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Frequency following and conduction speed as identifiers of major subsets among cuneothalamic neurons
EXPERIMENTAL
NEUROLOGY
64,190-201
(1979)
Frequency Following and Conduction Speed as Identifiers of Major Subsets among Cuneothalamic Neurons J. P. WALSH AND D. WHITEHORN' Department of Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, Vermont 05401 Received April 20, 1978; revision received November 3, 1978 Cuneothalamic relay (CTR) neurons display heterogeneity in terms of response properties and morphological characteristics. Subsystems have been proposed in which speed of conduction and afferent synaptic strength serve as distinguishing features. The present work examined the relevance of these two parameters to other commonly measured characteristics of CTR cells. Response properties of 63 CTR cells to electrical stimulation of the ipsilateral superficial radial nerve were studied using extracellular single-cell recording methods in chloralose-anesthetized cats. The stimulus was varied in amplitude and frequency. Conduction speed was inferred from antidromic latency to stimulating of the medial lemniscus just caudal to the thalamus, and from orthodromic first spike latency to superficial radial nerve stimulation. Afferent synaptic strength was estimated by determining maximum frequency following ability (FMAX). Other properties measured include number of spikes per discharge, threshold for electrical activation at the superficial radial nerve, depth of cell within the tissue, and a latency-intensity relation. Antidromic conduction time from the medial lemniscus had a significant inverse correlation only with cell depth. Orthodromic first spike latency was correlated (inversely) only with FMAX. The shape of the latency-intensity function was significantly linked to FMAX. Cells appeared to fall into three subsets tentatively identified with subsets proposed elsewhere on the basis of responses to natural stimulation.
INTRODUCTION The analysis of local circuit interactions within a nuclear complex requires the prior identifications of functional neuronal subsets. This Abbreviations: CTR-cuneothalamic relay, FMAX-maximum frequency following ability, L-I-latency-intensity, MLLAT-antidromic latency, L,-orthodromic latency. 1 This paper is based on part of a dissertation by Dr. Walsh in partial fulfillment of the requirements for the Ph.D. degree, who was supported by PHS Grant 5-TOl-GMOO439-14. This work was supported by grant NS09472 and Biomedical Research Support Grant S-SO7-RR05429 from the National Institutes of Health. We wish to thank Howard Lipson for help with computer programming. 190 0014-4886/79/040190-12$02.00/O Copyright 0 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.
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identification rests on the choice of sorting criteria to be used to carry out the classification. Ideally, one or a few properties are sought that predict clustering of other cellular characteristics. Towe (10, 11) described a population analysis approach that formalizes the search for classifying criteria. In that method a relatively large number of cellular properties are observed from each sampled neuron. The entire data matrix is then sorted and examined for covariance among the measured characteristics. This laboratory has previously used the population analysis approach (2) to show that intemeurons within the cuneate nucleus of cat comprise two groups, well defined by the maximum frequency of afferent stimulation which they can follow in a one-to-one fashion, the FMAX value. When cells are sorted in this manner, other properties (degree of convergence from peripheral and central sites, and the orthodromic latency of activation) can be predicted. Since cuneothalamic relay (CTR) neurons also vary among themselves in frequency following ability, Blum er al. (2) suggested that they also be divided into subsets on the basis of the FMAX value. Hand and Van Winkle (6) proposed a similar subclassification of the cuneothalamic projection system, citing anatomical, physiological, and phylogenetic evidence. They distinguished two systems by frequency following ability and speed of conduction: a fast, high-frequency following neolemniscal system, and a slow, low-frequency following paleolemniscal system. The present study tested the clustering of CTR properties caused by sorting cells on the basis of FMAX or conduction speed. Orthodromic latency from the periphery and antidromic latency from the medial lemniscus were used as the measures of conduction speed. Attempts at correlating other functional parameters with these measures revealed clustering around FMAX and orthodromic latency, but not around antidromic latency. METHODS Adult cats were anesthetized with a-choralose (50 mgkg, i.p., in propylene glycol, or i.v. saline under ether), paralyzed with gallamine triethiodide (Flaxedil), and artificially respired. Rectal temperature was monitored and maintained at 37.5”C. Carotid blood pressure was monitored. Single units were recorded extracellularly in the cuneate nucleus with 4.0 M NaCl-filled micropipets. Electrode tracks were 0 to 3 mm caudal to the obex. A bipolar stimulating electrode was placed on the ipsilateral superficial radial nerve in the region of the elbow. A bipolar recording
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electrode was positioned on the ipsilateral nerve in the region of the wrist. A concentric bipolar stimulating electrode was placed in the contralateral medial lemniscus just caudal to the ventral posterolateral nucleus of the thalamus (A6.0, L6.75). The final position of the electrode was chosen to maximize the size of the short-latency, high-frequency following potential evoked in the medial lemniscus by superficial radial nerve stimulation at the elbow. The search stimulus was an electrical pulse (50~ps duration) delivered through the electrode at the elbow. The intensity was such that all A-alpha and A-delta fibers were activated. Response properties were studied using an automated test sequence under control of an on-line PDP4/e computer. Fifty-microsecond stimulus pulses of varying voltage and frequency were applied to the superficial radial nerve at the elbow. The antidromic compound action potential at the wrist was digitized and integrated in real time to provide the principal measure of input intensity. Integrals included A-alpha and A-delta activity but not dorsal root or dorsal column reflex activity. Reflex activity was identified by its inability to follow stimulus frequencies of more than 5 Hz. The latencies of all spikes occuring between stimuli were measured and stored with the corresponding integral of the antidromic compound action potential at the wrist. CTR cells were identified by their ability to follow greater than 200-Hz stimulation from the medial lemniscus stimulation site on a one-for-one basis with short (~3 ms), invariant latency. Data were analyzed off-line by computer programs that calculated statistics for each cell and accumulated these statistics for the entire cell sample. The input intensity integral for each trial was normalized to the maximum integral observed among all the trials on that cell at the same stimulus frequency. This made possible comparisons between cells recorded in different preparations. RESULTS A total of 176 neurons was studied in 17 experiments. We identified 63 cells as CTR cells. Because our primary interest is in the relationship of conduction speed and frequency following ability to CTR cell function, results are presented in two sections in which the relationships between these parameters, and other functional parameters are separately described. Conduction
Speed
Antidromic Latency. CTR conduction speed was studied indirectly by measuring the antidromic latency (MLLAT) from the stimulation site at the
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thalamic end of the medial lemniscus. Figure 1 shows the distribution of MLLAT for 61 CTR neurons recorded in our experiments. Values were 0.9 to 2.1 ms with a mean of 1.42 ms. The corresponding conduction velocity range can be estimated by calculating the conduction distance using average sterotaxic coordinates of the stimulus and recording sites. If a straight-line conduction path is assumed the distance is 22 mm. With a 10% addition for fiber bending, the conduction velocity range obtained is 11 to 27 m/s. A series of basic parameters was obtained from each CTR cell. In addition to antidromic latency (MLLAT), these included latency of the tist spike evoked by stimulation of the superficial radial nerve (L,), threshold for activation by the nerve electrode (V,, measured as a percentage of maximum input intensity), number of spikes evoked per discharge in response to stimulation (S/D), depth of the cell in the tissue (as read from the microdrive), maximum frequency of nerve stimulation which the cell could follow on a one-for-one basis (FMAX), and a latency-intensity (L-I) relation. Both L, and S/D were determined with supramaximal stimulation. Not all parameters could be measured on every cell isolated. Table 1 contains a summary of the statistics for the parameters obtained from our sample. The distributions of L, and MLLAT resemble those reported previously (2,8). The coefficients of correlation between MLLAT and the other parameters, rML, are also contained in Table 1. With one exception, the parameters we examined did not correlate significantly with MLLAT. The correlation between MLLAT and depth of isolation is highly significant.
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.5 1:O 1.5 ANTIORONIC ML LRTENCY
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FIG. 1. Distribution of antidromic conduction latency for cuneothalamic relay neurons recorded in the cuneate nucleus. Stimulation was applied to the medial lemniscus approximately 24 mm rostra1 to the recording site. (N = 61; bin size = 0.1 ms).
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1
Statistics for Functional Parameters of Cuneothalamic Relay Cells mean + SD
Lib 6-M MLLAT (ms) S/D Depth (pm) FMAX (Hz) v, 6%
5.90 1.42 2.% 1331 58.6 35.1
Range
+ 3.05 f 0.32 + 1.72 -e 513 2 46.0 ” 18.2
rL, 0’)
3.1-18.8 0.9-2.1 1.0-8.3 350-3000 O-100 20-98
0.12 (42) -0.043(49) -0.377(60)* 0.087(29) 0.047(35)
0.12 (42) -0.023(42) 0.169(41) -0.622(29)* 0.188(35)
0 rML and r,,, are correlation coefficients between the indicated parameter and MLLAT and L,, respectively. Number of data pairs is in parentheses. P > 0.10 unless otherwise noted. * Abbreviations in Tables 1 and 2: L,-latency of first orthodromic spike, MLLAT-antidromic latency, S/D-spikes per discharge from nerve stimulation, FMAX-maximum frequency following ability, Vi-threshold. * Highly significant correlation (P < 0.001).
A plot of the antidromic latencies as a function of depth is shown in Fig. 2. The antidromic latencies varied inversely with depth; deeper cells tended to have shorter values. The slope- of the regression curve was 0.23 s/m. Cells deeper in the nucleus lie physically closer to the thalamus than more superficial cells. Therefore, if the decline in antidromic latency with increasing depth were due solely to decreasing conduction distance, then the reciprocal of this slope would represent the average conduction
0
I - 0.
600.
I
I
1600. 2L)oo. DEPTH (MICRONS1
I
3200.
FIG. 2. Antidromic medial Iemniscus latency versus depth of isolation for cuneothalamic relay neurons. Depth is distance below the dorsal surface of the medulla as indicated by the electrode microdrive. (N = 60).
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velocity of the cell sample. However, the velocity calculated in this way, 5 m/s, is much smaller than the range of velocities we observed. More likely, cells situated deeper in the nucleus have higher conduction velocities. Orthodromic Latency. Conduction speed in the dorsal column-medial lemniscal system is more frequently inferred from orthodromic latency from the periphery (L,). Table 1 also shows the coefficients of correlation between L, and the other measured parameters, rL,. Again, in all but one case, the value ofrL, is not statistically significant. The correlation between L, and FMAX is highly significant. A plot of these data is shown in Fig. 3. We observed an inverse relationship between L, and FMAX. A comparable result for all cuneate neurons was shown by Blum et al. (2). Changes In Orthodromic
Latency with Intensity
The latency-intensity relation we examined evolved from the measurement originally described by Towe and Morse (12). In this measurement we plotted the latency of every spike after stimulation of the superficial radial nerve versus the relative intensity of that stimulus as indicated by the compound action potential recorded on the nerve. If a cell failed to respond to a stimulus trial, a symbol was placed at a latency of 0.0 ms.
0
91 0
0
8
20
40
60
80
100
F MRX [ HZ I FIG. 3. Orthodromic first spike latency versus maximum frequency following ability (FMAX) for cuneothalamic relay neurons. Stimulation was on superficial radial nerve at the elbow. (N = 29).
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Two distinct cell types within the cuneate nucleus have been defined by the form of the L-I relationship (11, 12). Cells with large latency changes, graded for a wide range of intensities, are termed “integrative,” and cells which, when brought into activity, display little or no change in latency with further increases in intensity are called “obligatory.” We require that the spike discharge pattern of obligatory cells remain unchanged with increasing intensity as well. The L-I relation provides an independent measure of synaptic properties for comparison with FMAX. Data typical of our experiments are shown in Fig. 4. For 19 cells, stimulus intensities near threshold were distributed finely enough that we could clearly distinguish integrative (13 cells) from obligatory (six cells) responses. The mean values of L, and MLLAT did not differ between the integrative and obligatory groups. Frequency
Following
Ability
In the present study, 50% of the CTR cells for which the FMAX value could be determined followed stimulus repetition rates of 100 Hz or greater, and 35% failed to respond to rates above 5 Hz. On the basis of prior work (2), and for convenience, cells were divided into two groups on the basis of their FMAX value: those with FMAX greater than 20 Hz, and those with FMAX less than 20 Hz. Table 2 shows the mean values of measured parameters for the two FMAX groups as well as the probability of significance of the measured difference in means based on the unpaired t-test. The high-frequency following group had significantly shorter values of orthodromic latency (L,). There may also have been some tendency for this group to have larger values of spikes per discharge as well. The two FMAX groups did not differ with respect to MLLAT, threshold (V,), or depth. We were able to clearly identify both FMAX and the character of the latency-intensity curve for 14 CTR cells. Six cells had obligatory L-I responses, and eight were integrative. All six of the obligatory cells had FMAX values greater than 20 Hz, and five of the integrative cells had values less than 20 Hz. The remaining three integrative cells had FMAX values greater than 20 Hz. The Fisher exact test for independence indicated that these differences were statistically significant (P < .05). Highfrequency following cells tended to have obligatory synaptic connections, and cells with low FMAX values were more likely to be integrative. DISCUSSION Our results support the concept that there are two components to the cuneothalamic projection in cat. As proposed by Hand and Van Winkle (6)
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0
u o,# ox +o500 o*o+o
n 0
3% 0 0 + +t 0 00 000 A Ad
OL-
.o
.2
.8 INPlJ?
1.0
MEis6ITY
FIG. 4. Latency-intensity plots for two cuneothalamic relay neurons. Latency of every spike recorded from the neuron versus normalized stimulus input intensity. Stimulation applied to the superficial radial nerve. The ordinal position of each spike in a response train is indicated by the symbols (A = first, q = second, 0 = third, + = fourth, 0 = fifth, n = sixth). A-obligatory response pattern, B-integrative response pattern.
198
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AND WHITEHORN TABLE
2
Comparison of Response Properties of Cuneothalamic Relay Cells Divided on the Basis of Maximum Frequency Following Ability (FMAX) FMAX 2 20 Hz (N = 21)
Lb (W MLLAT S/D
4.64 + 0.95
(ms)
vt (%)
Depth (pm)
1.54 k 0.31 3.57 2 1.9 36.2 k 21.0 1123 2 331
FMAX < 20 Hz (N = 11) 9.75 1.43 2.43 38.8 1364
k k zt 2 f
4.1 0.45 1.5 14.0 408
Statistical significance” P < 0.001 P > 0.10 0.05
< 0.10
P > 0.10 P > 0.10
a Statistical significance based on unpaired t-test for differences in means. b Abbreviations as in Table 1.
and by Blum et al. (2), one subset of projection neurons is characterized by a high degree of synaptic security, short latency of activation, and little convergence. The other subset displays low synaptic security, longer (and more variable) latencies, and considerable convergence. Three orthodromic activation parameters, FMAX, first-spike latency, and the latency-intensity curve, appear to be the most useful sorting criteria for identifying these subsets. It is, therefore, important to consider factors influencing those measures. Are FMAX Differences among Cells a Valid Measure? The use of FMAX values rests on the assumption that the range of values observed (1 to 100 Hz) actually reflects differences in synaptic properties. It can be suggested however that some (those associated with long latencies) or all of the low FMAX values are from cells whose major input is not the superficial radial nerve. The low FMAX value (and long latency) would then only reflect incomplete activation of effective input to the cell and not an intrinsic property of the conduction pathway and synaptic mechanisms. Because we did not stimulate other peripheral nerves we cannot directly discount this possibility. However, several lines of evidence argue against this suggestion. First, Blum et al. (2) found that many cells with low FMAX values were classified as “insensitive” when a search for a sensitive receptive field was carried out over the entire limb and body surface. If such cells had major input from other nerves the cells should have been activated during this procedure. Second, if the suggestion were true, one would expect that stimulation of the dorsal columns, which contain afferents from all peripheral nerves, would produce dramatically better frequency following compared to
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peripheral nerve stimulation. Although the frequency response of activity within the medial lemniscus is improved when stimulation is applied to the entire dorsal columns, activity still declines significantly with increasing frequency (14). Furthermore, Bromberg (personal communication) reports finding individual cuneate neurons that have low FMAX values upon stimulation of the dorsal columns. Mechanisms Rejected in FMAX and the Latency-Intensity Relationship. FMAX and the form of the L-I relationship are closely, although not absolutely, related in our results. Generally a high FMAX value was found in conjunction with an obligatory L-I relationship, but we did observe several neurons with high FMAX values and integrative L-I curves. This suggests that the two measures do not reflect the same aspects of synaptic connectivity. FMAX could be related to transmitter release, to a summation of inhibitory influences, or to polysynaptic connections to the neurons. The last explanation is not a complete answer because a series circuit involving high security synapses might still display high FMAX characteristics at the terminal neuron. Polysynaptic circuitry also cannot explain the low FMAX values for cells with short latencies (Fig. 3). Several factors could also influence the two forms of the L-I relationship. Towe and Morse (12) in their initial discussions of this measurement identified two important influences: the time constant of the postsynaptic membrane and the degree of dispersion of the arrival of the afferent volley. Generally, a short time constant and small degree of dispersion would be associated with an obligatory L-I relationship. We further suggest that the L-I relationship also reflects the degree of convergence upon the cell from primary afferent fibers. From this point of view, cells with an obligatory L-I relationship receive input from only a few afferent fibers. These would, in addition, be expected to be of similar size and to reside close to one another within the peripheral nerve so that they all become active at the same level of stimulation (1). An integrative L-I relationship would arise from the progressive activation of large numbers of afferent fibers, all making effective endings upon the cell under study, but differing in size or placement within the nerve. Lack of Correlation with Axonaf Size. Our data suggest that axonal size, as measured by antidromic medial lemniscus latency, is not a major organizing principle in the cuneothalamic projection system. We found only one, relatively minor, correlation between axonal size and any of the other measures examined. J&rig (personal communication) was also unable to correlate axonal size with cell type defined by natural stimulation. In contrast, axonal size may be important in the functional organization of the somatosensory thalamus (13).
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Comparison of Cell Groups Defined by FMAX and by Natural Stimulation. The three most obvious cell types defined from our results are
(i) high FMAX value, obligatory L-I curve; (ii) high FMAX value, integrative L-I curve; and (iii) low FMAX value, integrative L-I curve. Do these correspond to cell groups defined on the basis of natural stimuli? Cells with high FMAX values, short orthodromic latencies, and obligatory L-I relationships are likely to correspond to the P cell class described by Per1 et al. (9) and studied most recently by Jtinig et al. (8). The P cells are identified by their ability to follow high frequency vibration at the periphery, a test nearly identical to that used to determine FMAX values. The short orthodromic latency is consistent with an input from highly sensitive afferent fibers with rapid conduction both within the peripheral nerve and the dorsal columns (3, 7). We also can tentatively suggest that slowly adapting cuneate neurons (8, 9) may correspond to our high FMAX value, integrative L-I relation group, and rapidly adapting, hair-sensitive cells (8,9) could be the same as our low FMAX , integrative cells. Because second-order, slowly adapting neurons normally receive continuous impulse trains in the lo- to lOO-Hz range (4, 5) they would require high frequency following ability for relatively long periods of time. The rare occurrence of this cell type in our sample agrees with the small proportions (10 to 20%) found in previous samples based on natural stimulation (8, 9). Rapidly adapting cells need not have high FMAX capability. Because the natural input for this group consists of short bursts of afferent spikes at high frequency, a good transient response would be adequate. REFERENCES 1. BEMENT, S. L. 1977. Electrical and thermal sensitivity of peripheral nerve fibers. Sot. Neurosci.
Abstr.
3: 476.
2. BLUM, P., M. B. BROMBERG, AND D. WHITEHORN. 1975. Population analysis of single units in the cuneate nucleus of the cat. Exp. Neurol. 48: 57-78. 3. BROMBERG, M. B., AND D. WHITEHORN. 1974. Myelinated fiber types in the superficial radial nerve of the cat and their central projections. Brain Res. 78: 157- 163. 4. BURGESS, P. R., D. PETIT, AND R. M. WARREN. 1968. Receptor types in cat hairy skin supplied by myelinated fibers. J. Neurophysiol. 31: 833-848. 5. CHAMBERS, M. R., K. H. ANDRES, M. Y. v. DUBRING, AND A. IGGO. 1972. The structure and function of the slowly adapting type II mechanoreceptor in hairy skin. Q. J. Exp. Physiol. 57: 417-445. 6. HAND, P. J., AND T. VAN WINKLE. 1977. The efferent connections of the feline nucleus cuneatus. J. Camp. Neural. 171: 83-110. 7. HORCH, K. W., P. R. BURGESS, AND D. WHITEHORN. 1976. Ascending collaterals of cutaneous neurons in the fasciculus gracilis of the cat. Brain Res. 119: 1- 17.
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8. JKNIG, W., T. SCHOULTZ, AND W. A. SPENCER. 1977. Temporal and spatial parameters of excitation and afferent inhibition in cuneothalamic relay neurons. J. Neurophysiol. 40: 822-835. 9.
10. 11. 12. 13.
PERL, E. R., D. G. WHITLOCK, AND J. GENTRY. 1962. Cutaneous projections of second-order neurons of the dorsal column system, J. Neurophysiol. 25: 337-358. Tows, A. L. 1965. Neuronal popuiation analysis in the cerebral cortex. Pages 143- 156 in P. W. NYE, Ed., Proceedings, Symposium on Information Processing in Sight Sensory Systems. California Institute of Technology, Pasadena. TOWE, A. L. 1968. Neuronal population behavior in the somatosensory system. Pages 552-574 in D. R. KENSHALO, Ed., Skin Senses. C. C Thomas, Springfield, Ill. TOWE, A. L., AND R. W. MORSE. 1962. Dependence of the response characteristics of somatosensory neurons on the form of their afferent input. Exp. Neural. 6: 407-425. TSUMOTO, T. 1974. Characteristics of the thalamic ventrobasal relay neurons as a function of conduction velocities of medial lemniscal fibers. Exp. Brain Res. 21: 211-224.
14. WHITEHORN, D., J. P. WALSH, 0. LIMCACO, AND M. C. MACKEY. 1974. Frequency response properties of the dorsal column nuclei. The Physiologist 17: 358.
NEUROLOGY
64,190-201
(1979)
Frequency Following and Conduction Speed as Identifiers of Major Subsets among Cuneothalamic Neurons J. P. WALSH AND D. WHITEHORN' Department of Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, Vermont 05401 Received April 20, 1978; revision received November 3, 1978 Cuneothalamic relay (CTR) neurons display heterogeneity in terms of response properties and morphological characteristics. Subsystems have been proposed in which speed of conduction and afferent synaptic strength serve as distinguishing features. The present work examined the relevance of these two parameters to other commonly measured characteristics of CTR cells. Response properties of 63 CTR cells to electrical stimulation of the ipsilateral superficial radial nerve were studied using extracellular single-cell recording methods in chloralose-anesthetized cats. The stimulus was varied in amplitude and frequency. Conduction speed was inferred from antidromic latency to stimulating of the medial lemniscus just caudal to the thalamus, and from orthodromic first spike latency to superficial radial nerve stimulation. Afferent synaptic strength was estimated by determining maximum frequency following ability (FMAX). Other properties measured include number of spikes per discharge, threshold for electrical activation at the superficial radial nerve, depth of cell within the tissue, and a latency-intensity relation. Antidromic conduction time from the medial lemniscus had a significant inverse correlation only with cell depth. Orthodromic first spike latency was correlated (inversely) only with FMAX. The shape of the latency-intensity function was significantly linked to FMAX. Cells appeared to fall into three subsets tentatively identified with subsets proposed elsewhere on the basis of responses to natural stimulation.
INTRODUCTION The analysis of local circuit interactions within a nuclear complex requires the prior identifications of functional neuronal subsets. This Abbreviations: CTR-cuneothalamic relay, FMAX-maximum frequency following ability, L-I-latency-intensity, MLLAT-antidromic latency, L,-orthodromic latency. 1 This paper is based on part of a dissertation by Dr. Walsh in partial fulfillment of the requirements for the Ph.D. degree, who was supported by PHS Grant 5-TOl-GMOO439-14. This work was supported by grant NS09472 and Biomedical Research Support Grant S-SO7-RR05429 from the National Institutes of Health. We wish to thank Howard Lipson for help with computer programming. 190 0014-4886/79/040190-12$02.00/O Copyright 0 1979 by Academic Press, Inc. AU rights of reproduction in any form reserved.
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identification rests on the choice of sorting criteria to be used to carry out the classification. Ideally, one or a few properties are sought that predict clustering of other cellular characteristics. Towe (10, 11) described a population analysis approach that formalizes the search for classifying criteria. In that method a relatively large number of cellular properties are observed from each sampled neuron. The entire data matrix is then sorted and examined for covariance among the measured characteristics. This laboratory has previously used the population analysis approach (2) to show that intemeurons within the cuneate nucleus of cat comprise two groups, well defined by the maximum frequency of afferent stimulation which they can follow in a one-to-one fashion, the FMAX value. When cells are sorted in this manner, other properties (degree of convergence from peripheral and central sites, and the orthodromic latency of activation) can be predicted. Since cuneothalamic relay (CTR) neurons also vary among themselves in frequency following ability, Blum er al. (2) suggested that they also be divided into subsets on the basis of the FMAX value. Hand and Van Winkle (6) proposed a similar subclassification of the cuneothalamic projection system, citing anatomical, physiological, and phylogenetic evidence. They distinguished two systems by frequency following ability and speed of conduction: a fast, high-frequency following neolemniscal system, and a slow, low-frequency following paleolemniscal system. The present study tested the clustering of CTR properties caused by sorting cells on the basis of FMAX or conduction speed. Orthodromic latency from the periphery and antidromic latency from the medial lemniscus were used as the measures of conduction speed. Attempts at correlating other functional parameters with these measures revealed clustering around FMAX and orthodromic latency, but not around antidromic latency. METHODS Adult cats were anesthetized with a-choralose (50 mgkg, i.p., in propylene glycol, or i.v. saline under ether), paralyzed with gallamine triethiodide (Flaxedil), and artificially respired. Rectal temperature was monitored and maintained at 37.5”C. Carotid blood pressure was monitored. Single units were recorded extracellularly in the cuneate nucleus with 4.0 M NaCl-filled micropipets. Electrode tracks were 0 to 3 mm caudal to the obex. A bipolar stimulating electrode was placed on the ipsilateral superficial radial nerve in the region of the elbow. A bipolar recording
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electrode was positioned on the ipsilateral nerve in the region of the wrist. A concentric bipolar stimulating electrode was placed in the contralateral medial lemniscus just caudal to the ventral posterolateral nucleus of the thalamus (A6.0, L6.75). The final position of the electrode was chosen to maximize the size of the short-latency, high-frequency following potential evoked in the medial lemniscus by superficial radial nerve stimulation at the elbow. The search stimulus was an electrical pulse (50~ps duration) delivered through the electrode at the elbow. The intensity was such that all A-alpha and A-delta fibers were activated. Response properties were studied using an automated test sequence under control of an on-line PDP4/e computer. Fifty-microsecond stimulus pulses of varying voltage and frequency were applied to the superficial radial nerve at the elbow. The antidromic compound action potential at the wrist was digitized and integrated in real time to provide the principal measure of input intensity. Integrals included A-alpha and A-delta activity but not dorsal root or dorsal column reflex activity. Reflex activity was identified by its inability to follow stimulus frequencies of more than 5 Hz. The latencies of all spikes occuring between stimuli were measured and stored with the corresponding integral of the antidromic compound action potential at the wrist. CTR cells were identified by their ability to follow greater than 200-Hz stimulation from the medial lemniscus stimulation site on a one-for-one basis with short (~3 ms), invariant latency. Data were analyzed off-line by computer programs that calculated statistics for each cell and accumulated these statistics for the entire cell sample. The input intensity integral for each trial was normalized to the maximum integral observed among all the trials on that cell at the same stimulus frequency. This made possible comparisons between cells recorded in different preparations. RESULTS A total of 176 neurons was studied in 17 experiments. We identified 63 cells as CTR cells. Because our primary interest is in the relationship of conduction speed and frequency following ability to CTR cell function, results are presented in two sections in which the relationships between these parameters, and other functional parameters are separately described. Conduction
Speed
Antidromic Latency. CTR conduction speed was studied indirectly by measuring the antidromic latency (MLLAT) from the stimulation site at the
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thalamic end of the medial lemniscus. Figure 1 shows the distribution of MLLAT for 61 CTR neurons recorded in our experiments. Values were 0.9 to 2.1 ms with a mean of 1.42 ms. The corresponding conduction velocity range can be estimated by calculating the conduction distance using average sterotaxic coordinates of the stimulus and recording sites. If a straight-line conduction path is assumed the distance is 22 mm. With a 10% addition for fiber bending, the conduction velocity range obtained is 11 to 27 m/s. A series of basic parameters was obtained from each CTR cell. In addition to antidromic latency (MLLAT), these included latency of the tist spike evoked by stimulation of the superficial radial nerve (L,), threshold for activation by the nerve electrode (V,, measured as a percentage of maximum input intensity), number of spikes evoked per discharge in response to stimulation (S/D), depth of the cell in the tissue (as read from the microdrive), maximum frequency of nerve stimulation which the cell could follow on a one-for-one basis (FMAX), and a latency-intensity (L-I) relation. Both L, and S/D were determined with supramaximal stimulation. Not all parameters could be measured on every cell isolated. Table 1 contains a summary of the statistics for the parameters obtained from our sample. The distributions of L, and MLLAT resemble those reported previously (2,8). The coefficients of correlation between MLLAT and the other parameters, rML, are also contained in Table 1. With one exception, the parameters we examined did not correlate significantly with MLLAT. The correlation between MLLAT and depth of isolation is highly significant.
I-
I
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.O
:
.5 1:O 1.5 ANTIORONIC ML LRTENCY
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2:0 [NSEC
)
2.5 I
FIG. 1. Distribution of antidromic conduction latency for cuneothalamic relay neurons recorded in the cuneate nucleus. Stimulation was applied to the medial lemniscus approximately 24 mm rostra1 to the recording site. (N = 61; bin size = 0.1 ms).
194
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AND WHITEHORN TABLE
1
Statistics for Functional Parameters of Cuneothalamic Relay Cells mean + SD
Lib 6-M MLLAT (ms) S/D Depth (pm) FMAX (Hz) v, 6%
5.90 1.42 2.% 1331 58.6 35.1
Range
+ 3.05 f 0.32 + 1.72 -e 513 2 46.0 ” 18.2
rL, 0’)
3.1-18.8 0.9-2.1 1.0-8.3 350-3000 O-100 20-98
0.12 (42) -0.043(49) -0.377(60)* 0.087(29) 0.047(35)
0.12 (42) -0.023(42) 0.169(41) -0.622(29)* 0.188(35)
0 rML and r,,, are correlation coefficients between the indicated parameter and MLLAT and L,, respectively. Number of data pairs is in parentheses. P > 0.10 unless otherwise noted. * Abbreviations in Tables 1 and 2: L,-latency of first orthodromic spike, MLLAT-antidromic latency, S/D-spikes per discharge from nerve stimulation, FMAX-maximum frequency following ability, Vi-threshold. * Highly significant correlation (P < 0.001).
A plot of the antidromic latencies as a function of depth is shown in Fig. 2. The antidromic latencies varied inversely with depth; deeper cells tended to have shorter values. The slope- of the regression curve was 0.23 s/m. Cells deeper in the nucleus lie physically closer to the thalamus than more superficial cells. Therefore, if the decline in antidromic latency with increasing depth were due solely to decreasing conduction distance, then the reciprocal of this slope would represent the average conduction
0
I - 0.
600.
I
I
1600. 2L)oo. DEPTH (MICRONS1
I
3200.
FIG. 2. Antidromic medial Iemniscus latency versus depth of isolation for cuneothalamic relay neurons. Depth is distance below the dorsal surface of the medulla as indicated by the electrode microdrive. (N = 60).
CUNEOTHALAMIC
RELAY
195
NEURONS
velocity of the cell sample. However, the velocity calculated in this way, 5 m/s, is much smaller than the range of velocities we observed. More likely, cells situated deeper in the nucleus have higher conduction velocities. Orthodromic Latency. Conduction speed in the dorsal column-medial lemniscal system is more frequently inferred from orthodromic latency from the periphery (L,). Table 1 also shows the coefficients of correlation between L, and the other measured parameters, rL,. Again, in all but one case, the value ofrL, is not statistically significant. The correlation between L, and FMAX is highly significant. A plot of these data is shown in Fig. 3. We observed an inverse relationship between L, and FMAX. A comparable result for all cuneate neurons was shown by Blum et al. (2). Changes In Orthodromic
Latency with Intensity
The latency-intensity relation we examined evolved from the measurement originally described by Towe and Morse (12). In this measurement we plotted the latency of every spike after stimulation of the superficial radial nerve versus the relative intensity of that stimulus as indicated by the compound action potential recorded on the nerve. If a cell failed to respond to a stimulus trial, a symbol was placed at a latency of 0.0 ms.
0
91 0
0
8
20
40
60
80
100
F MRX [ HZ I FIG. 3. Orthodromic first spike latency versus maximum frequency following ability (FMAX) for cuneothalamic relay neurons. Stimulation was on superficial radial nerve at the elbow. (N = 29).
196
WALSH
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WHITEHORN
Two distinct cell types within the cuneate nucleus have been defined by the form of the L-I relationship (11, 12). Cells with large latency changes, graded for a wide range of intensities, are termed “integrative,” and cells which, when brought into activity, display little or no change in latency with further increases in intensity are called “obligatory.” We require that the spike discharge pattern of obligatory cells remain unchanged with increasing intensity as well. The L-I relation provides an independent measure of synaptic properties for comparison with FMAX. Data typical of our experiments are shown in Fig. 4. For 19 cells, stimulus intensities near threshold were distributed finely enough that we could clearly distinguish integrative (13 cells) from obligatory (six cells) responses. The mean values of L, and MLLAT did not differ between the integrative and obligatory groups. Frequency
Following
Ability
In the present study, 50% of the CTR cells for which the FMAX value could be determined followed stimulus repetition rates of 100 Hz or greater, and 35% failed to respond to rates above 5 Hz. On the basis of prior work (2), and for convenience, cells were divided into two groups on the basis of their FMAX value: those with FMAX greater than 20 Hz, and those with FMAX less than 20 Hz. Table 2 shows the mean values of measured parameters for the two FMAX groups as well as the probability of significance of the measured difference in means based on the unpaired t-test. The high-frequency following group had significantly shorter values of orthodromic latency (L,). There may also have been some tendency for this group to have larger values of spikes per discharge as well. The two FMAX groups did not differ with respect to MLLAT, threshold (V,), or depth. We were able to clearly identify both FMAX and the character of the latency-intensity curve for 14 CTR cells. Six cells had obligatory L-I responses, and eight were integrative. All six of the obligatory cells had FMAX values greater than 20 Hz, and five of the integrative cells had values less than 20 Hz. The remaining three integrative cells had FMAX values greater than 20 Hz. The Fisher exact test for independence indicated that these differences were statistically significant (P < .05). Highfrequency following cells tended to have obligatory synaptic connections, and cells with low FMAX values were more likely to be integrative. DISCUSSION Our results support the concept that there are two components to the cuneothalamic projection in cat. As proposed by Hand and Van Winkle (6)
CUNEOTHALAMIC
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RELAY NEURONS
0
u o,# ox +o500 o*o+o
n 0
3% 0 0 + +t 0 00 000 A Ad
OL-
.o
.2
.8 INPlJ?
1.0
MEis6ITY
FIG. 4. Latency-intensity plots for two cuneothalamic relay neurons. Latency of every spike recorded from the neuron versus normalized stimulus input intensity. Stimulation applied to the superficial radial nerve. The ordinal position of each spike in a response train is indicated by the symbols (A = first, q = second, 0 = third, + = fourth, 0 = fifth, n = sixth). A-obligatory response pattern, B-integrative response pattern.
198
WALSH
AND WHITEHORN TABLE
2
Comparison of Response Properties of Cuneothalamic Relay Cells Divided on the Basis of Maximum Frequency Following Ability (FMAX) FMAX 2 20 Hz (N = 21)
Lb (W MLLAT S/D
4.64 + 0.95
(ms)
vt (%)
Depth (pm)
1.54 k 0.31 3.57 2 1.9 36.2 k 21.0 1123 2 331
FMAX < 20 Hz (N = 11) 9.75 1.43 2.43 38.8 1364
k k zt 2 f
4.1 0.45 1.5 14.0 408
Statistical significance” P < 0.001 P > 0.10 0.05
< 0.10
P > 0.10 P > 0.10
a Statistical significance based on unpaired t-test for differences in means. b Abbreviations as in Table 1.
and by Blum et al. (2), one subset of projection neurons is characterized by a high degree of synaptic security, short latency of activation, and little convergence. The other subset displays low synaptic security, longer (and more variable) latencies, and considerable convergence. Three orthodromic activation parameters, FMAX, first-spike latency, and the latency-intensity curve, appear to be the most useful sorting criteria for identifying these subsets. It is, therefore, important to consider factors influencing those measures. Are FMAX Differences among Cells a Valid Measure? The use of FMAX values rests on the assumption that the range of values observed (1 to 100 Hz) actually reflects differences in synaptic properties. It can be suggested however that some (those associated with long latencies) or all of the low FMAX values are from cells whose major input is not the superficial radial nerve. The low FMAX value (and long latency) would then only reflect incomplete activation of effective input to the cell and not an intrinsic property of the conduction pathway and synaptic mechanisms. Because we did not stimulate other peripheral nerves we cannot directly discount this possibility. However, several lines of evidence argue against this suggestion. First, Blum et al. (2) found that many cells with low FMAX values were classified as “insensitive” when a search for a sensitive receptive field was carried out over the entire limb and body surface. If such cells had major input from other nerves the cells should have been activated during this procedure. Second, if the suggestion were true, one would expect that stimulation of the dorsal columns, which contain afferents from all peripheral nerves, would produce dramatically better frequency following compared to
CUNEOTHALAMIC
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199
peripheral nerve stimulation. Although the frequency response of activity within the medial lemniscus is improved when stimulation is applied to the entire dorsal columns, activity still declines significantly with increasing frequency (14). Furthermore, Bromberg (personal communication) reports finding individual cuneate neurons that have low FMAX values upon stimulation of the dorsal columns. Mechanisms Rejected in FMAX and the Latency-Intensity Relationship. FMAX and the form of the L-I relationship are closely, although not absolutely, related in our results. Generally a high FMAX value was found in conjunction with an obligatory L-I relationship, but we did observe several neurons with high FMAX values and integrative L-I curves. This suggests that the two measures do not reflect the same aspects of synaptic connectivity. FMAX could be related to transmitter release, to a summation of inhibitory influences, or to polysynaptic connections to the neurons. The last explanation is not a complete answer because a series circuit involving high security synapses might still display high FMAX characteristics at the terminal neuron. Polysynaptic circuitry also cannot explain the low FMAX values for cells with short latencies (Fig. 3). Several factors could also influence the two forms of the L-I relationship. Towe and Morse (12) in their initial discussions of this measurement identified two important influences: the time constant of the postsynaptic membrane and the degree of dispersion of the arrival of the afferent volley. Generally, a short time constant and small degree of dispersion would be associated with an obligatory L-I relationship. We further suggest that the L-I relationship also reflects the degree of convergence upon the cell from primary afferent fibers. From this point of view, cells with an obligatory L-I relationship receive input from only a few afferent fibers. These would, in addition, be expected to be of similar size and to reside close to one another within the peripheral nerve so that they all become active at the same level of stimulation (1). An integrative L-I relationship would arise from the progressive activation of large numbers of afferent fibers, all making effective endings upon the cell under study, but differing in size or placement within the nerve. Lack of Correlation with Axonaf Size. Our data suggest that axonal size, as measured by antidromic medial lemniscus latency, is not a major organizing principle in the cuneothalamic projection system. We found only one, relatively minor, correlation between axonal size and any of the other measures examined. J&rig (personal communication) was also unable to correlate axonal size with cell type defined by natural stimulation. In contrast, axonal size may be important in the functional organization of the somatosensory thalamus (13).
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Comparison of Cell Groups Defined by FMAX and by Natural Stimulation. The three most obvious cell types defined from our results are
(i) high FMAX value, obligatory L-I curve; (ii) high FMAX value, integrative L-I curve; and (iii) low FMAX value, integrative L-I curve. Do these correspond to cell groups defined on the basis of natural stimuli? Cells with high FMAX values, short orthodromic latencies, and obligatory L-I relationships are likely to correspond to the P cell class described by Per1 et al. (9) and studied most recently by Jtinig et al. (8). The P cells are identified by their ability to follow high frequency vibration at the periphery, a test nearly identical to that used to determine FMAX values. The short orthodromic latency is consistent with an input from highly sensitive afferent fibers with rapid conduction both within the peripheral nerve and the dorsal columns (3, 7). We also can tentatively suggest that slowly adapting cuneate neurons (8, 9) may correspond to our high FMAX value, integrative L-I relation group, and rapidly adapting, hair-sensitive cells (8,9) could be the same as our low FMAX , integrative cells. Because second-order, slowly adapting neurons normally receive continuous impulse trains in the lo- to lOO-Hz range (4, 5) they would require high frequency following ability for relatively long periods of time. The rare occurrence of this cell type in our sample agrees with the small proportions (10 to 20%) found in previous samples based on natural stimulation (8, 9). Rapidly adapting cells need not have high FMAX capability. Because the natural input for this group consists of short bursts of afferent spikes at high frequency, a good transient response would be adequate. REFERENCES 1. BEMENT, S. L. 1977. Electrical and thermal sensitivity of peripheral nerve fibers. Sot. Neurosci.
Abstr.
3: 476.
2. BLUM, P., M. B. BROMBERG, AND D. WHITEHORN. 1975. Population analysis of single units in the cuneate nucleus of the cat. Exp. Neurol. 48: 57-78. 3. BROMBERG, M. B., AND D. WHITEHORN. 1974. Myelinated fiber types in the superficial radial nerve of the cat and their central projections. Brain Res. 78: 157- 163. 4. BURGESS, P. R., D. PETIT, AND R. M. WARREN. 1968. Receptor types in cat hairy skin supplied by myelinated fibers. J. Neurophysiol. 31: 833-848. 5. CHAMBERS, M. R., K. H. ANDRES, M. Y. v. DUBRING, AND A. IGGO. 1972. The structure and function of the slowly adapting type II mechanoreceptor in hairy skin. Q. J. Exp. Physiol. 57: 417-445. 6. HAND, P. J., AND T. VAN WINKLE. 1977. The efferent connections of the feline nucleus cuneatus. J. Camp. Neural. 171: 83-110. 7. HORCH, K. W., P. R. BURGESS, AND D. WHITEHORN. 1976. Ascending collaterals of cutaneous neurons in the fasciculus gracilis of the cat. Brain Res. 119: 1- 17.
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8. JKNIG, W., T. SCHOULTZ, AND W. A. SPENCER. 1977. Temporal and spatial parameters of excitation and afferent inhibition in cuneothalamic relay neurons. J. Neurophysiol. 40: 822-835. 9.
10. 11. 12. 13.
PERL, E. R., D. G. WHITLOCK, AND J. GENTRY. 1962. Cutaneous projections of second-order neurons of the dorsal column system, J. Neurophysiol. 25: 337-358. Tows, A. L. 1965. Neuronal popuiation analysis in the cerebral cortex. Pages 143- 156 in P. W. NYE, Ed., Proceedings, Symposium on Information Processing in Sight Sensory Systems. California Institute of Technology, Pasadena. TOWE, A. L. 1968. Neuronal population behavior in the somatosensory system. Pages 552-574 in D. R. KENSHALO, Ed., Skin Senses. C. C Thomas, Springfield, Ill. TOWE, A. L., AND R. W. MORSE. 1962. Dependence of the response characteristics of somatosensory neurons on the form of their afferent input. Exp. Neural. 6: 407-425. TSUMOTO, T. 1974. Characteristics of the thalamic ventrobasal relay neurons as a function of conduction velocities of medial lemniscal fibers. Exp. Brain Res. 21: 211-224.
14. WHITEHORN, D., J. P. WALSH, 0. LIMCACO, AND M. C. MACKEY. 1974. Frequency response properties of the dorsal column nuclei. The Physiologist 17: 358.