326

Brain Research, I 17 (1976) 326-330 ~i) Elsevier/North-HollandBiomedical Press, Amsterdam Printedin The Netherlands

Burst and doublet firing modes within spinal cord dorsal horn cells of the chicken (Gallus domesficus) JAMES A. HOLLOWAY, LOUIS E. WRIGHT and C. OVID TROUTH Department o[Physiology and Biophysics, College of Medicine, Howard University, Washington, D.C. 20059 (U.S.A.)

(Accepted August 13th, 1976)

In recent years, a vast quantity of information has been gathered concerning afferent input and integration in dorsal horn cells (DHC) 2,9,11,15,Is,Is. Most of this work has been done on cats and monkeys and in many respects, similar results have been obtained. In contrast to the vast quantity of information regarding the electrophysiological properties of spinal cord DHC in mammals, little or no equivalent data have been reported in birds. In these studies, the firing modes of some spinal cord DHC of the chicken have been examined extracellularly, focusing upon both the spontaneous firing modes and modification of these modes by natural stimuli. In 25 chickens lightly anesthetized with sodium pentobarbital (30 mg/kg i.m.), paralyzed with gallamine triethiodide and unidirectionally ventilated a, extracellular microelectrode recordings were obtained. This study represents a sample of 50 dorsal horn neurons. Glass capillary microelectrodes filled with a saturated solution of fast green dye in 3 M KCl were used for recording unit activity. Recordings were obtained 1° from cells in laminae IV-V| of the spinal cord gray matter 1, ipsilaterally throughout cord segments 21-28 (ref. 12). The sensory inputs of these neurons were determined by natural (adequate) stimuli applied to the ipsilateral hindlimb, body wall and the comb, with intact innervation. Following the analysis of the receptor inputs, the recording site was marked with dye by passing current through the recording electrode, utilizing a microiontophoresis programmer (Model 160, W.P. instruments, Hamden, Conn.). After marking, the spinal cord was blocked and fixed with l0 ~ formalin and 30 #m frozen transverse sections were made. Once a unit was isolated, the criteria used for identification of DHC were as follows: (a) in many cases, the presence of spontaneous discharge; (b) the ability to respond to several modalities of stimulation, i.e., light touch, feather manipulations, noxious pinch (which consisted of attaching a metal clip with a calibrated force of 600 g to the skin), joint manipulations, moderate to heavy pressure, and the application of ethyl chloride spray (cold) over the receptive field; (c) measurement of depth of recording from cord dorsum by means of microdrive micrometer readings; and (d) histological verification of electrode in gray matter rather than in white matter. These criteria were used since the presence of spontaneous

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100 MSEC Fig. 1. Alterations in firing modes with receptive field stimulation of chicken dorsal horn neuron C310-20. Record A represents spontaneous activity. Evoked activity is shown in records B-D. Reading from left to right, the inflection indicated by stimulus marker (bottom line of each trace) represents the approximate time stimulus was applied. The deflection (down) of the stimulus marker represents stimulus removal. The cell fired in very stereotyped triplets and some doublets; as light touch was applied to the receptive field, the firing mode changed: B, an increase in the number of spikes/burst; C, an increase in single spikes and an occasional broad doublet; D, a predominance of doublets with corresponding decrease in triplets. The changes in spike frequency seen here are characteristic of most dorsal horn neurons studied as the average firing rate increased.

discharge alone is not necessarily a differentiating characteristic of postsynaptic neuronal discharge 17. The majority of units encountered in these studies exhibited stereotyped doublet or triplet burst firing modes. The neuronal response illustrated in Fig. 1 is typical of that demonstrated by most cells that responded to adequate stimulation. Spontaneous activity is represented in Fig. 1A. Records B - D represent light touch of receptive field of the ipsilateral proximal thigh. Similar responses were obtained by feather movement over various angles or pressure by means of a blunt ( 1 m m dia. tip) glass probe on the ipsilateral thigh skin or body wall. Twelve per cent of the units studied responded in this fashion to noxious pinch of the ipsilateral foot pad. A m o n g these 1 2 ~ , 8 ~ responded also to the cooling effect of ethyl chloride spray. The interspike interval was 2 fi- 1 msec in a given cell. The triplet mode occurred most frequently, with an interburst interval of 60-70 -4- 2 msec. The less frequently occurring doublets almost always generated the following pattern: two pairs with interburst intervals of 20-30 q- 1 msec, followed by another doublet or pair of doublets

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100 M S E C Fig. 2. Effects of inhibitory stimulus of receptive field upon spontaneous firing pattern of celt C310-46. This cell characteristically fared in 2 msec triplets, as revealed in spontaneous activity preceding stimulus application, indicated by inflection of time marker in (A). The inhibition, occurring at 0.61 sec after stimulus application, was caused by a metal clip, calibrated at 600 g, attached to the distal portion of the comb. This stimulus was subjectivelyjudged as noxious by comparison of its effect on forearm skin of the experimenter. There was a considerable range in both duration of stimulus application prior to onset of inhibition, and duration of inhibition (see text). Records A and B represent continuous kymographrecordings and show (B) the pattern of spontaneous activity upon reappearance. Indeed, as the spontaneous activity reappears, the doublet pattern of varying intervals predominate. with intervals of 50-55 ± 2 msec; a few extend to 60 msec. When the D H C were synaptically driven by mechanical or thermal stimuli, three types of responses generally occurred: (1) the firing rate increased and the proportion of spikes occurring within some bursts (the "burst index")a, 7 increased. This mode of response is illustrated in Fig. 1B, where the common triplet-doublet mode is interrupted by an occasional quadruplet; (2) the firing rate increased but the burst mode or pattern was disrupted. Fig. 1C illustrates this pattern of response; (3) there was an increase in the frequency of doublets with a corresponding decrease in the frequency of triplets, as is illustrated in Fig. 1D. When a noxious stimulus was applied to the comb during the spontaneous activity of many units or, in some cases during evoked activity, these units were totally inhibited. The noxious stimulus was most effective when applied to the distal portion of the comb. Moderate to heavy pressure on the proximal, but not on the distal comb, also produced inhibition. Often this induced inhibition persisted 3-7 sec after the stimulus had been removed. Fig. 2 is a continuous kymograph recording of a spontaneously firing D H C that is inhibited by a noxious stimulus on the comb. The duration of stimulus application before the onset of inhibition was 0.56-0.69 sec with a mean of 0.66 sec; n ~ 25. However, in many instances there appeared to be little or no meaningful correlation between stimulus duration and duration of inhibition. Few (3) units were excited rather than inhibited by noxious pinch of the comb. One of the more pronounced features during maximal excitation was the increase in the number of isolated spikes. The burst index of triplets fell from a control (spontaneous activity) of 80 ~ to as low as 11 ~ at the time of maximal firing rates. It is also apparent in Fig. 1C that the doublets that occur during maximal firing rate are broader during this period. A frequently occurring phenomenon is that following the period of inhibition

329 caused by noxious stimuli of the comb, the reappearance of spontaneous activity is predominantly in the form of doublets of varying interburst intervals (Fig. 2B). In this figure, it is clear that the predominant pattern preceding the stimulus is of the triplet type. Almost all of the cells encountered throughout the various laminae (IV-VI) responded to polymodal input. Only on rare occasions did responses to unimodal input seem apparent. Previous studies have demonstrated the occurrence of burst firing patterns of neurons in various parts of the central nervous system (CNS) of mammals4-7 ,s, 13,14,19. Bursts are a common feature of many cortical neurons6. Calvin et al.e have shown that in neurons of the dorsal column nuclei in cat and man, natural stimuli evoked an increase in the number of isolated spikes with a dramatic fall in burst index at the time of the maximal firing rates (from a control of 93 % to 40 ~o) and that the interspike intervals of doublets which did occur were wider during this sequence. Somewhat similar results were observed in these studies (Fig. 1C) where the burst index oftriplets fell from a control of 88 % to 11%, with a predominance of doublets with broader interspike intervals. The results in these studies, however, show a dramatic rise in the doublet burst index. It was of interest that the afferent drive for most of the DHC studied came largely from the more phasic feather and touch receptors rather from those for deep proprioception; the phasic inputs, however, are more difficult to grade in a steady manner. This phasic responsiveness appears also from neurons in the main cuneate nuclei of mammals8. The fact that little physiological distinction could be made among the various laminae of the dorsal horn was of particular interest. If Wall's TM warning that the laminae should be regarded as zones of concentration rather than as absolute separate laminae of distinct specialization is heeded, then our data might support his observation; however, even these "zones of concentration" seem to be even more hazy in the chicken spinal cord. It may be that the operation of the nervous system, in addition to its patterns of cellular connections, depends upon its unique cell-firing rhythm; for it is not the location of cells that matter, but rather the rhythm at which they fire. Any given cell is important insofar as it contributes to the average behavior of a population of cells spread throughout the CNS. This burst firing mode may, in fact, represent a unique method of coding in the CNS. Supported by Grant NIGMS 1 TO 2 GM 05010-01 MARC. We are grateful to Ms. V. M. Boyd for typing the manuscript.

1 Brinkman, E. and Martin, A. H., A cytoarchitectonic study of the spinal cord of the domestic

fowl Gallus domesticus. I. Brachialregion, Brain Research, 56 (1975) 43-62. 2 Brown, P. B., Response of cat dorsal horn cells to variations of intensity, location and area of

cutaneous stimuli,Exp. Neurol., (1969) 249-265.

330 3 Burger, R. E. and Lorenz, F. W. Artificial respiration in birds by unidirectional air flow, Poultry Sci., 39 (1960) 236-237. 4 Calvin, W. H., Sypert, G. W. and Ward, A. A., Jr., Structured timing patterns within bursts from epileptic neurons in undrugged monkey cortex, Exp. Neurol., 21 (1968) 535-549. 5 Calvin, W. H., Ojemann, G. A. and Ward, A. A., Human cortical neurons in epileptogenic foci: comparison of interictal firing patterns to those of "epileptic" neurons in animals, Electroenceph. clin. Neurophysiol., 34 (1973) 337-351. 6 Calvin, W. H. and Loeser, J. D., Doublet and burst firing patterns within the dorsal column nuclei of cat and man, Exp. Neurol., 48 (1975) 406-426. 7 Fetz, E. E. and Wyler, A. R., Operantly conditioned firing patterns of epileptic neurons in the monkey motor cortex, Exp. Neurol., 40 (1973) 586-607. 8 Galindo, A., Krnjevic, K. and Schwartz, S., Patterns of firing in cuneate neurons and some effects of Flaxedil, Exp. Brain Res., 5 (1968) 87-t01. 9 Heavner, J. E. and DeJong, R. H., Spinal cord neuron response to natural stimuli. A microelectrode study, Exp. NeuroL, 39 (1973) 293-306. 10 Holloway, J. A. and Wright, L. E., Effects of peripheral afferent stimulation on dorsal horn neurons in the chicken, (Gallus domesticus), Exp. NeuroL, 49 (1975) 863-866. 11 Hongo, T. E. Janowska, E. and Lundberg, A., Convergence of excitatory and inhibitory action on interneurones in the lumbosacral cord, Exp. Brain Res., 1 (1966) 338-358. 12 Huber, J. F., Nerve roots and nuclear groups in the spinal cord of the pigeon, J. comp. Neurol., 65 (1936) 43-91. 13 Kandel, E. R. and Spencer, W. A., Electrophysiology of hippocampal neurons. II. After potentials and repetitive firing, J. Neurophysiol., 24 (1961) 243-259. 14 Kjerulf, T. D., O'Neal, J. T., Calvin, W. H., Loeser, J. D. and Westrum, L. E., Deafferentation effects in lateral cuneate nucleus of the cat: correlation of structural alterations with firing pattern changes, Exp. Neurol., 39 (1973) 86-102. 15 Price, D. D. and Wagman, I. H., Physiological roles of A and C fiber inputs to the spinal dorsal horn of Maeaca mulatta, Exp. Neurol., 29 (1970) 383-399. 16 Wagman, 1. H. and Price, D. D., Responses of dorsal horn cells of M. mulatta to cutaneous and sural nerve A and C fiber stimuli, J. NeurophysioL, 32 (1969) 803-817. 17 Wall, P. D., Repetitive discharge of neurons, J. NeurophysioL, 22 (1959) 305-320. 18 Wall, P. D., The laminar organization of dorsal horn and effects of descending impulses, J. Physiol. (Lond.), 188 (1967) 403-423. 19 Wyler, A. R., Fetz, E. E. and Ward, A. A., Jr., Spontaneous firing patterns of epileptic neurons in the monkey motor cortex, Exp. NeuroL, 40 (1973) 567-588.