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A novel class of unit in the substantia gelatinosa of the spinal cat
EXPERIMENTAL
57, 792406
NEUROLOGY
A Novel
Class
of Unit in the Substantia of the Spinal Cat IAN
~epartlrkcrkt
(1977)
of Biology
Massachusetts Caukbridge,
HENTALL
l
and Rcsearck Laboratory Ikkstitzkte of Techkkology, Massachusetts 021.39
Received
June
Gelatinosa
of Elfctrokkics,
9, 1977
Using platinum-tipped microelectrodes, 34 units of a kind not previously reported were found in the substantia gelatinosa of spinal, unanesthetized cats. Units were inhibited by innocuous mechanical stimuli, but after the withdrawal of such stimuli, a burst of activity lasting 5 to 20 s appeared with an initial delay (latent period) of 50 to 200 ms. In the absence of background activity, noxious stimuli had no immediate effect. However, in a period of about 20 min, units tested with noxious stimuli displayed a gradual increase in regular background firing and in the occurrence of unevoked bursts. Noxious stimuli were excitatory when applied ,to the units with this background activity. An approximately l-s period of diminished activity followed withdrawal of noxious stimuli. Consecutive identical stimuli produced bursts of activity with considerable variability; latency and burst length fluctuated. Also the probability of bursts fluctuated in units with low background activity. Poststimulus time histograms showed, under some conditions, a decrease in ‘the average number of impulses in the first 200 ms which roughly corresponded in time course to the negative dorsal root potential. It is argued that the initial latency and the decrease in activity in the histograms are temporal prolongations of the inhibitory process produced by innocuous stimuli. Moreover, based on these data, a mechanism for the generation of the negative dorsal root potential is proposed, involving dendroaxonic synaptic transmission from the substantia gelatinosa units to primary afferent terminals. Abbreviations : GABA--r-aminobutyric acid ; DRP-dorsal root potential. 1 This research was supported by a grant from Bell Telephone Laboratories. It was submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Biology, Massachusetts Institute of Technology. The author is grateful to Dr. Jerome Lettvin for valuable advice and encouragement on this project. Thanks are also due ‘to Drs. Howard Fields and Allan Basbaum for suggestions on the manuscript, to Mr. Eric Newman for his criticism of the dendroaxonic model, and to Mrs. Stephanie Bornstein for careful typing. The author’s present address is Department of Physiology S-762, University of California, San Francisco, California 94143. 792 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0014- -4886
SUBSTANTIA
GELATINOSA
793
INTRODUCTION Until recently there existed no direct information on the responses of single units in the substantia gelatinosa (26). Now Kumazawa and Per1 (13) have reported in monkeys, with chloralose or Nembutal as anesthetic agent, units situated in the lower sacral or coccygeal substantia gelatinosa which were specifically responsive to C-fiber stimulation. Also, in the substantia gelatinosa of cats anesthetized with chloralose, units whose basic response was inhibition by either noxious stimuli or weak nlechanical stimuli or both were briefly reported (6) ; in some units an adjacent excitatory receptive field was found. The present study, using spinal, unanesthetized animals, revealed a previously undocumented class of unit in the substantia gelatinosa. They were first described in a short preliminary report (11). The response of units of this class to cutaneous stimulation suggests that the negative dorsal root potential [ D.R.V of Lloyd and MacIntyre, (15)] arises, at least in part, from interaction 1,etween the dendrites of substantia gelatinosa neurons and afferent terminals. METHODS Tltc Prcparatiopt. Adult cats were made spinal by occluding under ether anesthesia the carotid and vertebral arteries. After this procedure, artificial respiration was conmenced and the anesthesia was discontinued. The animals were totally suspended by ear bars, a clamp on the L1 vertebral spine, and hip bars. Radiant heat maintained body temperature, as nieasured by a subscapular thermistor, at 37°C within 0.1 “C. Cord segments Lo and 1~; were exposed by laminectomy, the dura was cut back, and small pieces of pia-araclinoitl were torn an-a! to allow electrode penetration. Intravenous or intraperitoneal injection ol Flasetlil prevented refies movements. Rccordi~tg Mrfkotls. Glass nlicropipets, filled with iVood’s metal and with a S-pm layer of platinum black plated to their tips (7)) were used for recording.These particular electrodes were chosen because a high capacitance develops at the metal-tissue interface, due to the sponginess of platinum black (9) . In consequence, whereas the impedance measured at 1 kHz of the conmonl~~ used extracellular microelectrodes, fluid-filled glass inicropipets and unplatinized nletal, is generally above 1 MQ. impedances of the electrodes used in the present study were about 100 kf& This choice of electrode gave a lower thermal noise, because root-mean-square noise in a given frequency band is proportional to the square root of impedance (9). The achieved peak-to-peak noise of 20 pV enabled SO-PV nerve spikes to be analyzed.
794
IAN
I
IIENTALL
I
1 set FIG. 1. The electrical sign of the boundary between dorsal columns and lamina I. During the time indicated by the thick line the electrode tip was first pulled back 30 pm and ‘then returned the same distance across the boundary.
Conventional amplification and display techniques were used ; the signal was also recorded on tape for later processing. The position of the electrode tip during recording was determined by reading directly on the micromanipulator the distance from the boundary of the dorsal column and lamina I. In all preparations this boundary appeared as a region of change in characteristic background activity, occupying about 30 /*.ni in the dorsoventral direction (Fig. 1). Dorsally, in the white matter, multiple units with a predominantly positive spike were seen, firing at a moderately high rate; these responded to changes of limb position. In contrast, lamina I presented no activity or else a single spontaneously firing unit with a large receptive field to light touch. This method was verified in several cases by making small electrolytic lesions at recording sites, and subsequently reconstructing them in sections stained with cresyl violet. In laminae II and III, a complex multiple-unit response with the appearance of a doubling of thermal noise, also described by Wall (25)) appeared upon brushing a particular area of skin. Because this did not occur either in lamina I or in lamina IV, it provided a confirmatory marker of electrode position. To search for units an electrode was gradually lowered through the substantia gelatinosa while the skin was being brushed in the region which elicited the multiple-unit response. It took many penetrations to find one isolated single unit of the class described in this paper. .‘j’ti~rlr,lntion. Mechanical, thermal, and electrical stimuli were applied to the left hind limb, the hair of which had been clipped to less than 0.6 cm (0.25 in.). The mechanical stimuli can be classed as noxious or innocuous, aud as hand-delivered or automatic. Pinching with a hemostat and prodding with a hypoclern~ic needle constituted the noxious mechanical stinn~li. Innocuous stimuli included moving individual hairs with fine forceps,
brushing a group of hairs with a piece of cotton, and applying slight pressure to the skin with 3 blunt probe of a few square-millimeter tip area (referred to 3s “light touch”). I-land-delivered stimuli were sonietinies monitored by a strain gauge lvlmse output was recorded on FM tape. An electromeclm~ically driven pI-(JlJe lwlder, to which could he attached a liypoderiiiic needle or a blunt pTJlJe, cleliveretl automatic repeatable stimuli to the leg, which for this l~urlms~ WLS heltl rigidly in a clamp. A l-cm’, thermostatic hot plate, li~ltl against tlie skin and heated to ahut SO”C, 1IrOVided IlOSiollS therllld StilllUhtiOil. J’cJr direct f?leCtriCal SthIlhtiOll of afferents a pair of sulmttniieo~is nwtlles. rotighly 0.5 cm apart, was used.
123
4
A ,
lmsec
I
B
1
2
3
Fro. 2. Examples of J-unit responses to innocuous mechanical stimuli. The horizontal lines represent periods of stimulation. The time scale of :I applies to all records. -4 and B are from the same unit; C is from another unit. Multiple activity \vhich arose during stimulus application can also be seen. !-The first stimulus application (1) did not elicit a burst; subsequent applications ( 2, 3, and 4) produced a response and also inhibited the preceding response. Stimulation, consisting of brushing the hairs, took place each time at the same site un the skin. B-Prolonged light touch inhibited backgromld activity. The recurrence of activity twvard the end of stimulation may have hccn due lo atlalk~ticlii of affercnts. Release of the stimulus rcsultcd in a burst. C--.\ir initial apl)lication of light touch (1) \vas ineffecti\:c, but moving the stimulus lo an adjacent site (2) resulted in a burst, f~~llowcd upon further stimulation (3) by its inhibition. The burst did not appear again upon removing the third stimulus, although in other tests on this unit bursts continued after being inhibited.
796
IAN
HENTALL
RESULTS Classification and Location. Two classes of unit, as judged by evoked firing patterns, were discovered in the substantia gelatinosa. They were assigned the names J-unit and K-unit. The responses of K-units, to be treated in a later paper, occurred during the application of stimuli (allowing for a delay of less than 20 ms from Aa afferent conduction). J-units, in contrast, were mainly inactive before and during gentle mechanical stinulation and in the first 50 to ZOO ms following stimulus withdrawal (Figs. 2, 3), but they responded thereafter with a prolonged decaying burst, usually lasting 5 to 20 s (Figs. 4,6, 7). The 34 J-units observed possessed peak-to-peak amplitudes in the 0.1 to lo-kHz band of 50 to 200 pV. Their waveforms consisted of an initial negative phase and a smaller subsequent positive one. Their action potentials could be tracked through a vertical field of approximately 100 ~117. They were centered 150 to 350 ,.m below the white matter boundary in the lateral third of lumbar segments six and seven. Thus some units were in lamina II (the substantia gelatinosa proper) and others in lamina III, according to Ralston’s (19) mesurement of, respectively, 100 to 200 and 200 to 450 pm below the white matter for these laminae. Altcrafiom of Xcsponsiz~~ss. The variability in the time from stimulus withdrawal to burst onset (50 to 200 ms), referred to in the following as the latent period, and the variability in burst duration (50 to 20 s) existed with one unit’s responses. A considerable difference in these parameters
FIG. 3. The latency of burst appearance and of inhibition. The continuous line represents light touch stimuli monitored by a strain gauge. It is not precisely related to intensi,ty, but serves as an indicator of stimulus application. The arrows show when the stimuli were withdrawn. The two records are of consecutive tests after an initial burst elicitation, and they are almost continuous. The latent periods were approximately 70 and 150 ms. Inhibition occurred more rapidly than the response time of the strain gauge (20 ms). The receptive field lay in the midcalf region. One or two J-unit spikes are present at the start of stimulus withdrawal, but this is not a consistent phenomenon and js perhaps due to incomplete inhibition of the preceding burst.
:
b
.,
‘.
ICC
100
:
180
FIG. 4. The effect of cutaneous, electrical stimuli, 10 s apart. Each small dot reprcsents the interspike interval on a logarithmic scale. Stimuli consisted on 10-V. 0%ms pulses. The vertical lines mark the moments of stimulation. In some cases (e.g., a) it appears that a stimulus interrupted an ongoing burst; in others (e.g., b) ineffective stimuli clearly occurred in the absence of a burst.
often occurred between two consecutive tests with identical stimuli. When identical, well-spaced, brief stimuli at several times above threshold were delivered, bursts often failed to appear. Figure 4 shows, using electrical stimuli 10 s apart, that there was no definite pattern in the disappearance and reappearance of responsiveness. JVith the repetition rate increased to l/s, a period far less than the normal length of a burst, phases of responding alternated with inactive phases (Fig. 5). These phases lasted the same order of time as normal bursts. In the case of units with baseline activity higher than normal, the nuniber of iiq~ulses per response was variable (Fig. 6). It is possible to discern in the level of background activity in Fig. 6 one factor which correlated with the strength of the following burst: Lowered baseline activity always preceded the bursts of highest frequency. However, at higher rates of activity. there is no discernible correlation between the nun~her of impulses in a burst and background freqency.
FIG. 5. The effect of cutaneous, electrical stimulation, l/s. The logarithm of interspike interval is represented by dots, as in Fig. 4. Stimuli consisting of 10-V, 0.2-ms pulses were delivered throughout the duration of the record. The unit is the same as in Fig. 3.
1,000 10,000 1, 0
>C‘
I 100
200
FIG. 6. The effect of repetitive mechanical stimulation. Brief mechanical stimuli (< 20 ms) were given every 18 s. The unit was different from that in Figs. 4, 5. The dots represent the logarithm of the interspike interval, as in previous figures. No stimulus marker is necessary because each stimulus produced an observable burst. The most prolific bursts (indicated by A) were always preceded by the lowest background activity.
After several trials failed to elicit a burst from a spot of skin which hat1 recently been efficacious, then applying the light touch stimulus 2 to 3 cm distant could often reveal a newly competent spot. Alternate testing of the two sites demonstrated that this state remained for at least a minute. l)tlt continued testing was not possil,le without imposing a further change. Thus if the second spot was repeatedly testetl it became ineft%cti\-e, and the original or a third one could be found to produce bursts. This l)henomenon was observed in all 20 units for which it was tested. Inl~ibitim. During both spontaneous and evoked bursts a light touch to the receptive field inhibited the firing (Fig. 2). The onset of this effect was rapid (Fig. 3) and consistent with a delay due to the conduction velocity of AE afferents. On cessation of stimulation, the burst continued after the normal latent period (SO to 200 ms) (Figs. 2A, B, and 31 or did not return (Fig. 2C). Which of these occurred coulcl not be predicted from timing and the stimulus parameters. When sufficiently high to be nleasurably affected, background activity was also inhibited by light touch (Fig. 2B). Nanipulation of single guard hairs with forceps could also produce a burst and the subsequent inhibition, but this effect was not casil! obtained. S~~I~~POIIS Activity n)zd Noxiozrs Siin~ulation. Spontaneous activity took two forms: intermittent bursts, which were periods of high but generally decreasing frequency above .5impulses/s, and background activity, which maintained a fairly constant average rate below 5 impulses/s and changed only during periods of many minutes. Units whose receptive fields had not been subjected to noxious stimuli possessed no background activity and exhibited few spontaneous bursts. But in those units (10 altogether) whose receptive fields had been thus treated, frequent spontaneous bursts ancl a significant increase in background activity could be found about
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FIG. 7. The development of background activity and the effects of noxious stimulation. The small dots represent interspike intervals, as in Figs. 4, 5, 6. I
15 min after noxious stimulation (Fig. 7). Both heat at 50°C applied for 1 min and strong pinching with a hemostat could bring about this change. Six units which displayed this feature were held for a sufficient length of time to follow the development of spontaneous activity; the other four were first recorded after the receptive field had been given noxious stimulation in studying a previous unit. The short-term effect of applying noxious stimuli likewise depended 011 the unit’s history of stinmlation. Radiant heat or pinching did not influence those units with only slight or no spontaneous activity. However, an illcrease in impulse frequency took place during application of noxious stimuli to units firing spontaneously. Withdrawal of the stimuli led to a period of reduced activity lasting 1 to 2 s, followed by a burst (Fig. 7). Figure S shows the response of a unit which was in the initial stages of developing spontaneous activity, 15 min after inserting needles in the skin.
800
IAN
IIENTALL
-------lsec FIG. 8. The effect of tetanic electrical stimulation. A 1.3-s resulting from cutaneous electrical stimulation; 30-V, OS-ms 80 ms apart. The broken line indicates the time of stimulation; brief multiple responses were present at that time.
.
delay preceded a burst pulse width, 12 pulses, stimulus artifacts and
It responded with a long latent period (1.3 s) to strong tetanic electrical stimulation. J-Units and the Dorsal Root Potential (DRP). A correlation between
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0.8
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FIG. 9. Histograms of poststimulus responses to repetitive stimulation; bin width is 25 ms. A-Mechanical pulses, 0.95 s apart. B-Electrical stimulation, 10 s apart. C-Electrical stimulation, 1 s apart. B and C are from the data of Figs. 4 and 5, respectively; A is from the unit of Fig. 6, but studied under different conditions.
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FIG. 10. The dendroaxonic model of dorsal root potentials. D-Post synaptic depolarization caused by a presynaptic depolarization. H-Postsynaptic hyperpolarization caused by a presynaptic depolarization. The relative sizes of the boxes representing the J-unit regions depict the relative transmembrane conductances which, it is proposed, are directly related to their electrotonic influence on each other. Further details are in the text.
the activity of neurons in the substantia gelatinosa and the level of polarization of dorsal roots has been postulated (23). Therefore a relationship could exist between the time course of the dorsal root potential (DRP) and the poststimulus time histograms of J-unit impulses following a repeated stimulus. Figure 9 contains three histograms, two derived from the data in Figs. 4, 5 (Fig. 9B, C) and a third (Fig. 9A) taken from the study of Fig. 6 with the stimulus repetition period of 0.95 s. Electrical stimuli 10 s apart demonstrated average activity climbing to a plateau in 400 ms (Fig. 9B). In the same unit with a l-s period of repetition the peak rate was reached in 150 ms (Fig. SC) ; in this case the Lmit was firing almost continually, bursts lasting considerably longer than the repetition period (see Fig. 5). Short, mechanical pulses to the receptive field of a unit with high background activity produced only a 25 to 50-111s average for the rise to peak activity (Fig. 9A). In conclusion, the experimental condition which led to the results shown in Fig. 9C, namely, little background activity and one cycle of stimulation being several times shorter than a typical burst, permitted a decrease in firing to be measured whose tinle course resembled that of the cutaneously elicited negative DRP. The latter, as shown by Barron and Matthews [(l), see Fig. 1 of their paper], rises to a peak in 20 ms, decays in approximately the next 100 ms, and is evoked by mechanical and electrical stimuli similar to those used in the above tests.
802
IAX
IIEST.4LL
The Anatomical Correlate of J-Units. The reasons for ascribing J-unit activity to the cells of the substantia gelatinosa, as opposed to the other neuronal elements in this region, are two. First, the waveshape, a large negative phase and a smaller subsequent positive one, is typical of somatic rather than axonic spikes (3). In the present experiments the microelectrode was sometimesmoved deeper to record cells of laminae IV and V, where the waveshapes did not differ significantly from those of the J-units, except in amplitude. Moreover, on passing from white matter (axons) to lamina I, the spikes changed in predominant phase from positive to negative (Fig. 1). The second reason is that other possible generators of J-unit activity, namely, axons entering the region, may be excluded. No category of cutaneous afferent fiber possessesa delayed “off” response (5). The repetitive discharge of the dorsal root reflex lasts less than 100 ms (4). Descending fibers from the brain can obviously be ruled out in spinal animals. No known sensory cell of the spinal cord responds in the delayed off manlier (26)) thus excluding collaterals and displaced cells. The Influence of Cutaneous Ajjercnts on J-Units. Int~oc~~ous mechanical stilnulation has both inhibitory and excitatory effects on J-units. The results show that inhibition is the controlling effect during stimulation. The latent period is probably a temporal prolongation of the inhibition. This can be asserted primarily because after a burst is briefly inhibited by a second stimulus, the usual latent period precedes the continuation of the burst, though an excitatory influence can be presumed present continuously from the first burst excitation. The actual excitation of bursts finds a simple explanation in an unmasking by the decline of inhibition of a long-lasting excitatory influence which is present from the beginning of stimulation but whose exact nature, whether a long-lasting transmitter or a continuous interneuronal barrage, cannot be deduced from the present experiments. However, another possibility is that the decline of inhibition occurs while slowly conducted C-fiber impulses are still arriving and that these excite the burst; a long polysynaptic delay path could operate similarly. The short-term results of noxious stimulation have the appearance of positive feedback, with an initial overshoot to account for the phasic nature of the latent period, for a responsetakes place only with high background activity, and a reduction in noxious input causes J-unit activity to diminish for about a second. The axoaxonic synapseswhich impinge on afferents in the substantia gelatinosa (19) constitute a potential route for this feedback, though not a unique one (see below). With regard to the growth of background activity, nociceptive afferents, while initially silent, are known to fire spontaneously after excitation by
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inhil)ited 1)~ A films was ljroljoxxl 1,~ Rlentlell and \l’all (‘17). It is borne out by the data in the present lmljer, with the one qualification that the excitatory effect of nociceptivc atierents is not always seen, as just discussed. 1\‘all (23) earlier suggested that these same units caused negative and positive dorsal root potentials 1)~ a respective decrease or increase in their activity. But in spinal cats at least, the J-units do not respond until after a corresponding peripherally elicited negative DRP would have reached its peak 130 ins according to Earron and hlatthews (1) 1. TIowrver, the histogram of average reslmnse to l/s electrical stiiiiulatirm ( IGg. 9C) indicates that the negative DKI. ant1 the inliihitory process in J-units coultl llave some causal connection. l:ecause ilo other correlation to the negative 111~1’ has l)ecii fount1 in sljinal cord Ijliysiology, in impulse frecpcncies of dorsal horn cells (26). iii intracellular syiial)tic potentials of dorsal horn cells ( 12), in potassituii ion concentrations (lGj, or it1 afterllotentials (22), exe@ in the afferents themselves, the notion of a causal role for J-units in producing the DRP must he considered. The experiment of M’all (23), demonstrating the transmission of a passive DRP with dorsal columns cut between the stinlulated and recorded roots, suggests that an interaction among afferents cannot account for the phenoinenon. The association of J-unit inhibition and primary afferent depolarization mightt however, 1~ due to a state of hyperpolarization in J-unit distal dendrites causing the depolarization of afferent telodendra. There is an anatomical basis for such an interaction ; dendroasonic synapses have been observed in the spinal substantia gelatinosa (19). Also in the suljstantia gelatinosa of the spinal trigeniinal nucleus they have heen ohserved paired with axodendritic synapses (10). The events in the generation of a negative DRP can be modeled as follows (see also Fig. 10). P rior to stimulation, the distal region of J-unit dendrites steadily hyperpolarizes the afferent terminals. A barrage in the afferents then hyperpolarizes the distal dendritic region, while, through a different type of synapse, depolarization tends to be produced in the soma and proximal denclritic region. The mutual coupling of the distal tlendritic region and the afferent terminals is, in effect, a positive feedl>a&
804
IAN
HENTALL
Ioop. A prolonged relative depolarization of afferents, seen as the negative DRP, is a consequence of the activation of this loop. To function as described the model must incorporate two important requirements. First, because afferents are depolarized less during a DRP than during an action potential, the time constant of rise of the feedback to the afferents must be longer than the duration of the action potential, thus allowing the DRP to gradually increase. Second, current in the distal region must have a greater influence on the proximal region and soma than vice versa. A mismatch of impedances which the regions present to each other as sources and passive networks, due to differences in membrane area, represents a way this could be brought about; but clearly the geometrical and electrical factors in a precise formulation would be quite complex. The arrangement is necessary, because the distal hyperpolarization has to restrain spike initiation by the proximal depolarization, thus permitting the J-unit latent period, whereas the distal feedback loop cannot be too rapidly halted by the onset of a burst. To the extent that impulses are conducted electrotonically into the distal region, they would tend to hyperpolarize the afferents. This constitutes the route referred to earlier in the discussion for positive feedback onto noxious afferents, because hyperpolarization of a terminal probably results in an increase of its postsynaptic efficacy (24). The mechanism for initiating the decay of the DRP is not made explicit in the model; neither are such phenomena as the DRP from contralateral or descending fibers, for there is no relevant evidence on these questions. The passive DRP however, as a consequence of the model, must be transmitted by dendroaxonic activation of passive fibers converging onto the same cell as active fibers. The hypothesis explains two previously unaccountable results. First, Rudomin and Mufioz-Martinez (21) showed that DRPs could be generated by direct stimulation of the dorsal horn when intra-arterial injections of tetrodotoxin, which blocks sodium current, had abolished the DRP elicited by dorsal root stimulation. As is true of most synapses studied, the two postulated synapses of the feedback loop need not be dependent on sodium current for their activation. Second, negative DRPs are resistant to baracid (GABA), which biturate (8, 24), and the effect of y-aminobutyric depolarizes the dorsal root, is enhanced by barbiturate (18). Although it is possible that action potentials in substantia gelatinosa neurons are unaffected by barbiturate (24), an explanation ,derived from the presen,t hypothesis is that GABA is the inhibitory transmitter at the axodendritic synapse. Because GABA is a hyperpolarizing agent for neurons elsewhere in the central nervous system, causing increases in chloride ion permeability (20)) this explanation of its action is more parsimonious than one which confers upon it a depolarizing effect on afferent terminals. The fact that barbiturates diminish the positive dorsal root potential (14) accords well
SL-ESTANTIA
CELATI
KOSA
805
with the above scheme, if in fact the late llyperpolarization of the positive dorsal root potential is acconq~lished by impulses in J-units acting either through an axoaxonic route or by electrotonic conduction to the dendroaxonic synapse. REFERENCES D. H., A~UD B. H. C. MATTEINS;. 1938. The interpretation of potential in the spinal cord. J. Pllysiol. (Land.) 92 : 276-321. BESSOV, P., AND E. R. PERL. 1969. Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. /. Ncrwopll)‘siol. 32 : 1025-1043. BIS~~OIB, P. O., W. Buweq AND R. DAVIS. 1962. The identification of single units in central visual pathways. /. Physio/. (Land.) 162 : 409-431. BROOKS, C. McC., AXD K. Korzr~m. 1956. Origin of the dorsal root reflex. J. Ncwoplqsiol. 19 : 61-74. BUR;ESS, P. R., AND E. R. PERI.. 1973. Cutaneous mechanoreceptors and nociceptors. Pages 29-78 in A. ICCO, Ed., Handbook of Srrrsor~ Physiology, T’ol. Il. Springer-Verlag, Berlin. CERVERO, F., D. R. ENSOR, A. IGGO, AND V. MOLONY. 1977. Activity front single neuronrs recorded in the substantia gelatinosa Rolandi of the cat. J. Physiol. (Loud.) 169 : 37P-39P. DOWBEN, R. M., AND J, E. ROSE. 1953. A metal-filled microelectrode. S&rrcc 118: 22-24. ECCLES, J. C., AXD J. I>. h~AI.COr.hr. 1946. Dorsal root potential of the spinal cord. J. Nrzlropllysiol. 9 : 139-160. GESTIXAND, R. C., B. HO\VLAND, J. Y. LETTVIN, AXD W. H. PITTS. 1959. Comments on microelectrodes. Proc. I&. Radio E~rg. 47 : 1856-1862. GOBEL, S. 1976. Dendroaxonic synapses in the substantia gelatinosa glomeruli of the spinal trigeminal nucleus of the cat. J. Comj. Ncurol. 167: 165-176. HENTALL, I. D. 1976. Coding properties of substantia gelatinosa cells in the cat’s spinal cord. Q. Prog. Rep. Rcs. Lab. Electroll. M.I.?‘. 117: 125-126. HONGO, T., E. JANKO\VSKA, AND .4. LVNDBERG. 1968. Post-synaptic excitation and inhibition from primary afferents in neurones in the spine-cervical tract. J. Pl~~siol. (Loud.) 199 : 569-592. K~INAXA\VA, T., ASI) E. R. PEKI.. 1976. Differential excitation of dorsal horn and substantia gelatinosa marginal neurones by primary afferent units with fine (:\ and C) fibres. Pages 67-89 in Y. ZOTTERK~S, Ed., Srwwy Fwctiow of tlzc S/siu. Pergamon, Oxford. LLOYD, D. P. C. 1952. Electrotonics in dorsal nerve roots. Cold Spring Harbor Symp. Quarrt. Viol. 17 : 203-219. LLOYD, D. P. C., AND -1. K. MACINTYRE. 1949. On the origin of dorsal root potentials. J. Gcrr. Plty.siol. 32: 409-443. LOTIIRIAN, E. W., AND G. G. SOXJEN. 1975. Extracellular potassium activity, intercellular and extracellular potential responses in the spinal cord. J. Pkysiol. (Lorld.) 255 : 115-136. MENDELL, L. M., AND P. D. WALL. 1964. Pre-synaptic hyperpolarization: A rofe for fine afferent fibres. J. Pll)wiol. (Lond.) 172 : 279-294. NICOIL, R. A. 1976. The action of pentobarbital on the amino acid induced depolarization of primary afferents in the frog. Puoc. Il’rst. Plrarmml. SM. 19: 421423.
1. BAHRON,
changes
2.
3. 4. 5.
6.
7. 8. 9. 10. 11. 12.
13.
14. 15. 16.
17. 18.
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19. RALSTON,
22.
J., III. 1968. The fine structure of neurons in the dorsal horn of the cord. J. Comb. Nrwol. 132 : 275-302. AND R. HAMMERSCHLAG. 1972. Amino acid transmitters. Pages 131W. ALBERT, G. J. SIEGEL, R. KARZI~AN, AND B. W. -~GRANOFF, Eds., Basic Neurodwrristry. Little, Brown, Boston. RUDOMIN, P., AND J. MU~OZ-MARTINEZ. 1969. A tetrodotoxin-resistant primary afferent depolarization. Erp. Ncurol. 25 : 106115. RUDIN, D. O., AND G. EISENMAN. 1953. After potentials in spinal axons irt z~ivo.
23.
WALL,
20. 21.
H. cat spinal ROBERTS, E., 168 in R.
J. Ge.rt. Physiol. 36 : 643-657. 24.
25.
26.
P. D. 1962. The origin of a spinal cord slow potential. J. Physiol. (Lolrd.) 164 : 508-j26. WALL, P. D. 1964. Control of impulses at the firs’t central synapse in cutaneous pathways. Pages 92-118 in J. C. ECCLES, Ed., Pvogvrss iu Bvaitt Rrscarch, Vol. 12. Elsevier, Amsterdam/New York. WALL, P. D. 1965. Impulses recorded in the region of dendrites. J. Physiol. (LoII~.) 180 : 116-133. WALL, P. D. 1973. Dorsal horn electrophysiology. Pages 2533270 in A. IGGO, Ed., I-lamfbook of Srnsor~j Pll\lsiology, Vol. II. Springer-Verlag, Berlin.
‘
57, 792406
NEUROLOGY
A Novel
Class
of Unit in the Substantia of the Spinal Cat IAN
~epartlrkcrkt
(1977)
of Biology
Massachusetts Caukbridge,
HENTALL
l
and Rcsearck Laboratory Ikkstitzkte of Techkkology, Massachusetts 021.39
Received
June
Gelatinosa
of Elfctrokkics,
9, 1977
Using platinum-tipped microelectrodes, 34 units of a kind not previously reported were found in the substantia gelatinosa of spinal, unanesthetized cats. Units were inhibited by innocuous mechanical stimuli, but after the withdrawal of such stimuli, a burst of activity lasting 5 to 20 s appeared with an initial delay (latent period) of 50 to 200 ms. In the absence of background activity, noxious stimuli had no immediate effect. However, in a period of about 20 min, units tested with noxious stimuli displayed a gradual increase in regular background firing and in the occurrence of unevoked bursts. Noxious stimuli were excitatory when applied ,to the units with this background activity. An approximately l-s period of diminished activity followed withdrawal of noxious stimuli. Consecutive identical stimuli produced bursts of activity with considerable variability; latency and burst length fluctuated. Also the probability of bursts fluctuated in units with low background activity. Poststimulus time histograms showed, under some conditions, a decrease in ‘the average number of impulses in the first 200 ms which roughly corresponded in time course to the negative dorsal root potential. It is argued that the initial latency and the decrease in activity in the histograms are temporal prolongations of the inhibitory process produced by innocuous stimuli. Moreover, based on these data, a mechanism for the generation of the negative dorsal root potential is proposed, involving dendroaxonic synaptic transmission from the substantia gelatinosa units to primary afferent terminals. Abbreviations : GABA--r-aminobutyric acid ; DRP-dorsal root potential. 1 This research was supported by a grant from Bell Telephone Laboratories. It was submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Biology, Massachusetts Institute of Technology. The author is grateful to Dr. Jerome Lettvin for valuable advice and encouragement on this project. Thanks are also due ‘to Drs. Howard Fields and Allan Basbaum for suggestions on the manuscript, to Mr. Eric Newman for his criticism of the dendroaxonic model, and to Mrs. Stephanie Bornstein for careful typing. The author’s present address is Department of Physiology S-762, University of California, San Francisco, California 94143. 792 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0014- -4886
SUBSTANTIA
GELATINOSA
793
INTRODUCTION Until recently there existed no direct information on the responses of single units in the substantia gelatinosa (26). Now Kumazawa and Per1 (13) have reported in monkeys, with chloralose or Nembutal as anesthetic agent, units situated in the lower sacral or coccygeal substantia gelatinosa which were specifically responsive to C-fiber stimulation. Also, in the substantia gelatinosa of cats anesthetized with chloralose, units whose basic response was inhibition by either noxious stimuli or weak nlechanical stimuli or both were briefly reported (6) ; in some units an adjacent excitatory receptive field was found. The present study, using spinal, unanesthetized animals, revealed a previously undocumented class of unit in the substantia gelatinosa. They were first described in a short preliminary report (11). The response of units of this class to cutaneous stimulation suggests that the negative dorsal root potential [ D.R.V of Lloyd and MacIntyre, (15)] arises, at least in part, from interaction 1,etween the dendrites of substantia gelatinosa neurons and afferent terminals. METHODS Tltc Prcparatiopt. Adult cats were made spinal by occluding under ether anesthesia the carotid and vertebral arteries. After this procedure, artificial respiration was conmenced and the anesthesia was discontinued. The animals were totally suspended by ear bars, a clamp on the L1 vertebral spine, and hip bars. Radiant heat maintained body temperature, as nieasured by a subscapular thermistor, at 37°C within 0.1 “C. Cord segments Lo and 1~; were exposed by laminectomy, the dura was cut back, and small pieces of pia-araclinoitl were torn an-a! to allow electrode penetration. Intravenous or intraperitoneal injection ol Flasetlil prevented refies movements. Rccordi~tg Mrfkotls. Glass nlicropipets, filled with iVood’s metal and with a S-pm layer of platinum black plated to their tips (7)) were used for recording.These particular electrodes were chosen because a high capacitance develops at the metal-tissue interface, due to the sponginess of platinum black (9) . In consequence, whereas the impedance measured at 1 kHz of the conmonl~~ used extracellular microelectrodes, fluid-filled glass inicropipets and unplatinized nletal, is generally above 1 MQ. impedances of the electrodes used in the present study were about 100 kf& This choice of electrode gave a lower thermal noise, because root-mean-square noise in a given frequency band is proportional to the square root of impedance (9). The achieved peak-to-peak noise of 20 pV enabled SO-PV nerve spikes to be analyzed.
794
IAN
I
IIENTALL
I
1 set FIG. 1. The electrical sign of the boundary between dorsal columns and lamina I. During the time indicated by the thick line the electrode tip was first pulled back 30 pm and ‘then returned the same distance across the boundary.
Conventional amplification and display techniques were used ; the signal was also recorded on tape for later processing. The position of the electrode tip during recording was determined by reading directly on the micromanipulator the distance from the boundary of the dorsal column and lamina I. In all preparations this boundary appeared as a region of change in characteristic background activity, occupying about 30 /*.ni in the dorsoventral direction (Fig. 1). Dorsally, in the white matter, multiple units with a predominantly positive spike were seen, firing at a moderately high rate; these responded to changes of limb position. In contrast, lamina I presented no activity or else a single spontaneously firing unit with a large receptive field to light touch. This method was verified in several cases by making small electrolytic lesions at recording sites, and subsequently reconstructing them in sections stained with cresyl violet. In laminae II and III, a complex multiple-unit response with the appearance of a doubling of thermal noise, also described by Wall (25)) appeared upon brushing a particular area of skin. Because this did not occur either in lamina I or in lamina IV, it provided a confirmatory marker of electrode position. To search for units an electrode was gradually lowered through the substantia gelatinosa while the skin was being brushed in the region which elicited the multiple-unit response. It took many penetrations to find one isolated single unit of the class described in this paper. .‘j’ti~rlr,lntion. Mechanical, thermal, and electrical stimuli were applied to the left hind limb, the hair of which had been clipped to less than 0.6 cm (0.25 in.). The mechanical stimuli can be classed as noxious or innocuous, aud as hand-delivered or automatic. Pinching with a hemostat and prodding with a hypoclern~ic needle constituted the noxious mechanical stinn~li. Innocuous stimuli included moving individual hairs with fine forceps,
brushing a group of hairs with a piece of cotton, and applying slight pressure to the skin with 3 blunt probe of a few square-millimeter tip area (referred to 3s “light touch”). I-land-delivered stimuli were sonietinies monitored by a strain gauge lvlmse output was recorded on FM tape. An electromeclm~ically driven pI-(JlJe lwlder, to which could he attached a liypoderiiiic needle or a blunt pTJlJe, cleliveretl automatic repeatable stimuli to the leg, which for this l~urlms~ WLS heltl rigidly in a clamp. A l-cm’, thermostatic hot plate, li~ltl against tlie skin and heated to ahut SO”C, 1IrOVided IlOSiollS therllld StilllUhtiOil. J’cJr direct f?leCtriCal SthIlhtiOll of afferents a pair of sulmttniieo~is nwtlles. rotighly 0.5 cm apart, was used.
123
4
A ,
lmsec
I
B
1
2
3
Fro. 2. Examples of J-unit responses to innocuous mechanical stimuli. The horizontal lines represent periods of stimulation. The time scale of :I applies to all records. -4 and B are from the same unit; C is from another unit. Multiple activity \vhich arose during stimulus application can also be seen. !-The first stimulus application (1) did not elicit a burst; subsequent applications ( 2, 3, and 4) produced a response and also inhibited the preceding response. Stimulation, consisting of brushing the hairs, took place each time at the same site un the skin. B-Prolonged light touch inhibited backgromld activity. The recurrence of activity twvard the end of stimulation may have hccn due lo atlalk~ticlii of affercnts. Release of the stimulus rcsultcd in a burst. C--.\ir initial apl)lication of light touch (1) \vas ineffecti\:c, but moving the stimulus lo an adjacent site (2) resulted in a burst, f~~llowcd upon further stimulation (3) by its inhibition. The burst did not appear again upon removing the third stimulus, although in other tests on this unit bursts continued after being inhibited.
796
IAN
HENTALL
RESULTS Classification and Location. Two classes of unit, as judged by evoked firing patterns, were discovered in the substantia gelatinosa. They were assigned the names J-unit and K-unit. The responses of K-units, to be treated in a later paper, occurred during the application of stimuli (allowing for a delay of less than 20 ms from Aa afferent conduction). J-units, in contrast, were mainly inactive before and during gentle mechanical stinulation and in the first 50 to ZOO ms following stimulus withdrawal (Figs. 2, 3), but they responded thereafter with a prolonged decaying burst, usually lasting 5 to 20 s (Figs. 4,6, 7). The 34 J-units observed possessed peak-to-peak amplitudes in the 0.1 to lo-kHz band of 50 to 200 pV. Their waveforms consisted of an initial negative phase and a smaller subsequent positive one. Their action potentials could be tracked through a vertical field of approximately 100 ~117. They were centered 150 to 350 ,.m below the white matter boundary in the lateral third of lumbar segments six and seven. Thus some units were in lamina II (the substantia gelatinosa proper) and others in lamina III, according to Ralston’s (19) mesurement of, respectively, 100 to 200 and 200 to 450 pm below the white matter for these laminae. Altcrafiom of Xcsponsiz~~ss. The variability in the time from stimulus withdrawal to burst onset (50 to 200 ms), referred to in the following as the latent period, and the variability in burst duration (50 to 20 s) existed with one unit’s responses. A considerable difference in these parameters
FIG. 3. The latency of burst appearance and of inhibition. The continuous line represents light touch stimuli monitored by a strain gauge. It is not precisely related to intensi,ty, but serves as an indicator of stimulus application. The arrows show when the stimuli were withdrawn. The two records are of consecutive tests after an initial burst elicitation, and they are almost continuous. The latent periods were approximately 70 and 150 ms. Inhibition occurred more rapidly than the response time of the strain gauge (20 ms). The receptive field lay in the midcalf region. One or two J-unit spikes are present at the start of stimulus withdrawal, but this is not a consistent phenomenon and js perhaps due to incomplete inhibition of the preceding burst.
:
b
.,
‘.
ICC
100
:
180
FIG. 4. The effect of cutaneous, electrical stimuli, 10 s apart. Each small dot reprcsents the interspike interval on a logarithmic scale. Stimuli consisted on 10-V. 0%ms pulses. The vertical lines mark the moments of stimulation. In some cases (e.g., a) it appears that a stimulus interrupted an ongoing burst; in others (e.g., b) ineffective stimuli clearly occurred in the absence of a burst.
often occurred between two consecutive tests with identical stimuli. When identical, well-spaced, brief stimuli at several times above threshold were delivered, bursts often failed to appear. Figure 4 shows, using electrical stimuli 10 s apart, that there was no definite pattern in the disappearance and reappearance of responsiveness. JVith the repetition rate increased to l/s, a period far less than the normal length of a burst, phases of responding alternated with inactive phases (Fig. 5). These phases lasted the same order of time as normal bursts. In the case of units with baseline activity higher than normal, the nuniber of iiq~ulses per response was variable (Fig. 6). It is possible to discern in the level of background activity in Fig. 6 one factor which correlated with the strength of the following burst: Lowered baseline activity always preceded the bursts of highest frequency. However, at higher rates of activity. there is no discernible correlation between the nun~her of impulses in a burst and background freqency.
FIG. 5. The effect of cutaneous, electrical stimulation, l/s. The logarithm of interspike interval is represented by dots, as in Fig. 4. Stimuli consisting of 10-V, 0.2-ms pulses were delivered throughout the duration of the record. The unit is the same as in Fig. 3.
1,000 10,000 1, 0
>C‘
I 100
200
FIG. 6. The effect of repetitive mechanical stimulation. Brief mechanical stimuli (< 20 ms) were given every 18 s. The unit was different from that in Figs. 4, 5. The dots represent the logarithm of the interspike interval, as in previous figures. No stimulus marker is necessary because each stimulus produced an observable burst. The most prolific bursts (indicated by A) were always preceded by the lowest background activity.
After several trials failed to elicit a burst from a spot of skin which hat1 recently been efficacious, then applying the light touch stimulus 2 to 3 cm distant could often reveal a newly competent spot. Alternate testing of the two sites demonstrated that this state remained for at least a minute. l)tlt continued testing was not possil,le without imposing a further change. Thus if the second spot was repeatedly testetl it became ineft%cti\-e, and the original or a third one could be found to produce bursts. This l)henomenon was observed in all 20 units for which it was tested. Inl~ibitim. During both spontaneous and evoked bursts a light touch to the receptive field inhibited the firing (Fig. 2). The onset of this effect was rapid (Fig. 3) and consistent with a delay due to the conduction velocity of AE afferents. On cessation of stimulation, the burst continued after the normal latent period (SO to 200 ms) (Figs. 2A, B, and 31 or did not return (Fig. 2C). Which of these occurred coulcl not be predicted from timing and the stimulus parameters. When sufficiently high to be nleasurably affected, background activity was also inhibited by light touch (Fig. 2B). Nanipulation of single guard hairs with forceps could also produce a burst and the subsequent inhibition, but this effect was not casil! obtained. S~~I~~POIIS Activity n)zd Noxiozrs Siin~ulation. Spontaneous activity took two forms: intermittent bursts, which were periods of high but generally decreasing frequency above .5impulses/s, and background activity, which maintained a fairly constant average rate below 5 impulses/s and changed only during periods of many minutes. Units whose receptive fields had not been subjected to noxious stimuli possessed no background activity and exhibited few spontaneous bursts. But in those units (10 altogether) whose receptive fields had been thus treated, frequent spontaneous bursts ancl a significant increase in background activity could be found about
sI~I:s'I‘.\s'I‘I;\
(;lx..\'I'1sos.\
700
FIG. 7. The development of background activity and the effects of noxious stimulation. The small dots represent interspike intervals, as in Figs. 4, 5, 6. I
15 min after noxious stimulation (Fig. 7). Both heat at 50°C applied for 1 min and strong pinching with a hemostat could bring about this change. Six units which displayed this feature were held for a sufficient length of time to follow the development of spontaneous activity; the other four were first recorded after the receptive field had been given noxious stimulation in studying a previous unit. The short-term effect of applying noxious stimuli likewise depended 011 the unit’s history of stinmlation. Radiant heat or pinching did not influence those units with only slight or no spontaneous activity. However, an illcrease in impulse frequency took place during application of noxious stimuli to units firing spontaneously. Withdrawal of the stimuli led to a period of reduced activity lasting 1 to 2 s, followed by a burst (Fig. 7). Figure S shows the response of a unit which was in the initial stages of developing spontaneous activity, 15 min after inserting needles in the skin.
800
IAN
IIENTALL
-------lsec FIG. 8. The effect of tetanic electrical stimulation. A 1.3-s resulting from cutaneous electrical stimulation; 30-V, OS-ms 80 ms apart. The broken line indicates the time of stimulation; brief multiple responses were present at that time.
.
delay preceded a burst pulse width, 12 pulses, stimulus artifacts and
It responded with a long latent period (1.3 s) to strong tetanic electrical stimulation. J-Units and the Dorsal Root Potential (DRP). A correlation between
C @2
0.4
0%
0.8
1 set
FIG. 9. Histograms of poststimulus responses to repetitive stimulation; bin width is 25 ms. A-Mechanical pulses, 0.95 s apart. B-Electrical stimulation, 10 s apart. C-Electrical stimulation, 1 s apart. B and C are from the data of Figs. 4 and 5, respectively; A is from the unit of Fig. 6, but studied under different conditions.
I 2
1 spike
u
i-7
I sam
proximd
--I
and dendrilic
u
ONE J-“N’T distal
region
dendrdic
1
FIG. 10. The dendroaxonic model of dorsal root potentials. D-Post synaptic depolarization caused by a presynaptic depolarization. H-Postsynaptic hyperpolarization caused by a presynaptic depolarization. The relative sizes of the boxes representing the J-unit regions depict the relative transmembrane conductances which, it is proposed, are directly related to their electrotonic influence on each other. Further details are in the text.
the activity of neurons in the substantia gelatinosa and the level of polarization of dorsal roots has been postulated (23). Therefore a relationship could exist between the time course of the dorsal root potential (DRP) and the poststimulus time histograms of J-unit impulses following a repeated stimulus. Figure 9 contains three histograms, two derived from the data in Figs. 4, 5 (Fig. 9B, C) and a third (Fig. 9A) taken from the study of Fig. 6 with the stimulus repetition period of 0.95 s. Electrical stimuli 10 s apart demonstrated average activity climbing to a plateau in 400 ms (Fig. 9B). In the same unit with a l-s period of repetition the peak rate was reached in 150 ms (Fig. SC) ; in this case the Lmit was firing almost continually, bursts lasting considerably longer than the repetition period (see Fig. 5). Short, mechanical pulses to the receptive field of a unit with high background activity produced only a 25 to 50-111s average for the rise to peak activity (Fig. 9A). In conclusion, the experimental condition which led to the results shown in Fig. 9C, namely, little background activity and one cycle of stimulation being several times shorter than a typical burst, permitted a decrease in firing to be measured whose tinle course resembled that of the cutaneously elicited negative DRP. The latter, as shown by Barron and Matthews [(l), see Fig. 1 of their paper], rises to a peak in 20 ms, decays in approximately the next 100 ms, and is evoked by mechanical and electrical stimuli similar to those used in the above tests.
802
IAX
IIEST.4LL
The Anatomical Correlate of J-Units. The reasons for ascribing J-unit activity to the cells of the substantia gelatinosa, as opposed to the other neuronal elements in this region, are two. First, the waveshape, a large negative phase and a smaller subsequent positive one, is typical of somatic rather than axonic spikes (3). In the present experiments the microelectrode was sometimesmoved deeper to record cells of laminae IV and V, where the waveshapes did not differ significantly from those of the J-units, except in amplitude. Moreover, on passing from white matter (axons) to lamina I, the spikes changed in predominant phase from positive to negative (Fig. 1). The second reason is that other possible generators of J-unit activity, namely, axons entering the region, may be excluded. No category of cutaneous afferent fiber possessesa delayed “off” response (5). The repetitive discharge of the dorsal root reflex lasts less than 100 ms (4). Descending fibers from the brain can obviously be ruled out in spinal animals. No known sensory cell of the spinal cord responds in the delayed off manlier (26)) thus excluding collaterals and displaced cells. The Influence of Cutaneous Ajjercnts on J-Units. Int~oc~~ous mechanical stilnulation has both inhibitory and excitatory effects on J-units. The results show that inhibition is the controlling effect during stimulation. The latent period is probably a temporal prolongation of the inhibition. This can be asserted primarily because after a burst is briefly inhibited by a second stimulus, the usual latent period precedes the continuation of the burst, though an excitatory influence can be presumed present continuously from the first burst excitation. The actual excitation of bursts finds a simple explanation in an unmasking by the decline of inhibition of a long-lasting excitatory influence which is present from the beginning of stimulation but whose exact nature, whether a long-lasting transmitter or a continuous interneuronal barrage, cannot be deduced from the present experiments. However, another possibility is that the decline of inhibition occurs while slowly conducted C-fiber impulses are still arriving and that these excite the burst; a long polysynaptic delay path could operate similarly. The short-term results of noxious stimulation have the appearance of positive feedback, with an initial overshoot to account for the phasic nature of the latent period, for a responsetakes place only with high background activity, and a reduction in noxious input causes J-unit activity to diminish for about a second. The axoaxonic synapseswhich impinge on afferents in the substantia gelatinosa (19) constitute a potential route for this feedback, though not a unique one (see below). With regard to the growth of background activity, nociceptive afferents, while initially silent, are known to fire spontaneously after excitation by
a
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illLISt
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inhil)ited 1)~ A films was ljroljoxxl 1,~ Rlentlell and \l’all (‘17). It is borne out by the data in the present lmljer, with the one qualification that the excitatory effect of nociceptivc atierents is not always seen, as just discussed. 1\‘all (23) earlier suggested that these same units caused negative and positive dorsal root potentials 1)~ a respective decrease or increase in their activity. But in spinal cats at least, the J-units do not respond until after a corresponding peripherally elicited negative DRP would have reached its peak 130 ins according to Earron and hlatthews (1) 1. TIowrver, the histogram of average reslmnse to l/s electrical stiiiiulatirm ( IGg. 9C) indicates that the negative DKI. ant1 the inliihitory process in J-units coultl llave some causal connection. l:ecause ilo other correlation to the negative 111~1’ has l)ecii fount1 in sljinal cord Ijliysiology, in impulse frecpcncies of dorsal horn cells (26). iii intracellular syiial)tic potentials of dorsal horn cells ( 12), in potassituii ion concentrations (lGj, or it1 afterllotentials (22), exe@ in the afferents themselves, the notion of a causal role for J-units in producing the DRP must he considered. The experiment of M’all (23), demonstrating the transmission of a passive DRP with dorsal columns cut between the stinlulated and recorded roots, suggests that an interaction among afferents cannot account for the phenoinenon. The association of J-unit inhibition and primary afferent depolarization mightt however, 1~ due to a state of hyperpolarization in J-unit distal dendrites causing the depolarization of afferent telodendra. There is an anatomical basis for such an interaction ; dendroasonic synapses have been observed in the spinal substantia gelatinosa (19). Also in the suljstantia gelatinosa of the spinal trigeniinal nucleus they have heen ohserved paired with axodendritic synapses (10). The events in the generation of a negative DRP can be modeled as follows (see also Fig. 10). P rior to stimulation, the distal region of J-unit dendrites steadily hyperpolarizes the afferent terminals. A barrage in the afferents then hyperpolarizes the distal dendritic region, while, through a different type of synapse, depolarization tends to be produced in the soma and proximal denclritic region. The mutual coupling of the distal tlendritic region and the afferent terminals is, in effect, a positive feedl>a&
804
IAN
HENTALL
Ioop. A prolonged relative depolarization of afferents, seen as the negative DRP, is a consequence of the activation of this loop. To function as described the model must incorporate two important requirements. First, because afferents are depolarized less during a DRP than during an action potential, the time constant of rise of the feedback to the afferents must be longer than the duration of the action potential, thus allowing the DRP to gradually increase. Second, current in the distal region must have a greater influence on the proximal region and soma than vice versa. A mismatch of impedances which the regions present to each other as sources and passive networks, due to differences in membrane area, represents a way this could be brought about; but clearly the geometrical and electrical factors in a precise formulation would be quite complex. The arrangement is necessary, because the distal hyperpolarization has to restrain spike initiation by the proximal depolarization, thus permitting the J-unit latent period, whereas the distal feedback loop cannot be too rapidly halted by the onset of a burst. To the extent that impulses are conducted electrotonically into the distal region, they would tend to hyperpolarize the afferents. This constitutes the route referred to earlier in the discussion for positive feedback onto noxious afferents, because hyperpolarization of a terminal probably results in an increase of its postsynaptic efficacy (24). The mechanism for initiating the decay of the DRP is not made explicit in the model; neither are such phenomena as the DRP from contralateral or descending fibers, for there is no relevant evidence on these questions. The passive DRP however, as a consequence of the model, must be transmitted by dendroaxonic activation of passive fibers converging onto the same cell as active fibers. The hypothesis explains two previously unaccountable results. First, Rudomin and Mufioz-Martinez (21) showed that DRPs could be generated by direct stimulation of the dorsal horn when intra-arterial injections of tetrodotoxin, which blocks sodium current, had abolished the DRP elicited by dorsal root stimulation. As is true of most synapses studied, the two postulated synapses of the feedback loop need not be dependent on sodium current for their activation. Second, negative DRPs are resistant to baracid (GABA), which biturate (8, 24), and the effect of y-aminobutyric depolarizes the dorsal root, is enhanced by barbiturate (18). Although it is possible that action potentials in substantia gelatinosa neurons are unaffected by barbiturate (24), an explanation ,derived from the presen,t hypothesis is that GABA is the inhibitory transmitter at the axodendritic synapse. Because GABA is a hyperpolarizing agent for neurons elsewhere in the central nervous system, causing increases in chloride ion permeability (20)) this explanation of its action is more parsimonious than one which confers upon it a depolarizing effect on afferent terminals. The fact that barbiturates diminish the positive dorsal root potential (14) accords well
SL-ESTANTIA
CELATI
KOSA
805
with the above scheme, if in fact the late llyperpolarization of the positive dorsal root potential is acconq~lished by impulses in J-units acting either through an axoaxonic route or by electrotonic conduction to the dendroaxonic synapse. REFERENCES D. H., A~UD B. H. C. MATTEINS;. 1938. The interpretation of potential in the spinal cord. J. Pllysiol. (Land.) 92 : 276-321. BESSOV, P., AND E. R. PERL. 1969. Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. /. Ncrwopll)‘siol. 32 : 1025-1043. BIS~~OIB, P. O., W. Buweq AND R. DAVIS. 1962. The identification of single units in central visual pathways. /. Physio/. (Land.) 162 : 409-431. BROOKS, C. McC., AXD K. Korzr~m. 1956. Origin of the dorsal root reflex. J. Ncwoplqsiol. 19 : 61-74. BUR;ESS, P. R., AND E. R. PERI.. 1973. Cutaneous mechanoreceptors and nociceptors. Pages 29-78 in A. ICCO, Ed., Handbook of Srrrsor~ Physiology, T’ol. Il. Springer-Verlag, Berlin. CERVERO, F., D. R. ENSOR, A. IGGO, AND V. MOLONY. 1977. Activity front single neuronrs recorded in the substantia gelatinosa Rolandi of the cat. J. Physiol. (Loud.) 169 : 37P-39P. DOWBEN, R. M., AND J, E. ROSE. 1953. A metal-filled microelectrode. S&rrcc 118: 22-24. ECCLES, J. C., AXD J. I>. h~AI.COr.hr. 1946. Dorsal root potential of the spinal cord. J. Nrzlropllysiol. 9 : 139-160. GESTIXAND, R. C., B. HO\VLAND, J. Y. LETTVIN, AXD W. H. PITTS. 1959. Comments on microelectrodes. Proc. I&. Radio E~rg. 47 : 1856-1862. GOBEL, S. 1976. Dendroaxonic synapses in the substantia gelatinosa glomeruli of the spinal trigeminal nucleus of the cat. J. Comj. Ncurol. 167: 165-176. HENTALL, I. D. 1976. Coding properties of substantia gelatinosa cells in the cat’s spinal cord. Q. Prog. Rep. Rcs. Lab. Electroll. M.I.?‘. 117: 125-126. HONGO, T., E. JANKO\VSKA, AND .4. LVNDBERG. 1968. Post-synaptic excitation and inhibition from primary afferents in neurones in the spine-cervical tract. J. Pl~~siol. (Loud.) 199 : 569-592. K~INAXA\VA, T., ASI) E. R. PEKI.. 1976. Differential excitation of dorsal horn and substantia gelatinosa marginal neurones by primary afferent units with fine (:\ and C) fibres. Pages 67-89 in Y. ZOTTERK~S, Ed., Srwwy Fwctiow of tlzc S/siu. Pergamon, Oxford. LLOYD, D. P. C. 1952. Electrotonics in dorsal nerve roots. Cold Spring Harbor Symp. Quarrt. Viol. 17 : 203-219. LLOYD, D. P. C., AND -1. K. MACINTYRE. 1949. On the origin of dorsal root potentials. J. Gcrr. Plty.siol. 32: 409-443. LOTIIRIAN, E. W., AND G. G. SOXJEN. 1975. Extracellular potassium activity, intercellular and extracellular potential responses in the spinal cord. J. Pkysiol. (Lorld.) 255 : 115-136. MENDELL, L. M., AND P. D. WALL. 1964. Pre-synaptic hyperpolarization: A rofe for fine afferent fibres. J. Pll)wiol. (Lond.) 172 : 279-294. NICOIL, R. A. 1976. The action of pentobarbital on the amino acid induced depolarization of primary afferents in the frog. Puoc. Il’rst. Plrarmml. SM. 19: 421423.
1. BAHRON,
changes
2.
3. 4. 5.
6.
7. 8. 9. 10. 11. 12.
13.
14. 15. 16.
17. 18.
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19. RALSTON,
22.
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