14
Brain Research, 537 (1990) i4- 23
Elsevier BRES 16059
Phase-dependent modulation of primary afferent depolarization in single cutaneous primary afferents evoked by peripheral stimulation during fictive locomotion in the cat Jean-Pierre Gossard*, Jean-Marie Cabelguen** and Serge Rossignol Centre de Recherche en Sciences Neurologiques, Facultd de M~decine, Universit~ de Montreal, Montreal, Que. (Canada)
(Accepted 5 June 1990) Key words: Primary afferent depolarization; Fictive locomotion; Presynaptic inhibition; Cutaneous nerve; Cat
Previous results from our laboratory have shown with intra-axonal recordings that hindfoot cutaneous primary afferents are subjected to rhythmic depolarizations during fictive locomotion (L-PAD) suggesting that cutaneous presynaptic mechanisms are activated by the central locomotor program. In this study, we examined the transmission in pathways responsible for primary afferent depolarizations (PAD) of cutaneous fibres during spontaneous fictive locomotion in decorticate cats and in spinal cats injected with nialamide and L-DOPA. PADs were evoked (E-PADs) by electrical stimulation of peripheral nerves and recorded intra-axonally with micropipettes in identified superficialis peroneal (SP; n = 7) and tibialis posterior (TP; n = 17) cutaneous primary afferents. Results showed that the amplitude of E-PADs, which were superimposed on the L-PAD, was deeply modulated throughout the locomotor cycle; decreasing to reach a minimum during the flexor phase and increasing to a maximum during the extensor phase. The results were not statistically different in fibres of the two nerves and in both types of preparation. The amplitude of E-PADs was always maximum during the extensor phase whether there was a large L-PAD or not during that phase. This suggests that the presynaptic mechanisms activated by central locomotor networks (L-PAD) and those activated by peripheral inputs (E-PAD) may in part be controlled differently. The results thus show that the transmission in PAD pathways activated by cutaneous inputs is phasically modulated by the central pattern generator for locomotion. This strongly suggests that the presynaptic inhibition in cutaneous fibres evoked by the movement-related feedback during real locomotion could be similarly modulated.
INTRODUCTION It is now clear that there are phasic changes of polarization of primary afferent terminals accompanying rhythmic behaviors such as fictive scratching 4, fictive mastication 28 and fictive locomotion 2'3'6'13-15'17'23-25and real locomotion 13,47,4a. Presynaptic changes may be part of the premotoneuronal mechanisms involved in the phase-dependent modulation of transmission of cutaneous pathways 1'11'12'22'41 (see ref. 40 for review). Using Wall's method 'z , it was found that the excitability of cutaneous afferent terminals was decreased during the flexor phase in decerebrate and spinal cats injected with L - D O P A indicative of a relative hyperpolarization during that phase 3'6. O n the other hand, intra-axonal recordings in decorticate cats showed that the membrane potential of hindfoot cutaneous primary afferents is depolarized twice per fictive step cycle ( L - P A D ) with the largest depolarization occurring during the flexor phase 24. In these studies, it was assumed that the size of the afferent depolarization accompanying the rhythmicity repre* Present address: ** Present address: Correspondence: S. Succ. A, Montrdal,
sented the level of presynaptic inhibition even though the transmission in the primary afferent depolarization ( P A D ) pathways w was not specifically investigated. A recent study from our laboratory with dorsal root potential ( D R P ) recordings showed that the P A D pathways, activated by electrical peripheral stimulation, are phasically modulated during fictive locomotion in decorticate and spinal cats injected with nialamide and LD O P A 26. The D R P s evoked ( E - D R P s ) by the electrical stimulation of hindlimb cutaneous, muscle and mixed nerves were superimposed on the rhythmic fluctuations of D R P accompanying the fictive locomotor rhythm (L-DRP). In all cases, the amplitude of E - D R P was decreased during the flexor phase and increased during the extensor phase. This modulation pattern of E - D R P s amplitude could not be solely explained by the pattern of L - D R P (see, however, ref. 5). A n o t h e r study used electrical stimulation of different peripheral nerves to evoke antidromic discharges, known as dorsal root reflexes ( D R R s ) , recorded with chronically implanted nerve cuff electrodes in medial gastrocnemius nerve of
Department of Neurophysiology, Panum Institute, University of Copenhagen, Copenhagen, Denmark. Institut des Neurosciences UPMC-CNRS (UAl199), D6partement de Neurophysiologle Compar6e, Paris, France. Rossignol, Centre de Recherche en Sciences Neurologlques, Facultd de M~decine, Universit6 de Montr6al, C.P. 6128, (Quebec) Canada H3C 3J7.
0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
15 intact a n d d e c e r e b r a t e d cats w a l k i n g o n a t r e a d m i l l 16. T h e i r results s h o w e d t h a t t h e D R R s o c c u r r e d in c o r r e lation w i t h t h e f l e x o r p h a s e in b o t h p r e p a r a t i o n s . F r o m t h e s e studies, it is n o t c l e a r if P A D e v o k e d by s e n s o r y i n p u t is d i f f e r e n t l y m o d u l a t e d in d i f f e r e n t types o f afferents. M o r e o v e r , it w o u l d b e useful to d o c u m e n t m o r e p r e c i s e l y the r e l a t i o n b e t w e e n P A D e v o k e d f r o m p e r i p h e r a l i n p u t and t h e m e m b r a n e p o l a r i z a t i o n c h a n g e s (L-PAD)
accompanying
central
locomotor
networks.
Intracellular recordings of identified primary afferents may
disclose t h e
PAD
modulation
pattern
affecting
specific a f f e r e n t p a t h w a y s . In this study, w e e x a m i n e d t h e transmission
of cutaneous
PAD
p a t h w a y s with intra-
a x o n a l r e c o r d i n g s o f i d e n t i f i e d h i n d f o o t c u t a n e o u s prim a r y a f f e r e n t s d u r i n g s p o n t a n e o u s fictive l o c o m o t i o n in d e c o r t i c a t e cats and spinal cats i n j e c t e d with n i a l a m i d e a n d L - D O P A . A b r i e f d e s c r i p t i o n of t h e results was g i v e n p r e v i o u s l y 23.
MATERIALS AND METHODS
Preparation The experiments related in this paper were part of a larger experimental protocol performed on 30 cats and the procedures were explained in detail previously~. The transmission of PAD pathways was investigated successfully in 10 cats (2.8-4.6 kg). Under steroid anesthesia (Saffan: 1 ml/kg (Alphaxalone 9 mg/ml, Alphadolone 3 mg/ml)), a jugular and carotid cannulation, for fluid injection and arterial pressure monitoring, and a tracheotomy were followed by a complete decortication 33"3s. Two of these cats were subsequently spinalized at T13. Another cat was spinalized at T13 under barbiturate anesthesia (sodium pentobarbital 45 mg/kg) 3 days before the experiment to allow some recuperation of the spinal shock. On the day of the experiment, it was anaemically decerebrated under steroid anesthesia (Saffan: 1 ml/kg) by ligating the common carotid arteries and the basilar artery just cranial to the branch point of the posterior inferior cerebeUar arteries 36. Flexor (sartorius or tibialis anterior) and extensor (vastus lateralis or semimembranosus anterior) muscle nerves of the left hindlimb were dissected free and cut. Their central stumps were mounted on bipolar silver chloride electrodes for recording (Fig. 1A, ENG: electroneurogram). SP and TP nerves were exposed and enclosed in polymer cuff electrodes29 implanted at the ankle for stimulation (Fig. 1A). The pelvis and lumbar spine were fixed in a frame and, following a laminectomy exposing L s to S~ spinal segments, the dura and the pia were partially removed over the areas of penetration of the micropipette. An L 6 or 1-+7dorsal rootlet was cut and its proximal stump mounted on a bipolar silver chloride electrode for recording (Fig. 1A, DRP: dorsal root potential) '~. Paraffin oil pools were formed by drawing up skin flaps surrounding the spinal cord and the hindlimb nerves. The body and pool temperature were kept near 38 °C by means of a radiant heat lamp. After recovery from anaesthesia, the animals were paralyzed with gallamine triethiodide injection (Flaxedil: 20 mg/kg/h i.v.) and artificially ventilated. A bilateral pneumothorax was performed to stabilize the intra-axonal recordings. The end-expiratory pCO2 was maintained around 4%. Rhythmic activity could then be observed alternating in flexor and extensor electroneurograms (ENCrs) (fictive locomotion)34. When not present spontaneously, episodes of fictive locomotion could be initiated either by exteroceptive stimulation (tonic pinching) of various parts of the body or by trains of stimuli (300 Hz, 150 ms, 0.2 ms pulses) applied to cutaneous nerves27. Those stimuli were always ceased before recording. The
flctive locomotion in spinal animals was induced by the injection of nialamide (50 mg/kg) and L-DOPA (80 mg/kg) 27.
Recordings and analysis The intra-axonal recordings were performed with glass micropipettes filled with K + citrate (tip diameter less than 1/zm, impedance 15-35 M~) lowered with a 15° angle from vertical, close to the dorsal rootlets entrance at L 6 to S1 levels, 0.5 to 1.0 mm deep (Fig. 1A: Intra-axonal). Impaled axons were identified as being cutaneous by: (1) their ability to follow the electrical stimulation of the superficial peroneal (SP) or tibialis posterior (TP) nerves at a high frequency (over 700 I-Iz) with a short and constant latency as illustrated in the upper part of Fig. 1B; (2) the absence of a prepotential on the evoked spike; (3) their response to a natural stimulation of the receptor field such as fight brushing. We did not, however, fully characterize the receptive field and type because the necessary limb manipulations would have jeopardized the intraaxonal penetration. Because TP is a mixed nerve at the level of the ankle, all TP units that did not have a clear cutaneous receptive field were rejected. We also calculated the conduction velocity by dividing the latency by the nerve length measured at the end of the experiment. The threshold for nerve stimulation (T) was determined by the stimulus strength required to just evoke a deflection in the cord dorsum potential recording. When searching for identified axons, the stimulation strength was adjusted to a level high enough (50 x T) to recruit all types of fibres. The bandwidth used for intra-axonal AC recording (0.1 or 0.3-100 Hz) focussed on membrane potential fluctuation changes. The DRP signal was recorded with similar bandwidth whereas ENGs were recorded with a larger bandwidth (100 Hz to 10 kHz). The DC membrane potential was continuously monitored on an oscilloscope. Only axons with membrane potential more negative than -45 mV and with a stable DC signal were accepted for study. Through polymer cuff electrodes, stimuli (single square pulse, 0.2 ms, 1 to 30 x T) were delivered to SP and TP nerves in different parts of the locomotor cycle at every 2 or 3 cycles (Fig. 1C). The strength of the stimulation was usually adjusted to avoid a maximum E-PAD which would evoke DRRs. After each successful recording, the micropipette was drawn just outside the axon to record extracellular activity during a similar episode of fictive locomotion. The same stimulation was then given in different phases of the cycle to estimate the extracellular field potential (Fig. 1B: PAD extra). Data were recorded on a 7-channel FM recorder with frequency response of 0-5000 Hz at a speed of 19 em/s. The tapes were played back on an electrostatic printer (Gould ES-1000) and sections of data suited for analysis were digitized on a PDP 11/34 laboratory computer at 1 kHz. Interactive software 49 was used to detect the characteristics of the recorded signals. The locomotor cycle was defined as the interval between the onset of 2 successive bursts in a flexor nerve. Phase plots of E-PADs and E-DRPs amplitude were constructed by plotting the means of integrated amplitude of E-PADs and E-DRPs evoked by stimuli grouped in different phases of the cycle. The spontaneous activity in the control (non-stimulated) cycles corresponding to the phase of the stimulus in the stimulated cycle was subtracted from the evoked response. When present, the extracellular PAD amplitude was averaged and subtracted from the averaged amplitude of intra-axonal E-PADs for similar phases of the cycle (Fig. 1B). The ordinate was normalized to the largest response (100%). The ENGs (rectified and integrated), membrane potential and DRP signals of control cycles were averaged and normalized to 100%. Control cycles had similar durations so the signals would not be significantly distorted by the normalizing procedure. The extra-axonal activity was averaged and then subtracted from the intra-axonal membrane potential changes accompanying fietive locomotion (not illustrated) in order to estimate the effective axonal membrane potential variations (LPAD). In Figs. 2-4, the averaged signals were superimposed on the phase plot to facilitate viewing of the temporal relationships.
16
A
B ID
120 mV
t t DRP
INTRA-AXONAL
PAD
2 ms
,,,,,I NTRA
EXTRA I 0.5 mV
10ms PAl 909
C
0,5 mV SP U N I T
o,v DFILP 6
brtn
VLn STIM
,
ls
,
Fig. 1. A: schematic representation of experimental paradignl: DRP, recording of a proximal stump of a cut dorsal rootlet with a bipolar silver chloride electrode; intra-axonal, INTRA-AXONAL recording of cutaneous primary afferent with a glass micropipette; SP and TP, stimulating electrodes enclosed in polymer cuffs at the ankle around SP and TP nerves; ENG, proximal stumps of cut muscle nerves recorded with bipolar silver chloride electrodes. In the spinal grey matter: PAD pathways are simply represented by a single neuron i n t e r n e d between a SP and a TP fibre at the terminal level. B: ID, DC recording of the responses of a unit to stimulation of the SP nerve with a ldgh frequency (arrows: >700 Hz). Note the constant and short latency of the action potentials and the absence of prepotentials (2 superimposed sweeps). PAD: example of intra-axonal AC recording of PAD (Intra) evoked by a stimulation of TP nerve (single pulse, 2 x T). It is superimposed on the extra-axonal potential (Extra) evoked by the same stimulation in the same phase of the step cycle (4 sweeps each). C: from top to bottom: first trace, E-PADs (arrows) are superimposed on the L-PAD of a SP unit following fictive stepping; second trace, E-DRPs are superimposed on the L-DRP; third and fourth traces, locomotor activities alternating in flexor (Srtn) and extensor (VLn) ENGs; fifth trace, stimufiof TP nerve (single pulse, 2 x T) given at two different phases of the fictive step cycle.
Because the stimulated cycles are often shortened by the stimulation whereas the phase plot is relative to control cycles, values in the late phases of the plot may be absent. From the phase plots, the phase of the cycle of minimum and maximum ampfitude of E-PAD and the minimum E-PAD amplitude, expressed as a percentage of the largest value, were determined for each unit and averaged (Table I). These values were compared with the Student's t-test for SP and TP units and compared for different durations of fictive step cycles with regression analysis. RESULTS
Phasic modulation o f cutaneous P A D Fig. 1C shows a typical intra-axonal recording of a c u t a n e o u s unit in a decorticate cat during s p o n t a n e o u s fictive l o c o m o t i o n m o n i t o r e d as ~ t e r n a t i n g E N G activities in flexor sartorius (Srtn) and extensor vastus lateralis
(VLn) nerves. T h e m e m b r a n e potential of this SP unit innervating the hair on the t o p o f the fifth toe fluctuates at the r h y t h m of the fictive l o c o m o t i o n with two l o c o m o t o r - r e l a t e d depolarizations p e r cycle. T h e largest and most obvious d e p o l a r i z a t i o n occurs during the flexor phase (Srtn burst) and is followed by a trough of repolarization at the end of that phase. A smaller depolarization is sometimes a p p a r e n t (clearer in the 4th cycle) during the extensor phase ( V L n burst). This depolarization is, most of the time, o v e r l a p p e d by the larger depolarization o f the flexor phase. These characteristics were the most c o m m o n l y o b s e r v e d in an extensive study of m e m b r a n e potential changes of cutaneous p r i m a r y afferents of the h i n d l i m b during fictive locomotion in decorticate cats 24. In this figure, single stimuli of
17
~"
TABLE I Characteristics of PAD amplitude modulation
Max and Min, mean phase of the fictive step cycle for maximum and minimum E-PADs amplitude; Flexor phase, mean phase of the cycle for the end of flexor activity. Min/Max (%), mean minimum E-PAD amplitude expressed as a percentage of the largest value. Mean phases of the cycle have standard deviations. Unit
SP TP Total
Number of units
Phase of the cycle Max
Min
7 14 21
0.62 + 0.16 0.80 + 0.26 0.74 + 0.25
0.23 + 0.09 0.27 + 0.11 0.25 _+0.10
the TP nerve (2 x T) were given at the end of a Srtn burst and again in the middle of a VLn burst. It is readily seen from the raw data that the E - P A D (lst trace: arrows), superimposed on the L-PAD, is smaller in the first case, i.e. when the stimulation is given at the end of the flexor phase. E-DRPs elicited by the same stimuli can also be
SP UNITS A
STIM TPn (3 x T) PA 1105 N = 55
Srtn ~
VLR
J
Xcycle-- 1594 ms
Xcycle - 1350 ms
200 ms
200 ms SP UNIT
~
DRPL6
100~ l
.P
1
ol
~,w
"S/
0,
100% ~
a lOO~1~, \ "W 0
\,
~
...."/ .p../"'" /
DRPs 01 ""'"'" 0
PHASEOFCYCLE 1.0
0
Amplitude (Min/Max (%))
0.34 + 0.06 0.36 + 0.11 0.35 + 0.10
46.9 +_17.1 48.6 + 12.7 48.1 + 14.4
seen superimposed on the L - D R P (2nd trace). The E-PADs elicited in similar phases of the cycle were averaged and grouped in the phase plots (see Materials and Methods). Because the stimulation strength was usually low, the amplitude of extra-axonal E-PADs were either null or insignificant so that their subtraction (see Materials and Methods) turned out to have no substantial effect on the E-PADs amplitude. S P units
B
STIM TPn (2 x T)
Flexor phase
Y PHASEOFCYCLE
1i0
Fig. 2. Phase-dependent modulation of evoked PADs amplitude in two SP units during spontaneous fictive locomotion in decorticate preparations. From top to bottom: averaged integrated and rectified ENGs; averaged unit (L-PAD) and DRP (L-DRP) locomotor fluctuations; phase plots of the averaged amplitude of E-PADs and E-DRPs, normalized to the maximum responses in each case.
In Figs. 2-5, the averaged rectified E N G s (1st and 2nd traces) and the averaged L-PAD (3rd trace) and L-DRP (4th trace) are superimposed on the phase plot of the amplitude of E-PADs and E-DRPs. Complete phase plots were obtained for 7 SP units and two representative examples are given in Fig. 2. The results of the SP unit shown in Fig. 2A is the unit illustrated in Fig. 1C. The top phase plot shows that the averaged amplitude of E-PADs evoked by TPn stimulation (2 x T) in various phases of the cycle is modulated throughout the fictive step cycle. The amplitude begins to decrease in the middle of the flexor phase (Srtn activity), reaches a minimum at the end of that phase (36.3% of the largest response) then starts increasing to reach a maximum near the end of the extensor phase (VLn activity). It is noteworthy that the decrease of E-PADs amplitude coincides with the largest wave of the L-PAD (3rd trace) during the flexor phase and that the increase of E-PADs amplitude coincides with a second, although smaller, wave of the L-PAD during the extensor phase. The increase in E-PADs amplitude is not disrupted during the trough of repolarization in the L-PAD at the end of the flexor phase. In the phase plot underneath, the amplitude of E-DRPs follows a similar pattern of modulation as E-PADs and the relationship between the pattern of L-DRP and E - D R P modulation is also similar. The results of another SP unit, responding to slight pressure over the skin of the fifth toe, during spontaneous fictive locomotion in another decorticate cat is illustrated in Fig. 2B. The top phase plot of E-PADs
18 evoked by a TPn stimulation (3 × T) shows a very slight decrease in amplitude during the flexor phase when the L-PAD is the largest (3rd trace). This is followed by a rapid increase at the beginning of the extensor phase. The pattern of modulation in E-PADs amplitude has the same basic features as the previous unit but the magnitude of modulation (minimum: 67.9% of the largest response) is less important in this particular case. In the phase plot underneath, the E-DRPs amplitude reaches a minimum during the flexor phase and increases progressively at the beginning of the extensor phase as the E-PADs. Patterns of modulation such as those illustrated in Fig. 2 were seen in all SP units. Stronger TPn stimulation (15 x T) were used in two units and similar modulation patterns were found. Some characteristics of the amplitude modulation are given in Table I. E-PADs in SP units reached their maximum during the extensor phase and at the extensor-flexor transition (mean phase of the cycle: 0.62 + 0.16) and their minimum at the end of the flexor phase (mean phase of the cycle: 0.23 _+ 0.09). The mean phase of flexor ENG activity is given as a reference. The
mean minimum E-PAD amplitude, down to 46.9 +_ 17.1% of the largest value, shows that the E-PADs were deeply modulated during the fictive step cycle in all SP units. Note that there are important standard deviations. TP units
A previous study from our laboratory has shown that the pattern of L-PAD in cutaneous TP units had the same basic features as the pattern of L-PAD in SP units 24. Stimuli of the SP nerve evoked E-PADs in cutaneous TP units superimposed on the L-PAD. Complete phase plots were obtained in 13 TP units in decorticate cats and 3 examples are given in Fig. 3. In Fig. 3A, E-PADs were evoked in a TP unit innervating the pad of the 3rd toe by a SPn stimulation (3 x T) during spontaneous fictive locomotion in the same decorticate cat as in Fig. 2A. The top phase plot shows that the E-PADs amplitude decreases (48.0% of the largest response) towards the end of the flexor phase (Srtn activity) then increases during the extensor phase (VLn activity) up to the very beginning of the flexor phase. It is noteworthy in this case that the E-PADs amplitude increases during the extensor
TP UNITS A
C
B
STIM SPn (20xT)
STIM SPn (3xT) n L#~L
Srtn
PA 0504
PA 1910
f~r "
I~J~,
PA 3602
~ cycle= 1344 ms
cycle = 1402 ms
VLn
STIM SPn (20 x "I3
Xcycle=1090ms
~ 200ms
200 ms
200 ms
TP UNIT ~
DRPL6 100%
I00% 1~"' \
2
PADs l
4/.,
or" C3
O*
~. 1°°~ l DRPs
0~ 0
100%
......~,
"',.. c~
,, p
.-a~ --_
o
100% ............................. ,.-q Q .'" "o j ", . . . /o-..,
""', .
. '° ~ . ,PHASE OF CYCLE
I~o
O~
0
--,. ...... ~ ~ PHASE OF CYCLE 1.0
0
B
0 PHASE OF CYCLE 1'0
Fig. 3. Phase-dependent modulation of evoked PADs amplitude in 3 TP units during spontaneous fictive locomotion in dcoorti~t¢ preparations. Same display as in Fig. 2. In C, the averaged amplitudes of E-PADs and E-DRPs evoked in a TP unit during tonic VLn ENO activity are represented by a dashed line in the phase plots.
19 phase when the amplitude of the L-PAD is almost as large as the one during the flexor phase (3rd trace). The results of Fig. 3B are taken from a TP unit innervating the skin on the plantar-medial side of the foot during spontaneous fictive locomotion in another decorticate cat. A stronger SPn stimulation (20 x T) was used to evoke E-PADs and the top phase plot shows that the modulation of E-PADs amplitude followed the pattern previously described: it decreases (53.8% of the largest response) at the end of the flexor phase and peaks in the middle of the extensor phase. The modulation of EDRPs amplitude in the underneath phase plot follows a similar pattern. The results of the TP unit illustrated in Fig. 3C were obtained during spontaneous fictive locomotion in another decorticate cat. The amplitude of E-PAD evoked by a strong SPn stimulation (20 x T) decreases during the flexor phase and during the extensor phase. Note that the amplitude of L-PAD is comparable in both phases of the cycle with a clear trough separating the two depolarizations at the extensor-flexor transition (3rd trace). EPADs appeared to increase temporarily at the beginning of the extensor phase during the trough of repolarization in the L-PAD. In other words, the E-PAD's amplitude varies inversely with the L-PAD amplitude in this particular case. This interesting pattern was not representative of the sample, however, because in 7 other units (2 SP and 5 TP) where the L-PAD had comparable amplitude in both phases of the cycle, the amplitude of E-PADs increased progressively during the extensor phase (e.g. Fig. 3A). The L-DRP of Fig. 3C also shows comparable peaks of negativity in both phases of the cycle (4th trace) and the modulation of E-DRPs amplitude followed the previously described pattern. In this experiment, episodes of spontaneous fictive locomotion were alternating with episodes of tonic activities in the recorded ENGs. It was possible to evoke E-PAD with the same SPn stimulation (20 x T) while the VLn extensor ENG displayed weak tonic activity and the Srtn flexor ENG was silent. The averaged amplitude of E-PADs and E-DRPs are shown as a dashed line in each phase plot. The amplitude of E-PADs and E-DRPs during this extensor tonic activity equals the maximum values seen during fictive locomotion. In a different sequence in the same experiment, E-PADs could be evoked in another TP unit (not illustrated) innervating the pad of the 3rd toe with a weaker stimulation of SPn (2 x T) during fictive locomotion and during episodes of tonic VLn or tonic Srtn ENG activities while the antagonist muscle nerve was silent. The E-PAD amplitude obtained during tonic extensor ENG was again similar to the maximum value obtained during locomotion whereas the amplitude during the tonic flexor ENG
SPI NAL CAT STIM SPn (10xT) PA 7701 TAn ~ Xcyele = 3188 ms SmAn ~ 800 ms DRPL6 ~
TP UNIT ' ~ ~ ~ ' x ~ 100% - ~ . _ = 09
./~/,
PADs
Z
o
(3_ (/3 I.U rr E) UJ v O > UJ
0 100%
"L
DRPs 0
"Q,
0
o" ," "'e"
1
~L
,"'
PHASEOF CYCLE
1.0
Fig. 4. Phase-dependentmodulationof evoked PADs amplitudein a TP unit during fictive locomotion induced by nialamide and L-DOPA in a spinal cat. Same displayas in Fig. 2.
was just inferior to the minimum amplitude seen during locomotion. The amplitude of E-PADs did not fluctuate during tonic ENG activities and suggests there was no locomotion in non-recorded nerves as well. Characteristics of the modulation patterns of E-PADs amplitude of all TP units are detailed in Table I. Strong SPn stimulation (30 × T) were used in 6 TP units and gave similar results as illustrated in Fig. 3B. The maximum E-PAD always occurred towards the end of the extensor phase (mean phase of the cycle: 0.78 + 0.26) and minimum E-PAD, towards the end of the flexor phase (mean phase of the cycle: 0.25 + 0.11). The mean minimum E-PADs amplitude was 47.3 _+ 12.2% of the largest response. Note that there are important standard deviations as for SP units.
20 MIN
*
MAX
o
0 •
•
0
0 0 0
•
0
F--
0 0
Z
0 0
O O O O O O
• ,
\ '
o ,
'
'
'
'
~
i
1'.0 2.0 PHASE OF CYCLE Fig. 5. For each unit, the mean phases of the locomotor cycle where the amplitude of E-PADs is minimum (filled circles) and maximum (empty diamonds) are illustrated. The mean phase of the cycle of flexor activity of the normalized locomotor cycle was determined for each unit and connected to each other by a solid line. They were arbitrarily plotted from the shortest to the longest flexor phase. The mean values are listed in Table I.
Phasic modulation of cutaneous P A D in spinal cat Fig. 4 depicts the results from a cutaneous unit recorded in a spinal animal (3 days post-spinalization) displaying fictive locomotion induced by nialamide and L-DOPA. E-PADs were evoked by a SPn stimulation (10 x T) in a TP unit responding to blowing on the hair of the plantar surface of the foot around the toe pads. Note the typical long duration of the cycles during the L-DOPA rhythm ($ cycle = 3188 ms) displayed by the alternating activities of TAn (1st trace) and SmAn (2nd trace) averaged ENGs. The pattern of L-PAD in this cutaneous unit appears to be similar to the one observed in decorticate preparations 24 with a peak depolarization during the flexor phase followed by a trough and a second, smaller depolarization during the extensor phase. Also, as in the decorticate preparation, the amplitude of the E-PADs (top phase plot) was minimum at the end of the flexor phase and reached a maximum during the extensor phase at the peak of SmAn extensor E N G activity. The E - D R P modulation pattern, seen in the underneath phase plot, also shares similar characteristics as those obtained during spontaneous fictive locomotion in decortieate preparations. E-PADs were also evoked by SPn stimulation in 3 TP units in two acute spinal cats injected with nialamide and L-DOPA. The fictive locomotor bursts were not as
intense as in the previous case and the phases of stimuli could not be accurately determined but all E-PADs evoked in the middle of the flexor and extensor phases were put in two groups. The mean amplitude of E-PADs during the flexor phase was decreased relative to the one during the extensor phase in agreement with the modulation pattern described so far. In summary, the results showed that the modulation patterns of E-PADs amplitude in SP and TP units (conduction velocities > 55 m/s) had similar characteristics in decorticate and spinal cats. Furthermore, the mean phases of the locomotor cycle where E-PAD is minimum and maximum and the mean minimum EPADs amplitude were not statistically different for the two sets of units. These values were put together in the lower part of Table I and further illustrated in Fig. 5. The phases of the cycle for minimum (filled circles) and maximum (empty diamonds) E-PADs amplitude obtained for each unit are represented relative to the end of the flexor phase (solid line) within the locomotor cycle, arbitrarily plotted from the shortest to the longest flexor phase. The minimum E-PADs are distributed within the the flexor phase and the maximum E-PADs, within the extensor phase up to the extensor-flexor transition. Different cycle durations were obtained in different cats and linear regressions between cycle durations and the phases of minimum and maximum E-PAD showed no significant relationships (not illustrated). DISCUSSION Intra-axonal recordings have demonstrated that there is a phase-dependent modulation of the transmission in cutaneous PAD pathways during fictive locomotion. The results strongly suggest that these PAD pathways are modulated by the activity of central networks as part of the locomotor program as is the case for other reflex pathways (see ref. 40). The observation that modulation patterns of E-PADs amplitude have common characteristics during fictive locomotion in decorticate preparations and in spinal cats injected with nialamide and L-DOPA confirms that the spinal locomotor networks are able to phasically modulate P A D pathways 26. Supraspinal structures which are known to be rhythmically active during fictive locomotion a4 and some of them, known to influence interneurones mediating PAD 7, could have been involved in E-PAD modulation in decorticate preparations but their contribution to locomotor presynaptic mechanisms remains presently undetermined. Our results showed further that the patterns of E-PAD amplitude modulation did not differ for SP and TP cutaneous units even though they innervate different surfaces of the foot. This observation agrees with the
21 similarity of cutaneous reflexes elicited from those regions during real locomotion 18. Also, it is noteworthy that the modulation patterns were not significantly affected by the recruitment of intrinsic muscle fibres of the foot by TPn stimulation or by stronger stimulation recruiting smaller fibres (e.g. Fig. 3B). One possible explanation for this consistency is that E-PADs in cutaneous afferents are larger and more easily induced by volleys in cutaneous nerves than in muscle nerves, as described in the anaesthetized spinal cat 2°'21 (see ref. 44). Also, according to the same studies, depolarizing effects are already maximum with cutaneous volleys of 4-5 times threshold. The consistency of E-PAD modulation pattern with different sources of input might also indicate that the presynaptic changes depend more on the receiving cutaneous fibres than on the giving (stimulated) fibres (whether cutaneous or muscular or both) as suggested by our previous DRP study26. Because of the bias of microelectrode penetration towards large-caliber fibres (conduction velocities > 55 m/s), it is not excluded that small-diameter cutaneous fibres have different modulation patterns. The relation between the pattern of cyclic L-PAD and the modulation pattern of E-PADs amplitude does not appear to be straightforward. It is not a simple function of the membrane potential level (L-PAD) as for the modulation of motoneuronal EPSPs which tend to increase during the depolarized phase of the fictive step cycle45. Even though the exact mechanism generating the L-PAD is unknown, we may speculate for discussion purposes (see Introduction), that rhythmic changes of afferent polarization are due to the activation of the previously described PAD pathways 19-21 (see refs. 8, 30, 32, 44 for review). According to the concepts of presynaptic inhibition associated with those pathways, the size of afferent depolarization should represent the level of presynaptic inhibition: a large L-PAD or L-DRP during the flexor phase would thus indicate a relative increased presynaptic inhibition of the transmission in the receiving and giving (stimulated) fibres. Both phenomena would tend to decrease the amplitude of E-PAD. Conversely, a smaller L-PAD and L-DRP during the extensor phase would indicate relatively less presynaptic inhibition in the fibres, and hence, an increase of E-PAD. The level of polarization of receiving and giving fibres set by the locomotor networks could then account for the decrease of E-PAD during the flexor phase when there is a large L-PAD as seen in all units. However, this would not explain the increase of E-PAD when there is a large L-PAD during the extensor phase as observed in 7 units (e.g. Fig. 3A) and the absence of E-PAD increase during the trough of L-PAD following the flexor phase. Actually, the amplitude modulation of E-PAD of only one
unit could be explained by the underlying changes in L-PAD in that fibre (Fig. 3C). Occlusion could be another, non-exclusive, explanation. The recruitment of PAD interneurones by the central locomotor generators when the L-PAD is large would leave the stimulated peripheral input without significant effects. But this argument has the same limits as the changes of L-PAD during the extensor phase. Moreover, as seen in Fig. 1C, the amplitude of the E-PAD elicited during the flexor phase by a single TP stimulus may be larger than the amplitude of the L-PAD generated by the locomotor networks during that phase. Hence, it seems that neither the locomotor changes of polarization, nor occlusion, can account for the phasedependent modulation of E-PADs amplitude over the whole duration of the fictive step cycle for the majority of our cutaneous units. This conclusion actually suggests that the L-PAD and E-PAD could be generated by different interneuronal pathways and/or mechanisms. This was also suggested by dorsal root discharges not suppressed by GABA antagonists37 and by bicucullineresistant L-DRP and rhythmic antidromic discharges during fictive locomotion in decorticate cats (Gossard, unpublished observations). There is also the possibility of rhythmic changes in potassium activity as suggested by similar depolarizing waves recorded in slowly adapting lung stretch receptor afferents during fictive respiration as' 39. Also, sustained negative DC potential at the base of the dorsal horn and in the intermediate nucleus accompanying fictive locomotion in decerebrate cats suggested an increase of extracellular potassium that could depolarize afferent terminals 17. An interesting finding was that tonic efferent activities alter the amplitude of E-PADs (Fig. 3C) in decorticate cats as reported before with DRP recordings 26. The results suggest that there is no important shift in the effectiveness of PAD pathways between tonic (as in standing) and rhythmic (as in walking) efferent activities. Thus, there would be no special facilitation of PAD pathways during the activation of locomotor networks as it is the case for cutaneous reflex pathways in the forelimb 11.
Physiological implications The phase-dependent modulation of PAD pathways described in this study suggests that peripheral input would presynaptically inhibit the transmission in cutaneous pathways more during the extensor phase than during the flexor phase. During real locomotion, movementrelated afferent feedback would certainly be effective to elicit PAD and presynaptic inhibition as discussed before 15'26. DRPs of similar amplitude as the ones reported in this study (
Brain Research, 537 (1990) i4- 23
Elsevier BRES 16059
Phase-dependent modulation of primary afferent depolarization in single cutaneous primary afferents evoked by peripheral stimulation during fictive locomotion in the cat Jean-Pierre Gossard*, Jean-Marie Cabelguen** and Serge Rossignol Centre de Recherche en Sciences Neurologiques, Facultd de M~decine, Universit~ de Montreal, Montreal, Que. (Canada)
(Accepted 5 June 1990) Key words: Primary afferent depolarization; Fictive locomotion; Presynaptic inhibition; Cutaneous nerve; Cat
Previous results from our laboratory have shown with intra-axonal recordings that hindfoot cutaneous primary afferents are subjected to rhythmic depolarizations during fictive locomotion (L-PAD) suggesting that cutaneous presynaptic mechanisms are activated by the central locomotor program. In this study, we examined the transmission in pathways responsible for primary afferent depolarizations (PAD) of cutaneous fibres during spontaneous fictive locomotion in decorticate cats and in spinal cats injected with nialamide and L-DOPA. PADs were evoked (E-PADs) by electrical stimulation of peripheral nerves and recorded intra-axonally with micropipettes in identified superficialis peroneal (SP; n = 7) and tibialis posterior (TP; n = 17) cutaneous primary afferents. Results showed that the amplitude of E-PADs, which were superimposed on the L-PAD, was deeply modulated throughout the locomotor cycle; decreasing to reach a minimum during the flexor phase and increasing to a maximum during the extensor phase. The results were not statistically different in fibres of the two nerves and in both types of preparation. The amplitude of E-PADs was always maximum during the extensor phase whether there was a large L-PAD or not during that phase. This suggests that the presynaptic mechanisms activated by central locomotor networks (L-PAD) and those activated by peripheral inputs (E-PAD) may in part be controlled differently. The results thus show that the transmission in PAD pathways activated by cutaneous inputs is phasically modulated by the central pattern generator for locomotion. This strongly suggests that the presynaptic inhibition in cutaneous fibres evoked by the movement-related feedback during real locomotion could be similarly modulated.
INTRODUCTION It is now clear that there are phasic changes of polarization of primary afferent terminals accompanying rhythmic behaviors such as fictive scratching 4, fictive mastication 28 and fictive locomotion 2'3'6'13-15'17'23-25and real locomotion 13,47,4a. Presynaptic changes may be part of the premotoneuronal mechanisms involved in the phase-dependent modulation of transmission of cutaneous pathways 1'11'12'22'41 (see ref. 40 for review). Using Wall's method 'z , it was found that the excitability of cutaneous afferent terminals was decreased during the flexor phase in decerebrate and spinal cats injected with L - D O P A indicative of a relative hyperpolarization during that phase 3'6. O n the other hand, intra-axonal recordings in decorticate cats showed that the membrane potential of hindfoot cutaneous primary afferents is depolarized twice per fictive step cycle ( L - P A D ) with the largest depolarization occurring during the flexor phase 24. In these studies, it was assumed that the size of the afferent depolarization accompanying the rhythmicity repre* Present address: ** Present address: Correspondence: S. Succ. A, Montrdal,
sented the level of presynaptic inhibition even though the transmission in the primary afferent depolarization ( P A D ) pathways w was not specifically investigated. A recent study from our laboratory with dorsal root potential ( D R P ) recordings showed that the P A D pathways, activated by electrical peripheral stimulation, are phasically modulated during fictive locomotion in decorticate and spinal cats injected with nialamide and LD O P A 26. The D R P s evoked ( E - D R P s ) by the electrical stimulation of hindlimb cutaneous, muscle and mixed nerves were superimposed on the rhythmic fluctuations of D R P accompanying the fictive locomotor rhythm (L-DRP). In all cases, the amplitude of E - D R P was decreased during the flexor phase and increased during the extensor phase. This modulation pattern of E - D R P s amplitude could not be solely explained by the pattern of L - D R P (see, however, ref. 5). A n o t h e r study used electrical stimulation of different peripheral nerves to evoke antidromic discharges, known as dorsal root reflexes ( D R R s ) , recorded with chronically implanted nerve cuff electrodes in medial gastrocnemius nerve of
Department of Neurophysiology, Panum Institute, University of Copenhagen, Copenhagen, Denmark. Institut des Neurosciences UPMC-CNRS (UAl199), D6partement de Neurophysiologle Compar6e, Paris, France. Rossignol, Centre de Recherche en Sciences Neurologlques, Facultd de M~decine, Universit6 de Montr6al, C.P. 6128, (Quebec) Canada H3C 3J7.
0006-8993/90/$03.50 (~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)
15 intact a n d d e c e r e b r a t e d cats w a l k i n g o n a t r e a d m i l l 16. T h e i r results s h o w e d t h a t t h e D R R s o c c u r r e d in c o r r e lation w i t h t h e f l e x o r p h a s e in b o t h p r e p a r a t i o n s . F r o m t h e s e studies, it is n o t c l e a r if P A D e v o k e d by s e n s o r y i n p u t is d i f f e r e n t l y m o d u l a t e d in d i f f e r e n t types o f afferents. M o r e o v e r , it w o u l d b e useful to d o c u m e n t m o r e p r e c i s e l y the r e l a t i o n b e t w e e n P A D e v o k e d f r o m p e r i p h e r a l i n p u t and t h e m e m b r a n e p o l a r i z a t i o n c h a n g e s (L-PAD)
accompanying
central
locomotor
networks.
Intracellular recordings of identified primary afferents may
disclose t h e
PAD
modulation
pattern
affecting
specific a f f e r e n t p a t h w a y s . In this study, w e e x a m i n e d t h e transmission
of cutaneous
PAD
p a t h w a y s with intra-
a x o n a l r e c o r d i n g s o f i d e n t i f i e d h i n d f o o t c u t a n e o u s prim a r y a f f e r e n t s d u r i n g s p o n t a n e o u s fictive l o c o m o t i o n in d e c o r t i c a t e cats and spinal cats i n j e c t e d with n i a l a m i d e a n d L - D O P A . A b r i e f d e s c r i p t i o n of t h e results was g i v e n p r e v i o u s l y 23.
MATERIALS AND METHODS
Preparation The experiments related in this paper were part of a larger experimental protocol performed on 30 cats and the procedures were explained in detail previously~. The transmission of PAD pathways was investigated successfully in 10 cats (2.8-4.6 kg). Under steroid anesthesia (Saffan: 1 ml/kg (Alphaxalone 9 mg/ml, Alphadolone 3 mg/ml)), a jugular and carotid cannulation, for fluid injection and arterial pressure monitoring, and a tracheotomy were followed by a complete decortication 33"3s. Two of these cats were subsequently spinalized at T13. Another cat was spinalized at T13 under barbiturate anesthesia (sodium pentobarbital 45 mg/kg) 3 days before the experiment to allow some recuperation of the spinal shock. On the day of the experiment, it was anaemically decerebrated under steroid anesthesia (Saffan: 1 ml/kg) by ligating the common carotid arteries and the basilar artery just cranial to the branch point of the posterior inferior cerebeUar arteries 36. Flexor (sartorius or tibialis anterior) and extensor (vastus lateralis or semimembranosus anterior) muscle nerves of the left hindlimb were dissected free and cut. Their central stumps were mounted on bipolar silver chloride electrodes for recording (Fig. 1A, ENG: electroneurogram). SP and TP nerves were exposed and enclosed in polymer cuff electrodes29 implanted at the ankle for stimulation (Fig. 1A). The pelvis and lumbar spine were fixed in a frame and, following a laminectomy exposing L s to S~ spinal segments, the dura and the pia were partially removed over the areas of penetration of the micropipette. An L 6 or 1-+7dorsal rootlet was cut and its proximal stump mounted on a bipolar silver chloride electrode for recording (Fig. 1A, DRP: dorsal root potential) '~. Paraffin oil pools were formed by drawing up skin flaps surrounding the spinal cord and the hindlimb nerves. The body and pool temperature were kept near 38 °C by means of a radiant heat lamp. After recovery from anaesthesia, the animals were paralyzed with gallamine triethiodide injection (Flaxedil: 20 mg/kg/h i.v.) and artificially ventilated. A bilateral pneumothorax was performed to stabilize the intra-axonal recordings. The end-expiratory pCO2 was maintained around 4%. Rhythmic activity could then be observed alternating in flexor and extensor electroneurograms (ENCrs) (fictive locomotion)34. When not present spontaneously, episodes of fictive locomotion could be initiated either by exteroceptive stimulation (tonic pinching) of various parts of the body or by trains of stimuli (300 Hz, 150 ms, 0.2 ms pulses) applied to cutaneous nerves27. Those stimuli were always ceased before recording. The
flctive locomotion in spinal animals was induced by the injection of nialamide (50 mg/kg) and L-DOPA (80 mg/kg) 27.
Recordings and analysis The intra-axonal recordings were performed with glass micropipettes filled with K + citrate (tip diameter less than 1/zm, impedance 15-35 M~) lowered with a 15° angle from vertical, close to the dorsal rootlets entrance at L 6 to S1 levels, 0.5 to 1.0 mm deep (Fig. 1A: Intra-axonal). Impaled axons were identified as being cutaneous by: (1) their ability to follow the electrical stimulation of the superficial peroneal (SP) or tibialis posterior (TP) nerves at a high frequency (over 700 I-Iz) with a short and constant latency as illustrated in the upper part of Fig. 1B; (2) the absence of a prepotential on the evoked spike; (3) their response to a natural stimulation of the receptor field such as fight brushing. We did not, however, fully characterize the receptive field and type because the necessary limb manipulations would have jeopardized the intraaxonal penetration. Because TP is a mixed nerve at the level of the ankle, all TP units that did not have a clear cutaneous receptive field were rejected. We also calculated the conduction velocity by dividing the latency by the nerve length measured at the end of the experiment. The threshold for nerve stimulation (T) was determined by the stimulus strength required to just evoke a deflection in the cord dorsum potential recording. When searching for identified axons, the stimulation strength was adjusted to a level high enough (50 x T) to recruit all types of fibres. The bandwidth used for intra-axonal AC recording (0.1 or 0.3-100 Hz) focussed on membrane potential fluctuation changes. The DRP signal was recorded with similar bandwidth whereas ENGs were recorded with a larger bandwidth (100 Hz to 10 kHz). The DC membrane potential was continuously monitored on an oscilloscope. Only axons with membrane potential more negative than -45 mV and with a stable DC signal were accepted for study. Through polymer cuff electrodes, stimuli (single square pulse, 0.2 ms, 1 to 30 x T) were delivered to SP and TP nerves in different parts of the locomotor cycle at every 2 or 3 cycles (Fig. 1C). The strength of the stimulation was usually adjusted to avoid a maximum E-PAD which would evoke DRRs. After each successful recording, the micropipette was drawn just outside the axon to record extracellular activity during a similar episode of fictive locomotion. The same stimulation was then given in different phases of the cycle to estimate the extracellular field potential (Fig. 1B: PAD extra). Data were recorded on a 7-channel FM recorder with frequency response of 0-5000 Hz at a speed of 19 em/s. The tapes were played back on an electrostatic printer (Gould ES-1000) and sections of data suited for analysis were digitized on a PDP 11/34 laboratory computer at 1 kHz. Interactive software 49 was used to detect the characteristics of the recorded signals. The locomotor cycle was defined as the interval between the onset of 2 successive bursts in a flexor nerve. Phase plots of E-PADs and E-DRPs amplitude were constructed by plotting the means of integrated amplitude of E-PADs and E-DRPs evoked by stimuli grouped in different phases of the cycle. The spontaneous activity in the control (non-stimulated) cycles corresponding to the phase of the stimulus in the stimulated cycle was subtracted from the evoked response. When present, the extracellular PAD amplitude was averaged and subtracted from the averaged amplitude of intra-axonal E-PADs for similar phases of the cycle (Fig. 1B). The ordinate was normalized to the largest response (100%). The ENGs (rectified and integrated), membrane potential and DRP signals of control cycles were averaged and normalized to 100%. Control cycles had similar durations so the signals would not be significantly distorted by the normalizing procedure. The extra-axonal activity was averaged and then subtracted from the intra-axonal membrane potential changes accompanying fietive locomotion (not illustrated) in order to estimate the effective axonal membrane potential variations (LPAD). In Figs. 2-4, the averaged signals were superimposed on the phase plot to facilitate viewing of the temporal relationships.
16
A
B ID
120 mV
t t DRP
INTRA-AXONAL
PAD
2 ms
,,,,,I NTRA
EXTRA I 0.5 mV
10ms PAl 909
C
0,5 mV SP U N I T
o,v DFILP 6
brtn
VLn STIM
,
ls
,
Fig. 1. A: schematic representation of experimental paradignl: DRP, recording of a proximal stump of a cut dorsal rootlet with a bipolar silver chloride electrode; intra-axonal, INTRA-AXONAL recording of cutaneous primary afferent with a glass micropipette; SP and TP, stimulating electrodes enclosed in polymer cuffs at the ankle around SP and TP nerves; ENG, proximal stumps of cut muscle nerves recorded with bipolar silver chloride electrodes. In the spinal grey matter: PAD pathways are simply represented by a single neuron i n t e r n e d between a SP and a TP fibre at the terminal level. B: ID, DC recording of the responses of a unit to stimulation of the SP nerve with a ldgh frequency (arrows: >700 Hz). Note the constant and short latency of the action potentials and the absence of prepotentials (2 superimposed sweeps). PAD: example of intra-axonal AC recording of PAD (Intra) evoked by a stimulation of TP nerve (single pulse, 2 x T). It is superimposed on the extra-axonal potential (Extra) evoked by the same stimulation in the same phase of the step cycle (4 sweeps each). C: from top to bottom: first trace, E-PADs (arrows) are superimposed on the L-PAD of a SP unit following fictive stepping; second trace, E-DRPs are superimposed on the L-DRP; third and fourth traces, locomotor activities alternating in flexor (Srtn) and extensor (VLn) ENGs; fifth trace, stimufiof TP nerve (single pulse, 2 x T) given at two different phases of the fictive step cycle.
Because the stimulated cycles are often shortened by the stimulation whereas the phase plot is relative to control cycles, values in the late phases of the plot may be absent. From the phase plots, the phase of the cycle of minimum and maximum ampfitude of E-PAD and the minimum E-PAD amplitude, expressed as a percentage of the largest value, were determined for each unit and averaged (Table I). These values were compared with the Student's t-test for SP and TP units and compared for different durations of fictive step cycles with regression analysis. RESULTS
Phasic modulation o f cutaneous P A D Fig. 1C shows a typical intra-axonal recording of a c u t a n e o u s unit in a decorticate cat during s p o n t a n e o u s fictive l o c o m o t i o n m o n i t o r e d as ~ t e r n a t i n g E N G activities in flexor sartorius (Srtn) and extensor vastus lateralis
(VLn) nerves. T h e m e m b r a n e potential of this SP unit innervating the hair on the t o p o f the fifth toe fluctuates at the r h y t h m of the fictive l o c o m o t i o n with two l o c o m o t o r - r e l a t e d depolarizations p e r cycle. T h e largest and most obvious d e p o l a r i z a t i o n occurs during the flexor phase (Srtn burst) and is followed by a trough of repolarization at the end of that phase. A smaller depolarization is sometimes a p p a r e n t (clearer in the 4th cycle) during the extensor phase ( V L n burst). This depolarization is, most of the time, o v e r l a p p e d by the larger depolarization o f the flexor phase. These characteristics were the most c o m m o n l y o b s e r v e d in an extensive study of m e m b r a n e potential changes of cutaneous p r i m a r y afferents of the h i n d l i m b during fictive locomotion in decorticate cats 24. In this figure, single stimuli of
17
~"
TABLE I Characteristics of PAD amplitude modulation
Max and Min, mean phase of the fictive step cycle for maximum and minimum E-PADs amplitude; Flexor phase, mean phase of the cycle for the end of flexor activity. Min/Max (%), mean minimum E-PAD amplitude expressed as a percentage of the largest value. Mean phases of the cycle have standard deviations. Unit
SP TP Total
Number of units
Phase of the cycle Max
Min
7 14 21
0.62 + 0.16 0.80 + 0.26 0.74 + 0.25
0.23 + 0.09 0.27 + 0.11 0.25 _+0.10
the TP nerve (2 x T) were given at the end of a Srtn burst and again in the middle of a VLn burst. It is readily seen from the raw data that the E - P A D (lst trace: arrows), superimposed on the L-PAD, is smaller in the first case, i.e. when the stimulation is given at the end of the flexor phase. E-DRPs elicited by the same stimuli can also be
SP UNITS A
STIM TPn (3 x T) PA 1105 N = 55
Srtn ~
VLR
J
Xcycle-- 1594 ms
Xcycle - 1350 ms
200 ms
200 ms SP UNIT
~
DRPL6
100~ l
.P
1
ol
~,w
"S/
0,
100% ~
a lOO~1~, \ "W 0
\,
~
...."/ .p../"'" /
DRPs 01 ""'"'" 0
PHASEOFCYCLE 1.0
0
Amplitude (Min/Max (%))
0.34 + 0.06 0.36 + 0.11 0.35 + 0.10
46.9 +_17.1 48.6 + 12.7 48.1 + 14.4
seen superimposed on the L - D R P (2nd trace). The E-PADs elicited in similar phases of the cycle were averaged and grouped in the phase plots (see Materials and Methods). Because the stimulation strength was usually low, the amplitude of extra-axonal E-PADs were either null or insignificant so that their subtraction (see Materials and Methods) turned out to have no substantial effect on the E-PADs amplitude. S P units
B
STIM TPn (2 x T)
Flexor phase
Y PHASEOFCYCLE
1i0
Fig. 2. Phase-dependent modulation of evoked PADs amplitude in two SP units during spontaneous fictive locomotion in decorticate preparations. From top to bottom: averaged integrated and rectified ENGs; averaged unit (L-PAD) and DRP (L-DRP) locomotor fluctuations; phase plots of the averaged amplitude of E-PADs and E-DRPs, normalized to the maximum responses in each case.
In Figs. 2-5, the averaged rectified E N G s (1st and 2nd traces) and the averaged L-PAD (3rd trace) and L-DRP (4th trace) are superimposed on the phase plot of the amplitude of E-PADs and E-DRPs. Complete phase plots were obtained for 7 SP units and two representative examples are given in Fig. 2. The results of the SP unit shown in Fig. 2A is the unit illustrated in Fig. 1C. The top phase plot shows that the averaged amplitude of E-PADs evoked by TPn stimulation (2 x T) in various phases of the cycle is modulated throughout the fictive step cycle. The amplitude begins to decrease in the middle of the flexor phase (Srtn activity), reaches a minimum at the end of that phase (36.3% of the largest response) then starts increasing to reach a maximum near the end of the extensor phase (VLn activity). It is noteworthy that the decrease of E-PADs amplitude coincides with the largest wave of the L-PAD (3rd trace) during the flexor phase and that the increase of E-PADs amplitude coincides with a second, although smaller, wave of the L-PAD during the extensor phase. The increase in E-PADs amplitude is not disrupted during the trough of repolarization in the L-PAD at the end of the flexor phase. In the phase plot underneath, the amplitude of E-DRPs follows a similar pattern of modulation as E-PADs and the relationship between the pattern of L-DRP and E - D R P modulation is also similar. The results of another SP unit, responding to slight pressure over the skin of the fifth toe, during spontaneous fictive locomotion in another decorticate cat is illustrated in Fig. 2B. The top phase plot of E-PADs
18 evoked by a TPn stimulation (3 × T) shows a very slight decrease in amplitude during the flexor phase when the L-PAD is the largest (3rd trace). This is followed by a rapid increase at the beginning of the extensor phase. The pattern of modulation in E-PADs amplitude has the same basic features as the previous unit but the magnitude of modulation (minimum: 67.9% of the largest response) is less important in this particular case. In the phase plot underneath, the E-DRPs amplitude reaches a minimum during the flexor phase and increases progressively at the beginning of the extensor phase as the E-PADs. Patterns of modulation such as those illustrated in Fig. 2 were seen in all SP units. Stronger TPn stimulation (15 x T) were used in two units and similar modulation patterns were found. Some characteristics of the amplitude modulation are given in Table I. E-PADs in SP units reached their maximum during the extensor phase and at the extensor-flexor transition (mean phase of the cycle: 0.62 + 0.16) and their minimum at the end of the flexor phase (mean phase of the cycle: 0.23 _+ 0.09). The mean phase of flexor ENG activity is given as a reference. The
mean minimum E-PAD amplitude, down to 46.9 +_ 17.1% of the largest value, shows that the E-PADs were deeply modulated during the fictive step cycle in all SP units. Note that there are important standard deviations. TP units
A previous study from our laboratory has shown that the pattern of L-PAD in cutaneous TP units had the same basic features as the pattern of L-PAD in SP units 24. Stimuli of the SP nerve evoked E-PADs in cutaneous TP units superimposed on the L-PAD. Complete phase plots were obtained in 13 TP units in decorticate cats and 3 examples are given in Fig. 3. In Fig. 3A, E-PADs were evoked in a TP unit innervating the pad of the 3rd toe by a SPn stimulation (3 x T) during spontaneous fictive locomotion in the same decorticate cat as in Fig. 2A. The top phase plot shows that the E-PADs amplitude decreases (48.0% of the largest response) towards the end of the flexor phase (Srtn activity) then increases during the extensor phase (VLn activity) up to the very beginning of the flexor phase. It is noteworthy in this case that the E-PADs amplitude increases during the extensor
TP UNITS A
C
B
STIM SPn (20xT)
STIM SPn (3xT) n L#~L
Srtn
PA 0504
PA 1910
f~r "
I~J~,
PA 3602
~ cycle= 1344 ms
cycle = 1402 ms
VLn
STIM SPn (20 x "I3
Xcycle=1090ms
~ 200ms
200 ms
200 ms
TP UNIT ~
DRPL6 100%
I00% 1~"' \
2
PADs l
4/.,
or" C3
O*
~. 1°°~ l DRPs
0~ 0
100%
......~,
"',.. c~
,, p
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Fig. 3. Phase-dependent modulation of evoked PADs amplitude in 3 TP units during spontaneous fictive locomotion in dcoorti~t¢ preparations. Same display as in Fig. 2. In C, the averaged amplitudes of E-PADs and E-DRPs evoked in a TP unit during tonic VLn ENO activity are represented by a dashed line in the phase plots.
19 phase when the amplitude of the L-PAD is almost as large as the one during the flexor phase (3rd trace). The results of Fig. 3B are taken from a TP unit innervating the skin on the plantar-medial side of the foot during spontaneous fictive locomotion in another decorticate cat. A stronger SPn stimulation (20 x T) was used to evoke E-PADs and the top phase plot shows that the modulation of E-PADs amplitude followed the pattern previously described: it decreases (53.8% of the largest response) at the end of the flexor phase and peaks in the middle of the extensor phase. The modulation of EDRPs amplitude in the underneath phase plot follows a similar pattern. The results of the TP unit illustrated in Fig. 3C were obtained during spontaneous fictive locomotion in another decorticate cat. The amplitude of E-PAD evoked by a strong SPn stimulation (20 x T) decreases during the flexor phase and during the extensor phase. Note that the amplitude of L-PAD is comparable in both phases of the cycle with a clear trough separating the two depolarizations at the extensor-flexor transition (3rd trace). EPADs appeared to increase temporarily at the beginning of the extensor phase during the trough of repolarization in the L-PAD. In other words, the E-PAD's amplitude varies inversely with the L-PAD amplitude in this particular case. This interesting pattern was not representative of the sample, however, because in 7 other units (2 SP and 5 TP) where the L-PAD had comparable amplitude in both phases of the cycle, the amplitude of E-PADs increased progressively during the extensor phase (e.g. Fig. 3A). The L-DRP of Fig. 3C also shows comparable peaks of negativity in both phases of the cycle (4th trace) and the modulation of E-DRPs amplitude followed the previously described pattern. In this experiment, episodes of spontaneous fictive locomotion were alternating with episodes of tonic activities in the recorded ENGs. It was possible to evoke E-PAD with the same SPn stimulation (20 x T) while the VLn extensor ENG displayed weak tonic activity and the Srtn flexor ENG was silent. The averaged amplitude of E-PADs and E-DRPs are shown as a dashed line in each phase plot. The amplitude of E-PADs and E-DRPs during this extensor tonic activity equals the maximum values seen during fictive locomotion. In a different sequence in the same experiment, E-PADs could be evoked in another TP unit (not illustrated) innervating the pad of the 3rd toe with a weaker stimulation of SPn (2 x T) during fictive locomotion and during episodes of tonic VLn or tonic Srtn ENG activities while the antagonist muscle nerve was silent. The E-PAD amplitude obtained during tonic extensor ENG was again similar to the maximum value obtained during locomotion whereas the amplitude during the tonic flexor ENG
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was just inferior to the minimum amplitude seen during locomotion. The amplitude of E-PADs did not fluctuate during tonic ENG activities and suggests there was no locomotion in non-recorded nerves as well. Characteristics of the modulation patterns of E-PADs amplitude of all TP units are detailed in Table I. Strong SPn stimulation (30 × T) were used in 6 TP units and gave similar results as illustrated in Fig. 3B. The maximum E-PAD always occurred towards the end of the extensor phase (mean phase of the cycle: 0.78 + 0.26) and minimum E-PAD, towards the end of the flexor phase (mean phase of the cycle: 0.25 + 0.11). The mean minimum E-PADs amplitude was 47.3 _+ 12.2% of the largest response. Note that there are important standard deviations as for SP units.
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1'.0 2.0 PHASE OF CYCLE Fig. 5. For each unit, the mean phases of the locomotor cycle where the amplitude of E-PADs is minimum (filled circles) and maximum (empty diamonds) are illustrated. The mean phase of the cycle of flexor activity of the normalized locomotor cycle was determined for each unit and connected to each other by a solid line. They were arbitrarily plotted from the shortest to the longest flexor phase. The mean values are listed in Table I.
Phasic modulation of cutaneous P A D in spinal cat Fig. 4 depicts the results from a cutaneous unit recorded in a spinal animal (3 days post-spinalization) displaying fictive locomotion induced by nialamide and L-DOPA. E-PADs were evoked by a SPn stimulation (10 x T) in a TP unit responding to blowing on the hair of the plantar surface of the foot around the toe pads. Note the typical long duration of the cycles during the L-DOPA rhythm ($ cycle = 3188 ms) displayed by the alternating activities of TAn (1st trace) and SmAn (2nd trace) averaged ENGs. The pattern of L-PAD in this cutaneous unit appears to be similar to the one observed in decorticate preparations 24 with a peak depolarization during the flexor phase followed by a trough and a second, smaller depolarization during the extensor phase. Also, as in the decorticate preparation, the amplitude of the E-PADs (top phase plot) was minimum at the end of the flexor phase and reached a maximum during the extensor phase at the peak of SmAn extensor E N G activity. The E - D R P modulation pattern, seen in the underneath phase plot, also shares similar characteristics as those obtained during spontaneous fictive locomotion in decortieate preparations. E-PADs were also evoked by SPn stimulation in 3 TP units in two acute spinal cats injected with nialamide and L-DOPA. The fictive locomotor bursts were not as
intense as in the previous case and the phases of stimuli could not be accurately determined but all E-PADs evoked in the middle of the flexor and extensor phases were put in two groups. The mean amplitude of E-PADs during the flexor phase was decreased relative to the one during the extensor phase in agreement with the modulation pattern described so far. In summary, the results showed that the modulation patterns of E-PADs amplitude in SP and TP units (conduction velocities > 55 m/s) had similar characteristics in decorticate and spinal cats. Furthermore, the mean phases of the locomotor cycle where E-PAD is minimum and maximum and the mean minimum EPADs amplitude were not statistically different for the two sets of units. These values were put together in the lower part of Table I and further illustrated in Fig. 5. The phases of the cycle for minimum (filled circles) and maximum (empty diamonds) E-PADs amplitude obtained for each unit are represented relative to the end of the flexor phase (solid line) within the locomotor cycle, arbitrarily plotted from the shortest to the longest flexor phase. The minimum E-PADs are distributed within the the flexor phase and the maximum E-PADs, within the extensor phase up to the extensor-flexor transition. Different cycle durations were obtained in different cats and linear regressions between cycle durations and the phases of minimum and maximum E-PAD showed no significant relationships (not illustrated). DISCUSSION Intra-axonal recordings have demonstrated that there is a phase-dependent modulation of the transmission in cutaneous PAD pathways during fictive locomotion. The results strongly suggest that these PAD pathways are modulated by the activity of central networks as part of the locomotor program as is the case for other reflex pathways (see ref. 40). The observation that modulation patterns of E-PADs amplitude have common characteristics during fictive locomotion in decorticate preparations and in spinal cats injected with nialamide and L-DOPA confirms that the spinal locomotor networks are able to phasically modulate P A D pathways 26. Supraspinal structures which are known to be rhythmically active during fictive locomotion a4 and some of them, known to influence interneurones mediating PAD 7, could have been involved in E-PAD modulation in decorticate preparations but their contribution to locomotor presynaptic mechanisms remains presently undetermined. Our results showed further that the patterns of E-PAD amplitude modulation did not differ for SP and TP cutaneous units even though they innervate different surfaces of the foot. This observation agrees with the
21 similarity of cutaneous reflexes elicited from those regions during real locomotion 18. Also, it is noteworthy that the modulation patterns were not significantly affected by the recruitment of intrinsic muscle fibres of the foot by TPn stimulation or by stronger stimulation recruiting smaller fibres (e.g. Fig. 3B). One possible explanation for this consistency is that E-PADs in cutaneous afferents are larger and more easily induced by volleys in cutaneous nerves than in muscle nerves, as described in the anaesthetized spinal cat 2°'21 (see ref. 44). Also, according to the same studies, depolarizing effects are already maximum with cutaneous volleys of 4-5 times threshold. The consistency of E-PAD modulation pattern with different sources of input might also indicate that the presynaptic changes depend more on the receiving cutaneous fibres than on the giving (stimulated) fibres (whether cutaneous or muscular or both) as suggested by our previous DRP study26. Because of the bias of microelectrode penetration towards large-caliber fibres (conduction velocities > 55 m/s), it is not excluded that small-diameter cutaneous fibres have different modulation patterns. The relation between the pattern of cyclic L-PAD and the modulation pattern of E-PADs amplitude does not appear to be straightforward. It is not a simple function of the membrane potential level (L-PAD) as for the modulation of motoneuronal EPSPs which tend to increase during the depolarized phase of the fictive step cycle45. Even though the exact mechanism generating the L-PAD is unknown, we may speculate for discussion purposes (see Introduction), that rhythmic changes of afferent polarization are due to the activation of the previously described PAD pathways 19-21 (see refs. 8, 30, 32, 44 for review). According to the concepts of presynaptic inhibition associated with those pathways, the size of afferent depolarization should represent the level of presynaptic inhibition: a large L-PAD or L-DRP during the flexor phase would thus indicate a relative increased presynaptic inhibition of the transmission in the receiving and giving (stimulated) fibres. Both phenomena would tend to decrease the amplitude of E-PAD. Conversely, a smaller L-PAD and L-DRP during the extensor phase would indicate relatively less presynaptic inhibition in the fibres, and hence, an increase of E-PAD. The level of polarization of receiving and giving fibres set by the locomotor networks could then account for the decrease of E-PAD during the flexor phase when there is a large L-PAD as seen in all units. However, this would not explain the increase of E-PAD when there is a large L-PAD during the extensor phase as observed in 7 units (e.g. Fig. 3A) and the absence of E-PAD increase during the trough of L-PAD following the flexor phase. Actually, the amplitude modulation of E-PAD of only one
unit could be explained by the underlying changes in L-PAD in that fibre (Fig. 3C). Occlusion could be another, non-exclusive, explanation. The recruitment of PAD interneurones by the central locomotor generators when the L-PAD is large would leave the stimulated peripheral input without significant effects. But this argument has the same limits as the changes of L-PAD during the extensor phase. Moreover, as seen in Fig. 1C, the amplitude of the E-PAD elicited during the flexor phase by a single TP stimulus may be larger than the amplitude of the L-PAD generated by the locomotor networks during that phase. Hence, it seems that neither the locomotor changes of polarization, nor occlusion, can account for the phasedependent modulation of E-PADs amplitude over the whole duration of the fictive step cycle for the majority of our cutaneous units. This conclusion actually suggests that the L-PAD and E-PAD could be generated by different interneuronal pathways and/or mechanisms. This was also suggested by dorsal root discharges not suppressed by GABA antagonists37 and by bicucullineresistant L-DRP and rhythmic antidromic discharges during fictive locomotion in decorticate cats (Gossard, unpublished observations). There is also the possibility of rhythmic changes in potassium activity as suggested by similar depolarizing waves recorded in slowly adapting lung stretch receptor afferents during fictive respiration as' 39. Also, sustained negative DC potential at the base of the dorsal horn and in the intermediate nucleus accompanying fictive locomotion in decerebrate cats suggested an increase of extracellular potassium that could depolarize afferent terminals 17. An interesting finding was that tonic efferent activities alter the amplitude of E-PADs (Fig. 3C) in decorticate cats as reported before with DRP recordings 26. The results suggest that there is no important shift in the effectiveness of PAD pathways between tonic (as in standing) and rhythmic (as in walking) efferent activities. Thus, there would be no special facilitation of PAD pathways during the activation of locomotor networks as it is the case for cutaneous reflex pathways in the forelimb 11.
Physiological implications The phase-dependent modulation of PAD pathways described in this study suggests that peripheral input would presynaptically inhibit the transmission in cutaneous pathways more during the extensor phase than during the flexor phase. During real locomotion, movementrelated afferent feedback would certainly be effective to elicit PAD and presynaptic inhibition as discussed before 15'26. DRPs of similar amplitude as the ones reported in this study (
