoracic and lumbar white rami of cats spinalized at Cr Under these conditions in some units much higher background activities (up to 60 Hz) were seen. Furthermore, afferent stimulation, both electrical and natural, decreased the background activity in some units but increased it in others and left it unchanged in a third group. Finally, the close correlation between spontaneous and reflex activity seen in our preparations did not seem to exist in the spinalized animal, where many units without background activity displayed evoked discharges.
I I I . Single Unit Responses and the Mass Discharge Reflex Latency in Relation to Conduction Velocity Following cutaneous nerve stimulation the three initial components of the sympathetic mass reflex discharge had latencies of about 40, 70 and 110 ms respectively. These three average lateneies were approximately 10 ms longer if muscle afferents were used (Schmidt and SchSnfuB, 1970). By determining the reflex latencies of single units in the cervical trunk it was possible to correlate the latencies of the spike discharges to the conduction velocity of the fiber under observation. In Fig. 7 A the latencies of the initial (solid triangles) and the occasional second and third spikes (open triangles) evoked b y high strength SP and SU activation were plotted against the conduction velocity of the sympathetic fibers. Each point plots the average latency of a single unit (see Legend). I t is seen that the shortest and most constant latencies were found in fibers having conduction velocities above 11 m/s. This fiber group never showed more than one discharge after a single peripheral stimulus, and, as mentioned above (Fig.2), practically never exhibited any spontaneous activity. From 11 m/s down to 2 m/s the initial latency was more variable, with a definite tendency to longer latencies as the conduction velocity decreased. At least part of this latency increase was due to the prolonged conduction time in the efferent axons: The average distance between the preganglionic neuron and the recording electrode was about 12 cm. Impulses travelling with conduction velocities of 10, 5, and 2.5 m/s will need 12, 24, and 48 ms respectively for this distance. Thus, all other conditions being constant, a total reflex latency of 40 ms in the 10 m/s fiber will prolong to 52 and 76 ms in the two slower fibers. There was a further increase in latencies within the fiber group having conduction velocities below 2 m/s and with those postganglionic fibers coming from accessory ganglionic neurons. (The latter fibers were arranged in the inset at the right hand side of Fig.7A outside the conduction velocity scale.) Since, in addition, the fibers of the last two groups tended to discharge more than once after a
W. Jiinig and R. F. Schmidt:
212
L
S U . SP 9 first spike /x following spikes
.
2~
304
z~
z~
me. 200
'~tk,
100
_ ..o ..~ .'~.,
0 ~number
'
)
A
,m,j~.a. zlL'A ~,L4~
'
b
b m;', I~"
I0 s~im.
0
100
200
300 ms
I
0
100
200
300 ms
Fig. 7A--C. l~e]lex latencies o/8ympa~hetic fibers alter vutaneous nerve stimulation iv relation to their conduction velocities. I n A the reflex lateneies following SIT or SP stimulation (40--80 T) are plotted in the ordinate against the conduction velocities of the activated fibers in the abscissa. Each solid triangle represents the average latency of a fiber which was activated in more than 30~ of all trial stimuli. I f a fiber discharged two or more spikes per stimulus the average latency of the second and further spikes was represented by an open triangle. The points in the inset at the righ~ hand side show reflex latencies of postganglionie fibers. The solid contour line histogram in B is the projection of the solid triangles in A onto the latency scale, i.e. it relays the number of the initial spike discharges to their latencies. The dotted line gives the projection of the second and further spikes. C is, on the same time scale as in B, the schematic drawing of the mass discharge recorded from the sympathetic trunk (Schmidt and Sch6nfuB, 1970; Fig. 2 G)
213
SympatheticFiber Activity PDP, GS 9 first spike [3 following spikes
L 3O0 on ~i
ms
u n
u
=
0%
o
200
IAunt~um
~.-
A lO0 i
o b
~
'
,
~
,
12m'/~
I
h
9
9
i6
number
I0 s!im. 0
100
200 ms
I
A~§ 0
100
300
C 200
300 ms
Fig.8A--C. Reflex laten~ies o/sympathetic fibers after mu~dr nerve stimulation in relat~ to their conduction velocities. A--C correspond f~ A--C in Fig. 7 except that, as indicated in the figure, muscle nerves were used
single stimulus, nearly all spike discharges recorded from 120 to 320 ms after the stimulus came from these fiber groups. Fig.SA shows the corresponding results obtained after high strength stimulation of the muscle afferents in the P D P and GS nerve. There was a slight but definite increase of the average latencies and, except in the high conduction velocity group, a wider scattering of latencies. Again the postganglionic axons from accessory ganglionic neurons are shown in the inset outside the conduction velocity scale. Neither in Fig. 7A nor in Fig. 8A spike latencies longer than 350 ms are seen. Obviously at this time after the stimulus the post-excitatory inhibition (silent period) had started in all sympathetic fibers of the cervical trunk.
214
W. J~nig and R. l~. Schmidt:
The Reconstruction of the Mass Response The data obtained in this investigation revealed that the three initial components of the mass discharge are mainly due to single spike discharges in units having different reflex lateneies and not to the repetitive discharges of a homogeneous fiber population. I n Figs. 7 A and 8 A a distinct threefold grouping is not apparent. If, however, as done in Figs.7B and 8B, the points in A are projected onto the ordinates and formed into a histogram relating the time after the stimulus to the number of spikes occurring at this time, the grouping is immediately apparent in both cases, although the number of fibers was rather small for such an attempt. Figs.TC and 8C are schematic drawings to the same time scale as in 7 B and 8B of the mass reflex response recorded in the cervical sympathetic trunk (ef. Schmidt and SchSnfug, 1970; Fig. 2). The lateneies and times to peak of the mass responses correspond well to the lateneies of the peaks of the histograms in Figs. 7 B and 8 B. The dotted contour lines in 7B and 8B show the contribution of the second and further spikes to the reconstructed mass response. I t is seen that their contribution to the second peak is small and that even during the third peak the mass response is mainly due to the first discharge in units with long reflex latencies and not to the repetitive discharging of short latency fibers. Furthermore, it is now obvious that the positive potential wave, seen after the negative deflections, was due to the uniform inhibition of the spontaneous discharges during this period, accompanied b y a complete absence of evoked s13ikes from 350 ms onwards. Discussion Biased or Unbiased Fiber Population? As reported above, the cervical sympathetic trunk contains about 370/0 nnmye]inated and 63~ myelinated fibers. Of our fibers 27.5~ had conduction velocities below 2 m/s. I t appears, therefore, t h a t we recorded from a larger proportion of myehnated fibers than a random sample would show. However, within the myelinated fiber group most probably no severe biasing has been done with the dissection methods used: The peak of the conduction velocity histogram of the myelinated fibers (ef. Fig.2) is at 4 - - 6 m/s with a range from 2 to 24 m/s, and such a fiber distribution is also suggested b y measurements of the conduction velocity and strength dependence of the preganglionic volley evoked b y preganglionie nerve stimulation (J/~nig, Sate and Schmidt, unpublished observations). Types of Fibers in the Cervical Sympathetic Trunk The overwhelming majority of the myelinated fibers in the cervical sym13athetic trunk is of preganglionic origin (s. Results, 13.201) and it is
SympatheticFiber Activity
215
assumed that no serious distortion has been introduced by the occasional inclusion of a vagal fiber. The unmyelinated fiber group, however, is much less homogeneous, the percentages being 5 1 o preganglionic, 320/0 postganglionic, and 17~ vagal fibers (Foley and DuBois, 1940; Foley, 1943, 1945). We had no possibility to discern between these three fiber groups, yet it appears likely, that of those fibers with rather high spontaneous activity and more than 2 evoked discharges per stimulus a considerable proportion was postganglionie, since in our sample the postganglionic fibers coming from accessory ganglionic neurons showed exactly this bchaviour. On the other hand the silent fiber group might contain postganglionic fibers, namely those descending from the superior cervical ganglion, which were cut off from their central connections. Similarly, efferent vagal fibers would show up in our silent fiber population. Despite these possible sources of error, we take the fact that the relative proportions of silent and active fibers in the 0--2 and 2--7 m/s range were approximately equal as first evidence that unmyelinated preganglionic fibers behaved similar to myelinated one's under the present experimental conditions. Significance of Spontaneous and Reflex Activity The experiments have shown that approximately 90~ of our preganglionic fibers belonged to either of two fiber types: the majority (69~ in our population) were silent and did n o t show evoked spike discharges, the rest exhibited low frequency spontaneous activity and answered with one or two spikes to peripheral stimulation. This distinct grouping and the high correlation between spontaneous and evoked activity rises a number of questions which at the present state of our knowledge have to be left unanswered. For instance, the varying proportion of silent to active fibers in the individual experiments (Fig. 1) might be purely accidental or might indicate that the number of active fibers is not a constant fraction of the total fiber population, but varies depending on the state of the animal. The other possibility is, that the fiber characteristics do not change because they are typical for efferent preganglionic fibers with different peripheral functions. Numbers of Fibers Participating in the Reflex The two initial components of the mass reflex (cf. Schmidt and SchSnfu$, 1970; Fig.2) are due to spike discharges in the myelinated efferent fibers (Figs. 7, 8). In this group evoked discharges were observed in 35~ of the fibers with conduction velocities from 2--7 m/s and in 200/0 of those with higher conduction velocities. Since the cervical sympathetic trunk contains some 5,000 myelinated fibers and since not
216
W. JEnig and R. F. Schmidt: Sympathetic Fiber Activity
every active fiber discharges regularly to each stimulus (Fig.4, s. also Schmidt and SchSnfuss, 1969, ~igs. 1, 3) it can be estimated t h a t a single peripheral stimulus of sufficient strength evokes a reflex discharge in 1,000 or less myelinated preganglionic fibers. The third component of the mass discharge reflects evoked activity in the unmyelinated fibers of the trunk (Figs. 7, 8). The t r u n k contains about 3,000 fibers of this t y p e of which more t h a n half are of preganglionie nature (Foley, 1943, 1945). I n our population 35~ of the nnmyelina$ed fibers showed reflex discharges, so t h a t it appears t h a t in the unmyelinated group again 1,000 or less fibers respond to an appropriate afferent volley.
References Beacham, W. S., Peri, E.R.: Background and reflex discharge of sympathetic preganglionic neurones in the spinal eat. J. Physiol. (Lond.) 172, 400--416 (1964). Foley, J. 0.: Composition of the cervical sympathetic trunk. Proe. Soc. exp. Biol. (N. Y.) 52, 212--214 (1943). -- The components of the cervical sympathetic trunk with special reference to its accessory cells and ganglia. J. eomp. Neurol. 82, 77--91 (1945). -- DuBois, F. S.: A quantitative and experimental study of the cervical sympathetic trunk. J. comp, Neurol. 72, 587--603 (1940). Iggo, A., Vogt, ~. : Preganglionic sympathetic activity in normal and in reserpine treated cats. J. Physiol. (Lond.) 150, 114--133 (1960). J~nig, W., Schmidt, R. F. : Die Aktivierung pr~iganglion~rer sympathetischer Fasern durch Haut- und Muskelafferenzen. Pfliigers Arch. 307, 131 (1969). Ranson, S. W., Billingsley, P. R. : The superior cervical ganglion and the cervical portion of the sympathetic trunk. J. comp. NeuroL 29, 313--358 (1918). Sato, A., Schmidt, R. F.: ]~uscle and cutaneous afferents evoking sympathetic reflexes. Brain Res. 2, 399--401 (1966). Schaefer, H.: Central control of cardiac function. Physiol. Roy. 44), Suppl. 4, 213--231 (1960). Schmidt, R. F., Sch6nfuss, K. : Characteristics of sympathetic reflexes induced by somatic nerve stimulation. Proc. XXIV. Int. Congr. Physiol. Sciences 7, 1172 (1968). -- -- An analysis of the reflex activity in the cervical sympathetic trunk induced by myelinated somatic afferents. Pfliigers Arch. 314, 175--198 (1970). Ansehrift der Verfasser: II. Physiologisches Institut der Universit~t Heidelberg 6900 Heidelberg, Bergheimer Strat~e 147
