EXPERIMENTAL

NEUROLOGY

Quantitative M. B.

48, 37-56 (1975)

Characteristics of Inhibition Nucleus of the Cat BROMBERG,

P. BLUM,

AND D. WHITEHORN

Department of Physiology arrd Eiophys-ics, College Uk~ersity of Vcmont, Burlington, Vermont Received

in the Cuneate r

Medicine, 05401

of

Jannary 31, 1975

Several properties of inhibition in the cuneate nucleus were investigated as an aid in deriving cuneate circuitry. Inhibition was examined by measuring changes in primary afferent terminal depolarization (PAD) and in the size of evoked medial lemniscal activity. Inhibition was produced by stimulation of several peripheral and central sites. Quantitative measurements were made of the inhibitory time course and degree of facilitation to conditioning trains of different lengths. The data suggest a dual organization of inhibition, dependent upon the conditioning site and characterized by the inhibitory time course. Additional information was derived from PAD measurements following simultaneous stimulation of two conditioning sites and from an investigation of a cortico-cuneate feedback loop. The proposed two inhibitory systems appear to converge for simultaneous activation did not produce simple summation of PAD. These results were substantiated when PAD was measured from single, identified cutaneous fibers. The time course of single fiber PAD was similar to that in a whole nerve preparation. Single fibers could be depolarized by conditioning several sites and with no apparent fiber class dependence. Several models of inhibitory circuitry are presented, and the features that must be taken into account are discussed.

INTRODUCTION Early single-cell studies of the dorsal column nuclei described the properties of the ipsilateral receptive fields, somatotopic arrangement of the cells 1 This paper is based on part of a dissertation by the first author in partial fulfullment of the requirements for the Ph.D. degree. The work was supported by a USPHS grant NS 09472 and NS 05082 from the National Institute for Neurological Diseases and Stroke. We wish to thank Dr. Arnold Towe for his critical comments, Marty Baring for helping with many of the experiments, Hanna Atkins for the illustrations, Peter McCarthy for the photography, and Louise Alhadeff for typing the manuscript. The present address of Dr. Bromberg is: Department of Physiology and Biophysics, School of Medicine, University of Washington, Seattle, Washington 98195. The present address of Dr. Blum is: Department of Neurology, College of Physicians it Surgeons, Columbia University, 630 West 168th Street, New York, New York 10032. 37 Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

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WHITEHORN

in these nuclei (19, 20, 25), and a descending cortical influence (22, 24, 32). In 1964, a circuit diagram to explain the excitatory and inhibitory actions observed was proposed by Anderson and co-workers (2-5). The model contained four types of cells (relay cells and three types of interneurons mediating presynaptic and postsynaptic inhibition) and dealt with inputs from the ipsilateral dorsal columns and contralateral cerebral cortex. Since then, additional influences have been demonstrated from the contralateral periphery (17, 21), the reticular formation, nonspecific thalamic nuclei, cerebellum (14, 15, 30) and the ipsilateral dorsolateral funiculus (16, 31), and attempts to incorporate some of these findings into circuit diagrams have been made (18, 23, 34, 38). The purpose of the present work was to reinvestigate properties of inhibition within the cuneate nucleus for the derivation of cuneate circuitry, Quantitative measurements were made of the inhibitory time courses, degree of facilitation produced by multiple stimuli, and nature of inhibition produced by simultaneous activation of several inputs. Inhibition was examined by measuring changes in primary afferent terminal depolarization (PAD) and changes in the size of evoked medial lemniscal activity. Studies from several laboratories suggest that the time course of PAD and/or lemniscal inhibition differs according to the site of activation. The variability observed in the inhibitory responses necessitated a formal comparison of inhibition evoked from several conditioning sites. We find significant differences in the shape of the inhibitory time course curves evoked from the ipsilateral periphery on the one hand and from the contralateral periphery and contralateral cerebral cortex on the other hand. The inhibitory effects of a cuneo-cortico-cuneate feedback loop described by Towe and Zimmerman (33) were investigated. Several models of inhibitory circuitry are presented, and the features that must be taken into account are discussed. The following paper (8) considers the properties of single cuneate cells in relation to the data and models discussed here. A preliminary report of this work has been presented (9). METHODS Data were obtained from experiments on 40 adult cats. The animals were anesthetized with alpha-chloralose (65 mg/kg, ip), paralyzed with Gallamine triethiodide (Flaxedil) and artificially respired. Blood pressure was monitored, and rectal temperature was maintained between 37 and 38 C by a servocontrolled heating pad. Exposed neural tissues were kept close to body temperature by means of a radiant heat source. Three experimental procedures were used : (i) measurement of changes in presynaptic excitability for a population of fiber terminals, (ii) measure-

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ment of changes in presynaptic excitability for single fiber terminals, and (iii) measurement of the reduction of evoked medial lemniscal discharge. Measurement of Presynaptic Excitability for a Popdation of Fibers. The time course of changes in PAD of ulnar nerve terminals located in the cuneate nucleus was measured by a condition-test (C-T) sequence (35). Conditioning volleys were applied to several peripheral and central sites (ipsilateral and contralateral forelimbs and contralateral cortex) prior to test stimuli (0.05 msec square pulses) delivered by an electrode in the cuneate nucleus at 1 Hz. The test electrode was located at the site of the largest P wave evoked by stimulation of the ipsilateral forelimb, usually 24 mm caudal to the obex and l-l.5 mm lateral to the midline. Brainstem movement were reduced by pneumothorax and by stabilization of the brainstem with low melting point wax (36). Monophasic compound antidromic potentials (crushed nerve recordings) produced by the test stimuli were amplified and fed into an on-line computer for measurement of the area under the antidromic wave. The analogue signal was converted to digital values by a ten bit analogue-to-digital converter at a sampling rate of 20 kHz. To compensate for dc offset voltages and for any diphasic (negative) part of the antidromic potential, a base line average of 64 points was taken before the beginning of each potential. In the integration process, the area was approximated by summing the digital values minus the base line average. The portion of the compound potential to be integrated was specified by the experimentor and activity from fibers conducting from 90-45 m/set contributed to the potentials which were investigated. The amount of PAD produced was described by the ratio of the average of four integrals of the conditioned antidromic test response to the average of four integrals of the unconditioned test: C-T x 100/T = percentage increase over test. The experimental sequence was to precede every conditioned test set of four by an unconditioned test set of four so that the effect of conditioning was always compared to a preceding control. Measurement of Presynaptic Excitability from Single Fibers. Changes in excitability in the terminals of single superficial radial nerve fibers in the cuneate nucleus were measured by a similar condition-test paradigm. Extracellular single fiber action potentials were recorded from one branch of the intact superficial radial nerve (11) with glass microelectrodes filled with 4 M NaCl (d-c resistance 10-20 megohm). A fiber was isolated and tested for antidromic activation with a test electrode in the cuneate nucleus. If a collateral was found, the excitability of the fiber’s terminals was studied by measurement of the test electrode current necessary to just fire the unit every time at a stimulation rate of 1 Hz. This measurement was taken as the current was increased to threshold values. Terminal excitability was increased by application of conditioning volleys to the same peripheral and central sites. The amount of PAD produced was determined by the ratio

40

BROMBERG,

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of a conditioned test to the average of two unconditioned tests, one before and one after the conditioned test, and expressed as a percentage reduction in threshold current. Reduction of Evoked Medial Lemniscal Response. Test stimuli (0.05 msec square pulses) were applied to one branch of the ipsilateral superficial radial nerve, and evoked activity was recorded with a monopolar electrode stereotaxically placed in the contralateral medial lemniscus at coordinates AP = 6.5, ML = 6, DV = 0. Adjustments were made in electrode position so that the maximum amplitude response was obtained. The lemniscal response was identified by its short latency (4 msec) and the high frequency following characteristics of its early component. The area under the lemniscal response was measured by the digital integration system described above. The lemniscal response was generally monophasic, although later components were sometimes biphasic. In such cases, only the early part of the response was measured. Reduction in the medial lemniscal response was expressed as a ratio of the average area of four conditioned-test responses to the average area of four test responses. Conditioning Sites. Three conditioning sites were stimulated electrically : (i) the ipsilateral forelimb by hook electrodes on either the superficial radial or ulnar nerves, (ii) the contralateral forelimb with bipolar electrodes in the central pad of the contralateral forepaw and by hook electrodes on the contralateral superficial radial nerve, and (iii) the contralateral cerebral cortex by bipolar silver ball electrodes straddling the lateral end of the exposed cruciate fissure. Stimulus parameters were 0.05 to 0.1 msec square pulses at a voltage sufficient to produce maximal inhibitory effect (except where noted). Both single stimulus and trains of four stimuli at 300 Hz were used. Calibration of the Digital Integration System The accuracy of the digital integration system was verified by showing that computed integral values were within 1 to 3% of the area values determined by planimetry for the same waves. Trial-to-trial consistency of measurements by the digital integration system was examined by use of simulated as well as experimental data. In the former instance, coefficients of variation of 0.4 to 1.0% for 120 consecutive trials were usual. In the latter instance, the variability was a function of response size (28) and was smaller in the antidromic nerve responses than in the evoked lemniscal responses. Variation in nerve potential ranged from 3% for large responses to 7% for the smallest response used, whereas the variation in lemniscal potential ranged from 5 to 15% for the large and small potentials respectively. RESULTS Previous work (14, 21, 30) suggests that the inhibitory time course varies with the site of conditioning. An increase in terminal excitability due

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to primary afferent depolarization (PAD) is assumed to be related to presynaptic inhibition (1). A decrease in the size of the lemniscal response is considered to reflect both presynaptic and postsynaptic inhibition (2). Preliminary observations indicated considerable variability in results from preparation to preparation and the need for systematic examination amenable to statistical analysis. Measurements of PAD were used for this purpose, and the reduction of the lemniscal response was explored less systematically. Time course curves of PAD were derived from measurements taken at selected points, chosen in a random order, in the C-T interval range from 12 to 350 nlsec. Intervals less than 12 msec were not examined because the stimulus artifact from the conditioning trains interfered with the recording of the antidromic wave. When four-stimuli conditioning trains were employed, the C-T interval was measured from the first of the four shocks. Itlhibitory Ejfects of Ipsilateral Superficial Radial Nerve Conditioning. The time course of PAD produced by electrical stimulation of the ipsilateral superficial radial nerve is shown in Fig. 1A. It is characterized by a rapid rise to peak value (usually within 25 msec) and a slow decline (lasting up to 350 msec) although a smooth decay was not always observed. A time course curve displaying a late increase in PAD is shown in Fig. 1D. This unusual curve was the only time course curve produced by ipsilateral superficial radial nerve conditioning in the particular preparation, but other time course curves produced by conditioning from the contralateral forelimb in the same preparation displayed the usual form. Curves with similar late peaks were observed in two other experiments. Infrequently, multiple peaks at shorter C-T intervals occurred that were distinguishable from the main peaks. Time course curves of PAD were generated by both single-stimulus conditioning and four-stimuli trains. The C-T intervals at which maximum PAD occurred, and the maximal values of PAD produced by both conditioning parameters are shown in Table 1A. These data are compared statistically in a later section. The size of the medial lemniscal response evoked by stimulation of one branch of the ipsilateral superficial radial nerve was reduced when the other branch was used to condition the nucleus. The time course of this reduction, shown in Fig. lA, was similar to the time course of PAD produced by conditioning the same site. Inhibitory Eflects of Contralateral Cortical Conditioning. Conditioning with the contralateral cortex produced PAD with a different time course, as shown in Fig. 1B. The time course curves show a characteristic slow rise, a prolonged peak and a smooth decline ending at C-T intervals of 250 to 300 msec. Cortical conditioning was less effective than superficial radial

42

BROMBERG,

BLUM

AND

WHITEHORN

A.

C-T INTERVAL 180

(msec)

9, B. :! \

I

R-O------* + ,,o---:&-4’

50

loo

I50 200 C-T INTERVAL

250 (msed

300

C.

9

-

2 E

.- _____ +------

3

50

100 I50 200 C-T INTERVAL

250 (msec)

100

T 250 (msec)

300

350

300

350

D. ;;;:! 160

2_;-2

100

I 0

I 50

150 200 C-T INTERVAL

(solid lines) and reduction of the medial lemniscal FIG. 1. Time course of PAD response (dashed lines) produced by conditioning different sites at various C-T inter-

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nerve conditioning in the same preparation. Data on the C-T interval of the peak and the magnitude of peak PAD for cortically conditioned time course curves are shown in Table 1B. Similarly, the medial lemniscus discharge was reduced by cortical conditioning. The time course curves show maximal reduction at C-T intervals of 4&60 msec in most preparations (Fig. 1B). A major difference, however, is the presence of an apparent facilitation at early intervals in some lemniscal responses. Inhibitory Efects of Contralateral Forelimb Conditioning. Conditioning with the contralateral forelimb produced PAD time course curves quite different from those produced by ipsilateral forelimb conditioning but similar to those produced by the cortex. The peaks occurred at late C-T intervals (30-60 msec) , and the peak values were smaller than those produced by ispilateral forelimb conditioning (Fig. 1C). After the peak, PAD declined slowly and ended at C-T intervals of 200 to 300 msec. Listed in Table 1C are the maximum PAD values and the respective C-T intervals. The time course of reduction of the lemniscal response following contralateral forelimb conditioning (Fig. 1C) is similar to that for PAD evoked by stimulating the contralateral forepaw. Statistical Comparisons. Repeated measurements of PAD and the change in the lemniscal response revealed substantial variability between preparations, within a preparation over time, and from trial-to-trial. The effects of trial-to-trial variability were reduced by use of the average of four consecutive integrals in the PAD and lemniscal time course measurements. Differences between time course curves produced by conditioning of three different sites were verified by statistical tests.2 This was accomplished by a comparison of the maximum amount of PAD produced from each conditioning site and the C-T interval at which maximum PAD occurred (data in Table 1). As discussed earlier, the time courses of PAD produced by conditioning with the ipsilateral forelimb, contralateral cortex and contralateral forelimb appeared to differ (Fig. 1). This was verified statistically. Multiple comparisons showed that the PAD time course curves produced by fourstimuli ipsilateral forelimb conditioning have peaks of significantly greater 2 Statistical comparisons were made using the nonparametric two-sample Wilcoxon test because the data were suspected to be nonnormal, and Bonferonni’s procedure was used because multiple comparisons were made. vals (abcissa). Left-hand ordinate scale is percentage increase in terminal excitability (PAD) with respect to test; right-hand ordinate scale is percentage decrease in area of medial lemniscal response with respect to test. Time course curves produced by conditioning with (A) the ipsilateral superficial radial nerve, (B) contralateral cerebral cortex, and (C) contralateral forepaw. D : Unusual time course curve with late peak produced by ipsilateral superficial radial nerve conditioning.

44

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TABLE C-T

A. Ipsilateral radial

B. Contralateral

C. Contralateral

WHITEHORN

1

INTERVALSANDASSOCIATED PAD VALUESFORTHE PEAKS PAD TIME COURSE CURVES PRODUCED BY CONDITIONING ATTHREE SITES

superficial nerve

cortex

forepaw

Four-Stimuli conditioninp C-T interval @ peak (msec)

PAD @ Peak (%I

12 12 12 12 12 12 12 12 20 25 25

1.58 136 138 220 114 208 149 144 170 118 138

mean 15.1 SD 7.9 80 80 20 27 40 60 60 50 32 -

153.9 33.8 109 116 134 238 115 115 11.5 133 115

mean 49.8 SD 22.0 48 75 60 65 75 75 -

132.0 40.6 12.5 109 124 112 107 109

mean 66.3 SD II.0

114.3 8.2

OF

Single-Stimulus conditionine C-T PAD interval @I Peak 63 peak (msec) (%I 22 2.5 40 15 20 25 15 20 2.5 20 mean 22.7 SD 7.1

218 1.50 215 176 127 126 125 122 119 119 149.7 39.4

magnitude occurring at earlier C-T intervals than those produced by fourstimuli conditioning with the contralateral cortex or the contralateral forelimb (P < 0.01).

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,,! 6”; , , , , 123456 CONDITIONING

SHOCK NUMBER

FIG. 2. Effects of increasing the length of the conditioning stimuli trains on the excitability of primary afferent terminals. Dashed line represents contralateral cortical conditioning at SO-msec C-T interval. Solid line represents ipsilateral superficial radial nerve conditioning at ZO-msec C-T interval. The comparisons were complicated since the magnitude of depolarization was dependent upon the number of stimuli employed in the conditioning volley. The effect of increasing the number of conditioning stimuli is shown in Fig. 2. Conditioning trains used in the time course measurements were limited to four stimuli at 300 Hz in order to avoid excessively long conditioning artifacts. The effectiveness of conditioning trains was further explored by comparing the maximum amount of PAD produced by single and four-stimuli conditioning trains delivered to the same conditioning site. For PAD produced by the ipsilaterai forelimb, the peak values were not significantly greater for four-stimuli trains when compared to singlestimulus conditioning. The C-T intervals at which peak PAD occurred, however, were significantly shorter when four stimuli were used (P < 0.01). Similar comparisons were made for the cortex-evoked PAD and four-stimuli trains produced significantly more PAD (P < 0.01). Single conditioning stimuli applied to the contralateral forepaw produced detectable amounts of PAD, whereas four-stimuli trains increased the amount of depolarization. Accurate measurements of single-stimulus PAD were confounded by trial-to-trial variability in terminal excitability. For this reason, single-stimulus conditioning was not routinely used, and no statistical comparisons were made. A bias should be noted in the measurements of the peak of the PAD time course curves. Intervals of 12 and 15 msec were the shortest used, and a peak occurring at a shorter interval could not be accurately measured. If a peak obtained with four-stimuli conditioning occurred at a C-T inter-

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BROMBERG,

BLUM

20

30 C-T

~oo!i--T-z



30 C-T

AND

40 INTERVAL

40 INTERVAL

WHITEHORN

50

60

70

60

(msecl

50 7070 (msec)

FIG. 3. Time course curves of PAD produced #by separate and simuftaneous stimulation of two PAD-producing inputs to the cuneate nucleus. A: Solid line, PAD evoked by maximal contralateral cortical conditioning. Dashed line, PAD evoked by maximal ipsilateral superficial radial nerve conditioning. Dotted and dashed line, PAD evoked by simultaneous maximal contralateral cortical and maximal ipsilateral superficial radial nerve conditioning. B : Solid Iine, PAD evoked by maxima1 cortical conditioning. Dashed line, PAD evoked by low-level ipsilateral superficial radial nerve conditioning. Dotted and dashed line, PAD evoked by simultaneous maximal cortical and low-level ipsilateral superficial radial nerve conditioning.

val shorter than 12 or 15 msec, the value at the true peak would be greater than the reported value, tending to separate further the one- and fourstimuli values and their respective C-T intervals. PAD Interaction. Since in an awake cat, activity may reach the dorsal column nuclei simultaneously from several inputs, the question of interaction of PAD produced in this way arises. The data presented above suggest that the cortex and contralateral forelimb produce equivalent effects. The interaction combinations chosen were therefore (i) the ipsilateral superficial radial nerve and cortex, and (ii) the ipsiIatera1 superficia1 radial nerve and the contralateral forepaw. Four stimuli were used at all conditioning sites, and stimulus current for the cortical and contralateral forepaw sites was maximal whereas the stimulus current for the ipsilateral superficial radial nerve was graded.

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The ipsilateral forelimb input was found to be the strongest PAD-producing input to the cuneate nucleus. Maximal conditioning with the superficial radial nerve produced more PAD than any other site examined for any C-T interval. There was no increase in PAD or any other change in the time course curve when either the contralateral cortex or the contralateral forepaw was stimulated simultaneously with a maximal volley to the superficial radial nerve (Fig. 3A). In order to observe interaction, the superficial radial nerve conditioning volley was reduced until it produced less PAD than either the cortex or the contralateral forepaw produced alone. Under these conditions, addition of either cortical or contralateral forepaw conditioning to the lowstrength superficial radial nerve conditioning caused summation of PAD. The summation was such that the simultaneously conditioned time course curve began to take the shape of the curves produced by either cortical or contralateral forepaw conditioning (Fig. 3B). The summation in the simultaneous conditioned curves was not the simple summation of the PAD produced by the separate curves but rather a partial summation, despite the ability of a maximal ipsilateral superficial radial nerve volley to produce more PAD. Interactions examined using the reduction of evoked lemniscal activity yielded the same general form of interaction, with ipsilateral superficial radial nerve-evoked inhibition dominating other inputs to the extent that interactions were not observable unless ipsilateral superficial radial nerve stimulation intensity was adjusted to low levels. Cortical Re,tle.zr Loop. The effects of a cortical reflex discharge can sometimes be observed in the cuneate nucleus of choloralose-anesthetized cats as a negative wave (N” ) (Fig. 4A) occurring 10-16 msec following stimulation of the ipsilateral forelimb and lasting approximately 16 msec (33). It is possible that this reflex-discharge, negative wave is followed by a positive wave whose potential changes are not readily observed because they are buried in the peripherally evoked P wave (Towe, personal communication, and 29). If the reflex N” and P” waves are analogous to the N and P waves evoked by direct cortical stimulation (3)) they may signal excitatory and inhibitory events in the cuneate nucleus. Such effects may be presumed to result from activation of the cortex by functional circuits, in contrast to activation by direct cortical stimulation. Experiments were conducted to determine whether PAD evoked by this reflex activity could be detected by comparing peripherally evoked PAD in an intact animal and in the same animal after inactivation of the pericruciate cortex and, hence, interruption of the reflex loop (Fig. 4B). Cortical function was halted by cooling the exposed sensorimotor cortex with ice chips (33) or by ablation of one or both hemispheres by suction. Time course curves were determined just before and immediately after these procedures.

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BROMBERG,

,

BLUM

AND

WHITEHORN

A.

I

:

,I 1

IL

C.

s;;j,k-r ^_____ ~ 0

50

100

150 200 C-T INTERVAL lmsecl

250

300

350

FIG. 4. Changes in cuneate slow waves and PAD following cerebral cortical ablation. A: Cuneate N and P waves elicited by ipsilateral superficial radial nerve stimulation. Note N” wave in intact preparation. B: N and P waves recorded after contralateral pericruciate cortex ablated by suction. Note absence of N” wave. Positivity is down. C: PAD time course curve evoked by ipsilateral superficial radial nerve conditioning with cortex intact (dashed line), and time course curve after bilateral cortical ablation (solid line).

Eleven preparations examined for influences of the cortical reflex discharge showed no consistent changes in either the time course or peak value of PAD when the cortex was inactivated (Fig. 4C). Changes were sought in PAD produced by maximal and less than maximal volleys to the superficial radial nerve as well as volleys consisting of one and fourstimuli trains. The results were no different when the cortex was removed early in the experiment (about 7-8 hr following initial anesthesia) or late (about 12 to 14 hr after anesthesia) or when the contralateral side or both sides of the cortex were ablated. When all combinations of conditioning stimulus parameters were considered, there was almost an equal number of experiments in which small increases and decreases in peak PAD occurred following cortical ablation. The observed changes occurred at short C-T intervals ; the remaining portions of the postablation C-T curves were almost identical to the corresponding portions of the preablation curves (Fig. 4C).

INHIBITION

85+

50

0

IN

100

CUNEATE

150

200

C-T INTERVAL

49

NUCLEUS

250

300

lmsecj

FIG. 5. PAD time course curves of single ipsilateral superficial radial nerve fiber (Field fiber). Solid line, PAD time course curve produced by ipsilateral ulnar nerve conditioning; Dashed line, PAD time course following contralateral cortical conditioning. Dotted and dashed line, time course curve following contralateral forepaw conditioning.

PAD of Single Agerent Terminals. Recordings from single, intact fibers of the superficial radial nerve provided an opportunity to compare the time course of single unit PAD with the time course of PAD of a population of terminals, and to determine the degree of convergence of PAD-producing inputs onto the terminals of single fibers. Further, the preparation afforded the opportunity to examine the relationship of the type of cutaneous receptor innervated by the isolated fibers to the degree of convergence and time course of PAD. The classification of cutaneous receptors innervated by the isolated fibers is based on a scheme previously described (10, 12). PAD time course curves for a field receptor fiber produced by conditioning with the ipsilateral ulnar nerve, cortex and contralateral forepaw are diplayed in Fig, 5. Similarity of these results to those shown earlier for a whole population of nerve terminals is evident. When the data for all single fibers are conTABLE

2

PATTERN OF DEPOLARIZATION (PAD) OF SINGLE PRIMARY AFFERENT FIBERS PRODUCED BY STIMULATION OF THREE CONDITIONING SITES”

Ulnar N. Cortex

+ + + -I-

+ +

a + Conditioning

Contralateral forepaw

No.

f + -

7 1 1 2

Receptor type

3 1 1 1

G-2, 2 Field, 1 Type II, 1 Unknown Field Unknown Unknown, 1 G-2

produced PAD ; - Conditioning

did not produce PAD.

50

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sidered, the mean interval at which peak PAD occurred in 23 msec for ulnar nerve conditioning (n = 9)) 49 msec for cortical conditioning (n = 6) and 57 msec for conditioning with the contralateral forepaw (n = 3). The magnitude of depolarization is also greater for the ipsilateral forelimb input than for the other two inputs. These values compare favorably with the corresponding values in Table 1. PAD could be produced in primary afferent terminals from multiple sites. The excitability of all but one of the units tested (26 out of 27) could be increased by ulnar nerve conditioning. Eleven units were tested for convergence of PAD-production from all three conditioning sites and seven were found to be so activated (Table 2). Only one unit had a PADproducing input from the ulnar nerve alone, and three fibers lacked a PADproducing input from either the cortex or the contralateral forepaw. Although the sample is small, the data shown in Table 2 suggest that convergence of PAD-producing inputs is not restricted to specific receptor types. DISCUSSION Inhibition of long duration is a prominent feature of the cuneate nucleus. It can be measured either as a reduction in the number of spikes per discharge of cuneate cells, or as a reduction in the size of a volley passing through the nucleus, or as an increase in the excitability of primary afferent terminals. The latter two methods were used here to quantitatively examine the properties of inhibition produced by several inputs to this nucleus. Anesthetics. All experiments in this study were performed on cats anesthetized with alpha-chloralose, The time course curves of inhibition measured here are in basic agreement with the results reported by other investigators who used barbiturate anesthetics (Nembutal) and unanesthetized, decerebrate cats (4, 21). In contrast, in studies conducted in the spinal cord of the cat, increases in excitability of primary afferent terminals were observed with chloralose, but not with Nembutal (13). Comparison of PAD and the Reduction in the Lemniscal Response. PAD and lemniscal reduction time course curves produced by conditioning the same sites have similar shapes (Figs. lA, B, C). The reduction in the area under the evoked lemniscal activity is considered to be caused by both presynaptic and postsynaptic inhibition (2), whereas the increase in excitability of primary afferent terminals is considered to be a measure of presynaptic inhibition (1, 35). Either the lemniscal inhibition we observe reflects predominantly presynaptic inhibition or the time courses of presynaptic and postsynaptic inhibition are similar. Although primary afferent hyperpolarization was not observed, an early facilitation was found in some of the cortically evoked lemniscal reduction

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time course curves (Fig. 113). A similar phenomenon has ‘been reported by RosCn (27). The facilitation probably reflects direct or subthreshold cortical excitation of some cuneothalamic relay cells, since the majority of cortically evoked first spike latencies occur between 5 and 15 msec (Bromberg, unpublished data). These excitatory effects occur before cortically evoked inhibition has fully developed. Cortical Feedback Loop. Excitatory and inhibitory influences of the cerebral cortex on cells in the cuneate nucleus have been studied by direct cortical stimulation (22, 24, 32). The demonstration of a cortical reflex loop (33) suggestsa method of examining presynaptic changesproduced by corticofugal elements activated by normal means. However, the contribution of cortical reflex activity to peripherally evoked PAD appears to be negligible since no consistent or dramatic change in the time course of PAD was observed when the cortex was ablated (reflex loop interrupted). This result is explicable in light of the weak and subtle nature of the cortical influence as discussed below (see Fig. 3). Models of Cmeate Inhibitory Circuitry. The data for this paper were gathered with the intention of defining the features of inhibition in relation to cuneate circuitry. There is insufficient information to define a unique circuit diagram (29), but several candidates will be discussed.Although circuits are presented in terms of PAD, they apply as well to inhibition measured as a reduction in the lemniscal response. Any circuit diagram should take into account (i) the time course, (ii) magnitude, (iii) f acl‘l’t1 at’ion to conditioning stimuli, and (iv) frequency following abilities of inhibition (9, 37). In addition, in proposing a circuit model one should consider (v) that single primary afferent fibers receive convergent depolarizing inputs without regard to fiber type, and (vi) that saturation of the inhibitory mechanisms occurs with low levels of the ipsilateral input (4). Several arrangements of cuneate circuits with respect to inhibitory processesare presented in Fig. 6. The results of the present work stress the dual nature of presynaptic inhibition (14, 21). The ipsilateral forelimb input to the cuneate nucleus activates a rapidly acting PAD-generating system. Stimulation of a wide variety of peripheral and central inputs activates a second, more slowly acting PAD-generating system. These two systems (rapid and slow) are considered separately (Fig. 6A, B) and combined (Fig. 6C). The rapidity of onset and fast rise time of the rapid system suggest a direct activation of the PAD-generating processesby dorsal funiculi fibers. Three possiblemechanismsfor producing PAD are diagrammed in Fig. 6A1, -2, and -3. Figure 6A-1 shows the interneuron (afferent collateral) model suggested by Anclersen et al. (5). Figure GA-2 assumesan ionic mechanism involving changes in the concentration of potassium due to the afferent volley (6, 7, 26), and Fig. GA-3 suggestsaxo-axonic synapsesbe-

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FIG. 6. Possible arrangements of inhibitory circuitry in cuneate nucleus, A: Three different circuits to account for the rapid production of PAD by dorsal funicular activity. Al, depolarization via interneuron. AZ, depolarization produced by increased extracellular potassium concentration. A3, depolarization produced by direct axoaxonic synapses between afferent fibers. B: Three different locations for interneurons implicated in the production of slow time course PAD and activated by stimulation of off-focus peripheral and descending central systems. C: Circuitry combining rapid and slow PAD-generating systems. Cl, independence at interneuronal level. CZ, convergence at interneuronal level. Further details in text. DF: ipsilateral dorsal funiculus. CFP : contralateral forepaw. Cblm: cerebellum. RF: reticular formation. Ctx : cerebral cortex. Dark cells : interneurons. Stippled cells: cuneothalamic relay cells. Dark presynaptic terminals : depolarizing synapses.

tween primary afferent fibers although evidence for such a mechanism is lacking. The temporal characteristics of the elements involved in PAD produced by the ipsilateral input are illustrated in Fig. 7A, in which the interneuron model is used. The interneurons responsible for PAD are expected to have strong synaptic connections from dorsal funiculi fibers (ability to follow high frequency stimulation). The rapidity of onset and fast rise to peak PAD of the rapid system suggests that these interneurons should exhibit short latency, synchronous activity following peripheral stimulation. There is ample experimental evidence for cuneate cells with these properties (4, 8, 10). The following temporal properties are summarized in the figure: arrival time of activity in primary afferent fibers (lo), time course of unit activity of cuneate nonrelay cells following ipsilateral superficial radial nerve stimulation (8) and time course of ipsilateral forelimb evoked PAD (from Fig. 2A) . The slow PAD-generating system as defined here is activated by stimulation of a wide variety of neural structures, including the cerebral cortex, reticular formation, nonspecific thalamic nuclei, cerebellum, and off-focus

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periphery. Interneurons related to the slow system, if involved, are expected to be of the wide field type with poor frequency following abilities, and to show a wide temporal distribution of activity. Jabbur and Banna (21) failed to find cells in the cuneate nucleus with contralateral input to account for contralateral forepaw-evoked PAD and located the needed interneurons in the adjacent reticular formation. Experiments by Cesa-

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7. Temporal properties of neural elements involved in PAD. A : (a) tempomral distribution of primary affcrent fiber activity following ipsilateral superficial radial nerve stimulation, (b) latency distribution of all spikes evoked in nonrelay cells (interneurons) following ipsilateral superficial radial nerve stimulation and (c) time course of PAD produced by ipsilateral superficial radial nerve stimulation. B: (b) latency distribution of all spikes evoked in wide-field, nonrelay cells (interneurons) by contralateral forepaw stimulation, and (c) time course of PAD evoked by contralateral forepaw stimulation. Histogram bin sizes = 2 msec. DF: ipsilateral dorsal funiculus. CFP : contralateral forepaw. Dark cells : interneurons. Stippled cells : cuneothalamic relay cells. Dark presynaptic terminals : depolarizing synapses. FIG.

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Bianchi et al. (14) have further implicated the reticular formation in mediation of wide field inhibition in the cuneate nucleus. However, recent studies (8, 17) describe cuneate cells with wide field inputs. The various possible locations for interneurons mediating the wide field effects are shown in Fig. 6B 1-3, ,in which interneurons are placed in the reticular formation (Fig. 6B-l), in both the reticular formation and cuneate nucleus (Fig. 6B-2), and completely in the cuneate nucleus (Fig. 6B-3). Evidence to be presented elsewhere (8) suggeststhat Fig. 6B-3 is sufficient to account for the observed inhibition, Fig. 7B shows the temporal properties of the discharge of wide field cells in the cuneate nucleus (8) and the time course of contralateral forepaw-evoked PAD (from Fig. 1C). Two forms of circuitry combining both the rapid and slow systems are illustrated in Fig. 6C. Figure 6C-1 shows independence of the PAD producing elements while in Fig. 6C-2 PAD-producing-neurons are shared. An ipsilateral input to the wide field elements is included in Fig. 6C-1 (dashed line) to account for cuneate cells that have both ipsilateral and wide field inputs (8, 17). H owever, evidence to be presented elsewhere (8) suggests that activity of nonrelay cells with exclusive ipsilateral input is sufficient to explain the characteristic PAD time course of the rapid system. Both of these models would predict interaction between the two PAD generating systems (see Fig. 3A, B). In the present work, simultaneous stimulation of the ipsilateral input with either the contralateral forepaw or cortex showed the rapid system to be the dominant producer of PAD. No summation of PAD was produced during simultaneous activation when the rapid system was maximally activated. When the input to the direct system was reduced, only partial summation occurred, even though the primary afferent terminals were capable of greater depolarization. Partial summation may reflect interaction either at the interneurons (Fig. 6C-2) or at the primary afferent terminals themselves, since single unit PAD data demonstrate that primary afferent fibers are capable of prolonged depolarization through either system (Fig. 5). Note added in proof: Additional papers concerned with the influence potassium ions on the mechanism of PAD should be noted: K. Krnjevid and M. E. Morris. 1972. Extracellular K+ activity and slow potential changes in spinal cord and medulla. Canad. J. Physiol. Pharmecol. 50: 1214-1217; G. ten Bruggencate, H. D. Lux, and L. Liebel. 1974. Possible relationships between extracellular potassium activity and presynaptic inhibition in the spinal cord of the cat. PfEugers Arch. ges. Physiol. 349: 301-317; L. Vyklicky, E. SykovL, and N. Kiii. 1975. Slow potentials induced by changes of extracellular potassium in the spinal cord of the cat. Brain Res. 87: 77-80.

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Quantitative characteristics of inhibition in the cuneate nucleus of the cat.

EXPERIMENTAL NEUROLOGY Quantitative M. B. 48, 37-56 (1975) Characteristics of Inhibition Nucleus of the Cat BROMBERG, P. BLUM, AND D. WHITEHORN...
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