A CLAWEHCATEGN Q“? LONG-REM MAE-EWANEED NEfli‘fQAL ACTEWW' EN PQWEQE‘fiS QF THE SHEEP MfiéfiififiLEJI-s Thalia fico fire Dag?“ a? M. S. liflCfilGAN STEEE “WWW? John R. fiaigh‘a 5.968 "“ ‘ . “4:13;. Sign” '{ ~.'x'.-_a.* __ _ _> v _ ~12 LIBRAR; i mes‘s I‘Aichigan S in. J: ' A! University £m" v :1 I-- ——.—— gig. I . ABSTRACT A CLASSIFICATION 01' ION-MM MAINTAINED NEURAL.ACTIVITY IN PORTIONS OF THE SHEEP MEDULLA By John.R. Height A sampling of 57 single neural units exhibiting maintained firing properties was Obtained from 8 sheep. The sampling site was the caudal portion of the medulla which was chosen because of the rich variety of neuronal types present within a limited area. Some of the units ob- tained were drivable when appropriate mechanical stimuli were applied to the body surface. ‘Hos. had no Obvious driving stimulus. Consideration of the firing rates, averaged over 10 second inter- vals, gave rise to six distinct types of unit. These types are best characterized by the following description of their firing patterns: 1) regular and smooth; 2) regular but wobbly; 3) irregular, undulating; h) irregular, stepping; 5) irregular, erratic; and 6) irregular, decre- mental. Each type of unit described was distributed throughout all anatomical areas of the medulla that were considered. Interspike interval histograms computed from the same data also yielded six distinct types of interval distribution. Four of these were uninodal and were characterized by: 1) symmetric peak with "pre- modal" or fast intervals; 2) symmetric peak with little or no "pro-modal" activity; 3) asymmetric peak with "exponentially" decreasing tail; h) asymmetric peak with "ramp-like" tail. Two distributions were multi- modal having dominant features: 5) one symmetric major peak with one John R. Height or more minor peaks in the histogram tail and 6) two peaks, one symmetric and one asymmetric or both symmetric, all with short tails. These types were all found distributed throughout each discrete anatomical area that could be defined either histologically or electrophysiologically. Certain correlations of interval distribution type and anatomical location were noted. 'Units found in the afferent dorsal fiber tracts tend to produce histograms of the symmetric type with emphasis upon multiperiodicity in the tail. Pre-modal activity was common. Ordinary symmetric distributions - those without pre-modal activity or without multimodal tails - were missing altogether. Units in the somatic-sensory nuclei produce the ordinary symmetric interval distributions. The multiperiodic tailed variety were not found in this area. unit firing pattern types were evenly distributed in all other medullary-areas. It was noted that the type of interspike interval distribution produced remained constant regardless of the length of time sampled. Each maintained neuron appears committed to fire in an established pattern. units with.higher overall mean firing rates tend to produce more multi- periodic interval distributions. Units of slow to moderate firing rate generate symmetric histograms while units firing at extremely slow rates (less than 2 or 3 spikes per second) produce more of the asymmetric variety of interval distribution. A CIASSIFICATION 0F LONG-TERM.MAINTAINED NEURAI.ACTIVITY IN PORTIONS OF THE SHEEP MEDULLA A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biophysics 1968 ". . . . our theories should be based on the actual biological evidence at hand, so as to prepare the ground for the next step in experimentation, and not primarily on physical analogies." Ragnar Granit Receptors and Sensory Perception Chapter’B. 11 ACKNOWLEDGEMENTS I wish to thank Dr. J.I. Johnson, Jr., the laboratory director and chairman of this thesis comittee, both for his guidance and forbearance during the sometimes erratic course of this research. This research was supported by NIH grants GM 10890 and KB 05982 together with NASA contract NSG ,+75. 111 MILE 0F COMES LIST OF WWQOOOOOOOO.IOQOOOOOOOOOOQe00.0.0000...0.00.00.00.00.0 v LET 0F “MCCOOOOOOOOO.0.0...O...OOOOOOOOOOOOOOOOOOOOO00.0.... V1 Introduction..... ............. ........... ....... 1 Methods. 7 The SUchcts.......................................... Electrodes............................................ Recording and Processing Equipment.................... Histology and Track Localization...................... 00-4-44 ResultBOOIOO....00...OOOOOOOOOOOIOOOOO...0.00.00.00.00...0.0.0.... 9 munit PopflatimOOOO0....O.0OOOOOOOOOCCOOOCICOOOOOI 9 MAv.rg° Firm Bate Hof11880000000000000000...... 9 The hterspm. mamaHistmm...OOOOOOOOOOOOOOOOO1'1 matOIWOOOCOOOOOO.0.00.00.00.00.OOOOOOOOOOCOOO00.... l5 Discus81m000000000OOOOOOOOOOOOOOOOOOOOOOO.OOOOOOOIOOOOOOIOOOOOOOO l7 BiblimamOOO00.00.000.000.0.0.0.000...00.0.0.000000000000000000$ Appendix A: Abbreviations to be used in the following appendices. 33 An explanation of the unit identification system u8.d 111mg reseuchOOOOIO00.000.00.00...0.0.0.0.... 33 AppendixB: ACatalogue oftheUnits UsedinThis Study..........35 Appendix C: Tracims of the Electrode itacks.....................#0 AppendixD: TheAverage FiringRate Profiles.....................ll? AppendixE: Thelnterspike Interval Histograms...................66 Appendix]: Wmtails ornethoda...........................83 Appendix G: The Recipe for Dial-Urethane......................... 93 iv Table l. 2. h. LIST OF TABLES Page Maintained Unit Activity in the Central Nervous System: A Comparison of Various Regions and Animals................. 2 The Distribution of Interspike Interval Histogram 'hrpes Found in Various Medullary Regions as Determined H18tol%icdm0000000000000.0.0.0...OOOOOOOOOOOOOOOOOOOOOOOO20 The Distribution of Average Firing Rate Profile Categories Found in Various Medullary Regions as Determined HiatOlgicWOOO.OOOOOOOOOOOOOIOOOOOOOOOO...00.00.000.00... 21 A Cross-Comparison of the Occtn'rence of the Various Interspike Interval Histogram Types and the Average firm Rate HOfile CategwiesOOOOO00.0.0000.000.00.00.00... 22 LIST OF FIGURES Figure Page 1. The properties of the average firing rate profiles: schematized topographical properties plus the mean firing rate of each category together with the standard error associated with the mean rate........................ 10 2. A description of the various types of interspike interval histograms reported in the present work........... 12 3. A Thicnin.(lissl) stained section of the sheep medulla at the level of the gracile-cuneate complax........ 16 h. .A topographical comparison of the interspike interval histograms repcrted by various workers..................... 23 Cl. Electrode tracks 1+1 CZ. Electrode tracks... #2 C3. Electrode tracks........................................... ”3 Ch. Electrode tracks........................................... hh C5. Electrode tracks........................................... ”5 C5. Electrode tracks........................................... h5 D1. Average firing rate profiles............................... h9 D2. Average firing rate profiles............................... 51 D3. Average firing rate profiles............................... 53 Dh. Average firing rate profiles............................... 55 D5. Average firing rate profileS............................... 57 D6. .Average firing rate profiles............................... 59 D7. Average firing rate profileS............................... 60 D8. Average firing rate profiles............................... 62 D9. Average firing rate Profiles............................... 6h D100 Averwo firim rate PronleSOOOOOOOOOOOOOO.00...00.0.0.0...65 vi Interspike Interspike Interspike Interspike Interspike Interspike Interspike Interspike LIST OF.IIGUREB ( c ontinued) Page interval histograms 68 interval histograms............................. 7O interval histograms............................. 72 interval histograms............................. 7k interval histograms............................. 76 interval histograms 78 interval histograms............................. 80 inteflal msthOOOOOOOOOQOO0.000.000.0000...82 vii Introduction A recording electrode inserted into virtually any portion of the central nervous system will reveal spontaneous or what thfler (1956) (more aptly calls maintained activity. A recent review by Moore, Perkel and Segundo (1966) discusses maintained activity research throughout the vertebrate nervous system. Bishop's (1967) review covers maintained activities in the afferent portions of the nervous system. Table 1 compares some of the maintained firing properties of neurons found in various nervous system areas as reported by various authors. Ignoring maintained neural activity in favor of the experimentally more tractible dynamic properties of neural units may be a rather back- ward approach. Analysis of either steady-state or transient responses to stimuli cannot be considered adequately without first giving some attention to the neural baseline. It is therefore desirable to devise a means of dealing with the maintained or "spontaneous" nerve behavior in such a fashion as to facilitate comparisons with a given unit's driven responses, and with maintained activities from other neural regions (which may or may not be functionally related). .Hany investigators have gone further than merely noting the presence of maintained activity. As is well-known from the work of thtler (1956) and others (see Bishop, 1967), the receptive field properties of the mammalian (cat) retinal ganglion cell are predicated upon a continuous firing rate modulated by alterations in visual contrast. Similar organ- izations have been noted in other sensory systems, including the auditory (Katsuki, st 21., 1959; Greenwood and Maruyama, 1965) and the somatic sensory (Mountcastle and others, 1961a, 1961b; Gordon and Jukes, 196k). 1 egg Swan; ".333?qu mm .. w an” - 1: 13m Hosts; n38: Sousa—o; a 330m .. .. am e8 a nooBon sing gen: 33 m u o a." memo need "3150; van GmmC .353 a 30.5 833 3.33: pm - H m «Zoo 30m «8 83.3 magnum a Sass. Season 3 - n «we 338: 3850 you 333 and mm .8335 Agdopouoov .l I. 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"goofingiag bongo: Emmi 3.3% AogoomVnofimmu 3H8 oomfimofiflmv octagon BHpoHflHonH .Ho ovum can 390533 3ch no 093m SHAW-mu 09922 «6.3. 3.34 bonagvuopnw 3.983 H 02% Some neurologic models have been constructed which attempt to relate stimulated or driven and the maintained behavior of neurons. These models generally view the response to the stimulus as either a modulation or a steady—state change in the maintained baseline. lhth- ematical treatments have been presented; notable among these efforts is the work by Werner and Mountcastle (1963); Finkelstein and Grfisser (1965) ; Goldberg, 513 _a__1_. (1964); and Mountcastle, 33 5;. (1963). All of these authors have reported a power law relation between stimulus and response with maintained activity providing an irreducible minimum of nerve activity present at a zero stimulus level or at some state of physiological equilibrium. Other efforts have been concerned with the statistical behavior of spike trains, both in the stimulated and unstimulated (maintained) modes. Klang (1965); Gerstein and Mandelbrot (l96h); and Bodieck (1967) have approached the problem of spike train decoding in this manner. Such techniques do not stand alone, however. These models are useful for determining what a given neuron gagggt‘be doing during some time inter- val. Their predictive value has generally been nil. Such results can establish guidelines for interpreting experimental results when more data are present. Further discussion concerning the value of these techniques can be found in Bishop's review (1967) and in the paper by Gerstein and Mbndelbrot (196h). Many investigators have not been able to hold maintained units for appreciable periods of time. .Movements of tissue due to arterial pul- sations and respiratory activity often limit the long-term recording possibilities from a given unit. The movement of the tissue around the electrode causes, at best, spike amplitude changes which can make 5 amplitude discrimination from the baseline noise level very difficult. Progressively more disastrous in nature are the occasions where the unit is removed from the region of the electrode tip altogether. Finally, the moving tissue and the stationary electrode often conspire to stim- ulate the unit being studied, thus making, for example, "respiratory" units out of these whose primary concern in the organism's life is not respiration or the control thereof. (see Amassian, 196%) As a consequence, recording sessions with single units seldom ex- tend beyond a few minutes. It should be noted that differing brain regions present varying sets of difficulties in this regard. Rodieck and Smith (1906) and Bodieck (1967) have shown that it is possible to record from the retinal ganglion cell of the cat for periods of an hour or more. This is in a thoroughly paralyzed preparation. Amassian (196%), on the other hand, experienced considerable difficulty in holding single units for more that a minute or two while recording from the cat's medulla. Comparisons of maintained neural activity from different areas of the same brain are also lacking. To date most investigators have been interested in a particular functional area to the exclusion of surrounding areas. .Haintained activity analysis throughout the nervous system as a whole or even in a smaller but multifunctional portion has not been at- tempted. Some researchers have compared nervous activity in the system of their immediate interest with contiguous areas (see Cross and Silver, 1966; Evarts, l96h). In general, however, this is not attempted. In the present investigation I obtained a distributed sample of maintained neural activity over relatively long time intervals and from a functionally rich and varied portion of the nervous system. An elucidation or categorization of baseline activity is necessary before dealing with the dynamic responses of the nervous system to both external and internally generated stimuli. Therefore, the answer to the following question is needed. Can the sequential sampling of a unit's maintained firing rate and the distribution of interspike intervals pro- vide enough information to distinguish consistently and reproducibly between possible classes of units found in functionally distinct systems or structurally differentiated brain regions? The vehicle for this study was the posterior portion of the medulla of the domestic sheep, Ovis aries. This area of the medulla is desirable from the standpoint of this research because of the myriad of functional systems and distinct morphological entities present in a rather limited space. The sheep medulla was desirable because, unlike the medullas of smaller animals, it is relatively free from movements caused by either the animal's breathing or arterial pulsations. Methods The subJects. Eight female domestic sheep, Ovis aries, were used in this study. The animals were anesthetized with dial-urethane or nitrous oxide plus halothane (see Appendix I for details), tracheotomized and positioned for favorable exposure of the medulla. The caudal portion of the cranium and the dorsal aspects of the first two cervical vertebrae were removed and the exposed medulla was positioned using techniques similar to those employed by Johnson, 53 3;. (1968). Normal supportive measures including injections of normal saline to prevent dehydration, atropine to prevent excess secretion, and anesthetic as needed. The animals' temperatures were maintained at 3h-37ob. by means of a battery of infra-red lamps. Electrodes. The recording electrodes were modifications of either the Hubel (1957) tungsten microelectrode or the Baldwin, st 21- (1965) glass insulated tungsten electrode. Electrodes were commonly uninsulated for 30-50 microns back from the tip. These were found to perform ade- quately with shaft diameters of from 200 to 300 microns. This config- uration gave the needed physical strength to penetrate the overlying pia mater but had a small enough tip size to allow reliable access to single units. Recording andgprocessing equipment. Conventional recording equip- ment was employed throughout these experiments. A low level AC amplifier received the electrode signals and drove both an oscilloscope and an audio monitor. Signals deemed usable were recorded on magnetic tape using a good quality stereophonic recording machine The second tape channel was used for voice commentary and time marking. 7 On playback the recorded signals were smoothed and rectified. The spikes were converted to standard pulses by amplitude gates and fed into a multi-channel scalar (CAT hOOB) which was programmed to determine either the average firing rate per second (over 10 second intervals) or to construct the appropriate interspike interval histogram. Histologz;and track localization. At the termination of the ex- periments the animals were intercardially perfused with 0.9% saline followed by 10$ formalin in 0.9% saline. The medullas were removed, embedded in celloidin, sectioned and stained. Alternate sections were thionin stained (Nissl method) for cell bodies. The remaining sections were alternately stained with.hemstoxylin (wail or Sanides-Heidenhain method) for myelinated fibers. The small number of electrode punctures in any one medulla and the fact that punctures providing useful data showed heawy gliosis be- cause of the time spent in the puncture by the electrode made electrode track tracing relatively'simple and certain. A few units were definitehy localized as to position in the puncture by means of electrolytic lesions generated by passing small amounts of current through the recording electrode. Complete details as to all the methods employed in this study will be found in Appendix F. Results The unit population. Eight sheep provided 57 separate units ranging in overall firing frequency from 0.k to hh.7 Spikes per second. The mean frequency for the entire population was 12.3 spikes per second. Fifty percent of the units fell between 6 and 18 spikes per second. The units comprising this study were held for time intervals of from 80 to over 5000 seconds. Only six of the units were held for periods of less than 300 seconds while over one-half were held for 900 seconds or longer. With 22 units mechanical stimulation of the body surfaces gave rise either to alteration of the firing rates of the units themselves or to audible differences in the background firing activity as discerned from the audio monitor. Four units were observed to be related to the respiratory cycle of one animal. The remaining units were unaffected by any obvious stimulus, internal or external. The average firing rate profiles. The plotted average firing rate profiles (see Appendix D) fell into two major classes, regular and irregular. (see Figure 1) Regular (B) units were observed to maintain a more or less constant firing rate during the time the unit’s activity was sampled. The regular units were further subdivided, on the basis of topographical features of the profiles, into two subgroups, R1 (even) and 32 (wobbly). R1 (even) units fired with extreme regularity over each ten second sampling. The standard error of their ten second averages was 10% or less in most cases. 32 (wobbly) units tended to exhibit minor fluctuations about a central tendency. The standard errors of these units would often run to 20% or more. (in this text and in the figures the standard error is expressed as a percentage of the mean rate of firing.) 9 Type of Average Mean Firing Rate Firing Rate Pro- of Profile Sub- Mean Standard Schematic 1 Error of Profile Diagram of file group in Spikes Subgroup Expressed Profile per Second as 96 of Mean Rate R1 (even) 20 .h 8.7 I 15 R2 (wobbly) 1+.6 19 .h I 7 I1 (stepping) 10.5 55.8 % 11+ g a 8. m 12 (undulating) 9.7 29.3 a? 11 53‘ I3 (erratic) 5.0 35.9 ll !! n r! h I), (decremental) 11+ .9 35.8 t 7 Time (sec .) figure I. W properties of the averagegfiring rate profiles: scThema- tized topographical properties plus the mean firing rate of each category together with the standard error associated with the mean rate. ll Irregular (I) units were divided into four subcategories: I1 (stepping), 12 (undulating), 13 (erratic) and It (decremental). I1 (stepping) units characteristically Jumped from steady-state to steady- state taking a very few seconds to accomplish the change. 12 (undulating) units, on the other hand, did not achieve steady-state firing rates but continuously and slowly changed rate in a sinuous fashion, though most often with no regular periodicity. The I3 (erratic) units tended to change firing rate rather dramatically and did so continuously, often effecting a rate change of 100% or more within the space of a very few seconds. The final group, the 1h (decremental) exhibited a monotonic decrease in firing rate throughout their sampled lifetimes. Eventually these units always "died". Though the sample size employed in this study is not large enough to allow significant assessments as to the randomness or non-randomness of the distribution of firing rate profile types in various anatomical areas, it should be noted that no profile type was found to be excluded from any of the anatomically defined areas considered in this study, This point will be considered further in the next chapter. The interspike interval histograms. Three major types of inter- spike interval histogram (see Figure 2) emerged considered purely on the basis of their plotted shapes. These were distributions with: l. sym- metric peaks, 2. asymmetric peaks and 3. more than one peak. Each of these in turn could be further divided into subgroups. Taking the symmetric group first, tne essential difference was to be found in the presence or non-presence of short intervals, in suffi- cient quantity to establish a pre-modsl plateau in the finished histogram. Distributions lacking this feature were more nearly "normal" in shape. 12 Abbreviated Description of Histogram Schematic Diagram N Symbol Character of Histogram Type for Interval Histogram _* SOS-PT) Symmetric, unimodal with 11 pro-modal plateau. Hod- erate tail length. S(ST) Symmetric, unimodal with 10 no pro-modal plateau. underate tail. 3 4s MET) Unimodal, asymetric . Fast ,3 10 rise to peak with long, a heavy tail which falls off 3. exponentially. . e a MRI) Unimodal, asymstric. Fast ‘0 ’4 rise to peak with a "ramp- ‘3 like" tail. a ;§ M(Pl') Dailtimodal, symetric 10 maJor peak with.one or more minor peaks in tail. ‘ M(BI) Bimodal, either symetric- 9 symtric or asymetric- d/ symmetric with short tail. N No interval histogram plotted. 3 (See note at end of Appendix I .) fiL_ W dth Figure 2. A description of the various types of interspike interval histograms reported in the present work. 13 Both types exhibited fairly long tails. These will be referred to as S(P-Ml‘) and S(ST) respectively in the remainder of this report. Asymmetric interval distributions were characterized by fast linear rises in the short interval side of the peak followed by either a long, heavy "exponential" tail or by a "ramp-like" tail. These types will be henceforth abbreviated as A(ET) and A(RT) respectively. Finally, the two varieties of multimodal interval distribution will be referred to as M(PT) and “(B1). The first type typically displayed a highly symmetric maJor peak followed by a lengthly tail with one or more minor peaks superimposed upon it. M(BI) interval histograms had only two peaks. These were usually near to each other in size. With some M(BI)'s both peaks were symmetric and with others one peak was sym- metric while the other was not. When this was the case, the longer inter- val peak was generally the symmetrically shaped one. It is of interest to consider the nature of the spike trains that go into the makeup of each type of interspike interval histogram. It 13 found that the generating spike trains are similar for both the symmetric (S) distributions. Both contributing trains are characteristically smooth and even-firing. By triggering an oscilloscope with one spike and measuring the time to its successor, it is possible to obtain a fair estimate of the mean rate of the unit. The S(P-Ml‘) units would appear to slip occasionally and fire two spikes in rapid succession. These short intervals are not uniform, however, and the resulting histogram is consequently characterized by a pro-modal plateau. The highly peri- odic S(ST) distributions do not show this feature. Their interval histograms rise slowly from the zero level with no irregularities. 1h The spike trains comprising the asymmetric histograms present a different case. They fired erratically, almost in a ”Poisson" sequence. Triggering on one spike and measuring the time interval to the next spike does not reveal the mean firing rate as the interval variation is often several hundred percent. The long, heavy tails typical of these distributions are generated by the relatively frequently occurring long intervals. Both the A(ET) and A(RT) types have a very similar spike train makeup. Coming to the multimodal (M) types, it is found that these are usually composed of spike trains that exhibit bursting behavior. That is, a cluster of evenly spaced spikes is followed by a pause and than another cluster. When the pauses and the intervals between spikes in a burst are highly periodic, a M(PT) type interval histogram results. When the spike bursts are made of only two or three spikes and the pause between bursts is on the order of the intro-burst interval, a M(BI) interval histogram with two symmetric peaks is usually produced. (Occasionally the clustering consists of unequally spaced spikes within the cluster but with equally spaced intervals. This produces an asymmetric-symmetric type “(31) distribution that bears some simi- larity to the symmetric S(P«MT).nistogrem discussed earlier. The IE (decremental) average firing rate profile producing units were often observed to display an MKBI) type of interval distribution as well. These will be discussed in the next chapter. A consideration of anatomical location and interspike interval histogram type will also be left to the next chapter. Appendices D and E contain all of the average firing rate profiles and interspike interval histograms used in this study. Notes are included in these 1? appendices which point out some of the more interesting features of the plotted figures which have come under discussion in this chapter and will come under more discussion in the following chapter. Histology. An attempt was made to establish an anatomic locus for each unit used in this study. Of the eight sheep used, two were not available for this purpose. (see footnote 3 at end of Appendix B) The remaining six medullas were sectioned, stained and examined for electrode tracks. Traced units were assigned to four basic medullary locations. These were: a) the ascending afferent tracts overlying the dorsal column nuclei, b) the nuclei themselves including the spinal trigeminal, e) the medullary reticular areas and d) the "other" portions of the medulla. These included the cranial nerve nuclei, the ventral white matter and the ventral horns. The anatomical localizations were based on the descriptions of Palmer (1958) and Wondenberg (1968). Specific locations (in as much as these are possible to claim, see Akert and Welker, 1961) will be found in.Appendix C where all the track tracings are presented. Figure 3 shows a typical electrode track as it appeared in a thionin (Nissl) stained section. 16 Note electrode track passing A Thisnin.(Nissl) stained section of the sheep medulla at Figure 3. the level of the gracile-cuneate complex. through cuneate nucleus on the left side. Discussion As was Observed in the introductory comments of the preceding chapter, some of the units used in this study were held for periods of one and more hours. A question might arise: does such a unit maintain stationarity with respect to its statistical properties over long time intervals? During data playback the shape of the computed interspike interval histogram was monitored continuously. Once a recognizable form had been achieved, the overall shape of the plotted distribution did not change except for a few irregularly bimodal MtBI) producing units. As a further check upon stationarity, segmental records were made and com- pared in a manner similar to that employed by Amassian, gt 2;. (196h). Successive 10 minute samplings were compared as to the shape of the interval distribution produced. Minor variations were noted, but no unit could be said to change the type of interval histogram that it characteristically produced. The MKBI) type units, it will be remembered, were produced by units having two distinct types of spike train. One of these was an irregularly bursting train while the other, superficially at least, was rather even and regular in discharge rate. Observations made on these latter units indicated that they were strongly correlated with the Ih (decremental) firing rate profile producing units. These, while gradually diminishing in firing rate, appeared to fire with extreme regularity over shorter time intervals. However, the "slowing down" of these units was typified by a preference on the part of the unit for a certain modal interval. Ever longer intervals were interspersed among the "preferred" intervals such that a facade or more-or-less constant interval could be maintained. Eventually, this was no longer possible and the unit would shift to some 17 18 new model value with a longer interval. As a rule not more than two of these shifts were noted because the units in question would stop firing altogether, usually rather suddenly. Similar behavior, _i_._e_. the production of M(BI) interval histograms, was not Observed with the remaining types of irregularhy profiled units, at least not in appreciable quantity. For instance, two type 11 (step- ping) units displayed M(BI) type interval histograms. However so did two Bl (smooth) profile producing units. A better view of the trends is contained in the cross comparison tables presented below. All the units used in this study are compared as to: 1. Anatomical location and the type of interspike interval histogram produced. (Table 2) 2. Anatomical location and the type of average firing rate profile produced. (Table 3) 3. Intersgike interval histogram and average firing rate profile produced. (Table h) The data are presented as the number of observed units sharing any two properties as described above. The number in parentheses following each observed value is the "expected" value which would be obtained if the relation between the variables were completely independent. The significance of the "expected" value is predicted upon the randomness of the method in which the units were sampled in the medulla - a matter about which this study makes no claims. The basis for the calculation of the "expected" values arises from contingency table constructions, the details of which can be found, for example in Freund (1962). The obvious next step of determining the chi2 test for independence of the variables was not feasible because of the limited sample size. Proper Issage demands that the expectation values in each cell of the contin- égeency table be greater than five. 19 In spite of the testing limitations, trends can be noted. Table 3 shows that MKPT) interval histograms are preferentially produced by fibers of the dorsal fiber tracts. This indicates that such fibers tend, as a class, to fire in regular bursts. A few S(P-MEO or symmetric, pre-modal histograms are produced by the fibers in this area. These can be construed as units which fire short bursts of two spikes with regular intervals between bursts and irregular intervals within the burst. The other symmetric S(ST) type of interval histogram is appar- ently not produced by units comprising the primary afferents of the dorsal column tracts. Neurons in the somatic sensory nuclei would seem to favor S(P-MT) irregular bursting but symmetric firing patterns together with the S(ST) symmetries. Both of these are highly "periodic" in nature and can'be distinguished from each other only by the absence of the "pre-modal" or fast activity in the case of the S(ST) units. The regular bursting multimodal.M(PT) units are missing from these nuclear areas though they were common enough in the pre-synaptic areas. Reticular and "other" areas of the medulla appear to be distributed very close to expectation across the various interval histogram types. The average firing rate profiles receive the same treatment in Table 3. Again, significance tests cannot be applied, but it is reason- ably apparent that firing rate profile patterns are distributed very close to expectation throughout all areas of the medulla that were con- sidered as separate entities. Identical techniques were used to compare the occurrence of a given average firing rate profile category within a particular interspike interval histogram class. These results are presented in Table h. 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A topographical comparison of the interspike interval histograms reported by various workers . Data are presented from three studies in addition to the present effort. The six different types of interspike interval distribution dis- cussed in the present study are represented by the six figures under Height, 1968. these are presented in the order: S(M-PT) Symmetric peak, pre-modal fast activity. “(PM Multimodal with regular symmetric peaks. S(ST) Symmetric peak, moderate tail, no pre- modal activity. MET) Asymmetric peak, long tail. MET) Asymmetric peat, ”ramp-like" tail. MEI) Bimodal, irregular. (see rigure 2 for more details) Date from the work of other investigators have been expended or compressed to best fit the format used in this study. The unit identifications used are the author's own with the exception of the data from Amassian. Here the three digit number refers to the page upon which the figure is to be found and the single digit following the dash is the figure number used in the article. The uits from Kiang's study will be found on page 97 of his monograph. SEhe short table below directly compares each author's data with the categorization scheme proposed in this study. Under each author's name are listed the units reported by him arranged in accordance with the classification used to distinguish units in the sheep medulla. W of Unit Amassian Kaing Rodieck Height (Haight Class- (1961+) 1965) (1967) (1968) ification) # S(M-P'I') 2h1-5; 2&2-7 x x 681h5—11A MP1?) . x x x 68116-111. S(s'r) 2&2-8 x E; r 68115-1313 Mm) aha-9 299-25 A 681h5-16A-1 MR?) 1: 299-19 B; D 67136-123 “(B1) 1 x C 6811414-88 < a a. We . ._ .. f w ~1- 5; fl ‘ m rm: I C‘ :‘r- a" '- "1““:- h 4 9 5.." 5,..1 f *- é. ' a i 3“ m j ‘ EV 6‘?" ("if 3 T4" ’1 4"» “’ "1 I!“ 95%" i Q .3 . ‘K 1.: ruin. 3 ant” 1" ’.‘ I I ‘ «II 11.53 L: t. "LA 4 ‘ . - v. < u L "a" - x. . m . . .J , . .. 512 IIIHIISPIIIE IIIIEIWM IIISIIIIIIIAMS AMASSIAN, 1964 CAT, CUNEATE NUCLEUS 3000 241-5 § 1 N § _ ' .\ /”/\’\¢ 7-— ‘ \\ \ .\\\ L ;\ 0 32 64 FJVI 242-8 1 Q 1 O r J (N j h__rJ_. £0 60 1000 242-7 INIEBSPIIIE INTEIIIIAl IIISIIIEIIIIMS RODIECK, 1967 CAT, RETINAL GANGLION CELLS 242-9 16 KIANG, 1965 CAT, COCHLE AR NERVE 256 Kiang, 1965 spontaneous unit 299- 25 W E .0. - A J1 ' «Ll (o b 7L J ‘ 100 I 200 o 100 C U ' <0 i A 160 200 ‘ 160 200 512 256 Kiang. 1965 spontaneous unit 299-19 INTERVAL IN MILLISECONDS 4. I; 166 INTERVAL IN MILLISECONDS X 100 INIIIISPIIIE lNIEIIIIIIl HISIIIEIIIIMS HAIGHT,1968 SHEEP, MEDULLA OP 68145-11A 5‘31 58145” arm 0 . I} V ' F It: 5,. N. j m ' Laws ‘ L 0 LE ii. 0 250 500 1“ 68145-130 2914.. f i i L I 1.; 1500 I [J : I 1 1 “a; A‘- ‘M I 0 250 500 0 500 1000 67136-128 m- 58‘44‘88 b ,J'L N . 250 500 0 125 250 INTERVAL IN MILLISECONDS 25 statistical dependence or independence of the properties which produce a given type of interspike interval distribution and those which are reaponsible for a specific pattern of average firing rates are, in themselves, independent of sampling prejudices. Therefore, the Observed versus the expected differences seen in Table h can.be viewed with less suspicion than was possible in Tables 2 and 3. Examination of Table h will show that S(P—MT) or the symmetric, pre-modal type of interval histogram is heavily "over represented” in the R2 (wobbly) profile class. The bursting M(PT) or multiperiodically tailed interval histograms are represented heavily in the R1 (even) average firing rate profiles. They are also present in excess within the 11 (stepping) category but are totally absent in the 32 (wobbly) and 12 (undulating) profile groups. This is contrary to random or independent expectation. Further perusal of table h shows that over or under-representation is the case in about one-half of the cells with the remainder being rather close to the calculated expectation. Finer anatomical distinctions may bring out other properties, allowing for more concrete assignments of spike train character with both function and anatomy. In the main, however, Tables 2-h do not serve as much to answer questions as to pose new ones. On an overall basis the interspike interval histograms described in the present study are not appreciably different from those described by other workers. Figure h compares the "typical" interval histograms discussed in this study with those reported by three other investigators. The findings of this study and the reports of others, see Braitenberg, at al., 196%; Amassian, £3.2i1: 196k; Herz, £3.2l-’ 196h; and Rodieck, 1967) indicate the following: 26 l. The variety of interspike interval histograms found by manipulating neuronal spike data is limited to a few basic types which can be char- acterized by: a) symmetry or asymmetry of the major peak, b) the number of peaks present, 0) the length and weight of the histogram tail and d) the presence or absence of pre- modal or fast activity. 2. These basic qualities are not mutually ex- clusive. Host investigators have arrived at descriptive schema which incorporate these features to a greater or lesser degree. 3. The limited number of histogram types found in the central nervous system are found in all sites thus far considered. Unfortunately, data are lacking with respect to the average firing rate profiles. Mbst workers do not discuss long-term sequential properties of the units with which they are working. When they do, it generally is not in terms of easily analyzed sequential plots such as are used in the present study. (see Bodieck, 1967 for an exception.) One must not lose track of the fact that it is the spike train which is received and acted upon by the recipient neuron and not the interSpike interval histogram. Keeping the foregoing in mind plus the preJudicial sampling techniques employed in this study, the comments which follow summarize the essential findings. In the portions of the sheep medulla considered a limited number of interspike interval patterns were found. This limited repertoire was found distributed throughout the anatomical areas that were delineated either histologically or electrophysiologically. Certain correlations of interval histogram type and anatomical location were noted: 1. Units producing multiperiodic interval histo- grams appear to be most prevalent in the afferent dorsal fiber tracts of the medulla. 27 2. Symmetric peaked histograms with or with- out pre-modal or semi-burstlike activity are found predominantly in the dorsal column nuclei. Multimodal types such as are found in the overlying afferent tracts are not found in these nuclei. 3. There are no Obvious trends to be noted concerning the nature of the unit pro- ducing a given interval histogram type in other areas of the medulla, though all observed histograms were noted in these regions. With respect to the average firing rate profiles it can be said that if these measures are related to function, it is not solely on the basis of anatomical location. Each profile type is to be found in each medullary area as nearly as can be determined from the present effort. It would be desirable to be able to make a quantitative statement about the prdbabilities of finding maintained activity of a specific kind at a particular medullary locus - or, indeed, anywhere else in the nervous system. The results of the present research indicate that, using the techniques described herein, such statements cannot be made with high degrees of confidence. It should be emphasized that while relative representation of both profile and histogram categories defined in this study do vary from site to site, maintained activity appears to be present everywhere. It is perhaps significant that such activity is not associated exclusively with the proprioceptive steady-state units of the somatic afferent system or with heart and respiratory units. Maintained activity was commonly found associated with units whose re- sponse to external stimuli were decidedly transient in nature. This tends to indicate that functional subdivisions within a given anatomical area are going to have to be considered before the role of maintained neural processes is completely understood. 28 Future work might be concerned with a more equal emphasis upon all portions of the medulla or other brain areas that happen to be under investigation. The more ventral portions of the medulla, for example, were rather neglected in this study. With larger unit samples statistical methods might be utilized to better advantage. This study has shown that certain tendencies do exist; more information is needed to determine whether these tendencies are important either neurophysiologically or behaviorally. Mbre research also needs to be done on the effects of anesthetic agents. The difficulties experienced in comparing the efforts of various authors is in great part due to differential anesthetic effects. In this regard, Evart's (196k, 1968) work with awake and naturally sleeping animals is encouraging in that it presages the eventual establishment of a technique which utilizes a reproducible, yet biologically natural state of the animal for examining nervous behavior. Sometimes arti- ficial agents must be used. Paralytic agents for retinal research provide an example. It would be desirable to be able to quantify some of the effects of these agents in such a manner that they could be used to insure experimental reproducibility. 10. 11. Bibliography Akert, K. and W.I. Welker. 1961. Pimblems and Methods of Anatomical Localization. Chapter 17 in Electrical Stimulation of the Brain. Ed. by D.E. Sheer. University of Texas Press, Austin, Texas. 251. Amassian, V E.., J. Macy, Jr., R ..J Waller, H .8. leader and M. Swift. 1961}. Transformation of Afferent Activity at the Cunsate Nucleus. In Information Processing in the Nervous System. Ed. by R. W. Gerard and J ..W Dwff. Procedings of the International Union of Physiolog- ical Sciences, International Congress Series Number 3&9. kcerpta Medics Foundation, New York. 3: 235. Baldwin, HAL, 6. Frank and J3. Iettvin. 1965. Glass-Coated Tungsten Microelectrodes. Science. 1&8; 1&2. Bishop, P .0. 1967. Central Nervcus System: Afferent Mechanisms and Perception. In Annual Review of Physiolog, Volume 29. Ed. by V ..E Hall. Annual Reviews, Inc., Palo Alto, California. 1:27. Braitenberg, V., G. Gambardella, G. Ghigo and U. Vota. 1965. Observations on Spike Sequences from Spontaneously Active PurkinJe Cells in the Frog. Kybernetik. 2: 197. Cross, B.A. and J .A. Silver. 1966. Electrophysiological Studies on the Hypothalamus. British Medical Bulletin. 22: 25h. Bvartc, E .v. 196k . Temporal Patterns of Discharge of pyramidal Tract Neurons During Sleep and Waking in the Monkey. Journal of Neurophysiolfl . 27: 152 . Everts, By. 1968. Relation of Pyramidal Tract Activity to Force hherted During Voluntary Movement . Journal of NeurOphysi 01g. 31: l . Pinkelstein, D. and 0.-J. Grflsser. 1965. Frog Retina: Detection of Movement. Science. 150: 1050. Freund, J .E. 1962. Mathematical Statistics. Prentice-Hall, Inc. , Englewood Cliffs, New Jersey. 232. Gerstein, 0.1.. and B. Mandelbrot. 196:. Random Walk models for the Spike Activity of a Single Neuron. Biopfisical Journal. h: hl. Goldberg, J .14., 3.0. Adrian and F.D. Smith. 1961}. Reaponse of Neurons of the Superior Olivary Complex of the Cat to Acoustic Stimuli of Long Duration. Journal of NeurOphysiolog. 27: 706. 29 13. 1h . 15. 16. 17. 18. 19. 20. 21. 22. 23. 21+. 25. 26. 30 Gordon, G. and M.G.M. Jukes. 1961b. Dual Organization of the Extero- captive Components of the Cat's Gracile Nucleus . Jom'nal of Wei- ol L {London} . 173: 263 . Granit, R. 1962. Receptors and Sensory Perception. Yale University Press, New York and London. Greenwood, D. and N. minivans. 1965. Excitatory and Inhibitory Response Areas of Auditory Neurons in the Cochlear Nucleus. Journal of Neuroplwsiolgg. 28: 863. Herz, A., 0. Creuzfeldt and J. Fuster. 196k. Statistische Eigen- schaften der Neuronactivitlt im Ascendierenden Visuallen System. gybernetik . 2: 61 . Hubel, D. 1957. Tungsten Microelectrode for Recording from Single Units. Science. 125: 519. Johnson, J.I. Jr., W.I. Walker and B.H. Pubols, Jr. 1968. Somato- topic Organization of Raccoon Dorsal Column Nuclei. Journal of CemEative Neurolog. 132: 1. Katsuki, Y., T. Watanabe and N. Suga. 1959. Interaction of Auditory Neurons in Response to TWO Sound Stimuli in the Cat. Journal of Neurophysiom. 22: 603. Kiang, N. Y.-S . 1965. Discharge Patterns of Single Fibers in the Cat's Auditory Nerve. M.I.T. Research Monograph No. 35. The M.I.T. Press, Cambridge, Massachusetts. Kuffler, 14.14., R. Fitzhugh and H.B. Barlow. 1956. Maintained activ- ity in the Cat's Retina in Light and Darkness . Journal of General Physiology. ho: 368. Mbrtin, A.R. and C.L. Branch. 1958. Spontaneous Activity of Betz Cells 5n Cats with Midbrain lesions. Journal of Neuroflsiolog, 21: 3 . Moore, 6., D. Perkel and J. Segundo. 1966. Statistical Analysis and Functional Interpretation of Neuronal Spike Data. In Annual Review of Raysiolg, Volume 28. Ed. by V.B. Hall, A.C. Giese and B.R. Son- nenschein. 93. Mountcastle, V.B. 1961a. Some Functional Properties of the Somatic Afferent System. In Sensory Consummation. Ed. by W.A. Roeenblith. The M.I.T. Press, Cambridge, Massachusetts. l$03. Mountcastle, V.B. 1961b. Duality of Function in the Somatic Afferent System. In Brain and Behavior. Ed. by M.A.B. Brazier. American Institute of Biological Sciences, Washington, D.C. 67. Mountcastle, V.B., GJ‘. Poggio and G. Werner. 1963. The Relation of Thalemic Cell Response to Peripheral Stimuli Varied Over an Intensive Continuum. Journal of Neurophysiology. 26: 807. 27. 28. 30. 31. 32. 33. 31 Palmer, A.C . 1958. Anatomical Arrangement of the Grey Matter in the Medulla, Pons and Midbrain of the Sheep . Zentralblatt fir Veterinflr- medizin. 5: 953. Pfeiffer, R.R. and N. I.-S. Kiang. 1965. Spike Discharge Patterns of Spontaneous and Continuously Stimulated Activity in the Cochlear Nucleus of Anesthetized Cats. Biophysical Journal. 5: 301. Poggio, or. and L.J. Viernstein. 1961;. Time Series Analysis of Im- pulse Sequences of 'nialamic Somatic Sensory Neurons. Journal of Neurophysiolgg. 27: 517. Rodieck, 13.14. and P.S. Smith. 1966. Slow Dark Discharge Rhythm of Cat Retinal Ganglion Cells. Journal of Neurophysiolggz. 29: 9&2. Rodieck, s.w. 1967. Maintained Activity of Cat Retinal Ganglion Cells. Journal of Neurgphysiolog. 30: 10113. Werner, o. and V.B. Mountcastle. 1963. The Variability of Central Neural Activity in a Sensory System and Its Implications for the Central Reflection of Sensory Events. Journal of NeurOplweiolog. 26: 958. Woudenberg, R. 1968 . goJectionsfif Machanoreceptive Fields in Cune- ate-Gracile and S inal Trigeminalt Nuclear Regions in Sheep. Unpublished Eater's Thesis, EEcEigan State University, East Lsnsfnngichigan. APPENDICE I. II. Appendix A Abbreviations to be used in.the following appendices .Al. N., alar nucleus (dorsal motor nucleus of the.Xth cranial nerve). Ar. Post., area postrema. Cu.-Gr. N., cuneate-gracile nuclear complex. Cu. N., cuneate portion of cuneate-gracile nuclear complex. Ex. Cu. N., external cuneate nucleus. Gel. Su., gelatinous substance. Gr. N., gracile portion of cuneate-gracile nuclear complex. H.N., hypoglossal nucleus. I.0.N., inferior olivary nuclear complex. IHR.N., lateral reticular nucleus. MLL.F., dorsal portion of median longitudinal fascicle. Pyr. Dec., pyramidal decussation. R.F., reticular formation Sp. Trg. N., spinal trigeminal nucleus. Sp. Trg. Tr., spinal trigemdnal tract. S. Tr., solitary tract. ‘Vn,.Hn., ventral horn of caudal medulla. ‘Vn. Wh., ventral white matter of caudal medulla. .An explanation of the unit identification system used in this research. The first two identifying digits represent the year in which the recording session was run. The next digit denotes the category of animal, marsupials being zero, ruminants being one, and so on. The next two digits stand for the "nth" animal in a given series. Following the hyphen is the puncture number. The letters denote the number and positions of units obtained within a given puncture. Occasionally'twOror more units were recorded simultaneously from the same electrode position. When this happened a second hyphen followed by a number was used to separate the units involved. 33 314 Hence: Unit 67129-2L»rerers to an experiment run.in 1967 upon a sheep which happened to be the 29th in its series. The unit Obtained came from the second electrode puncture and was the Lth response. Unit 681h5-lfic-3 would.likewise refer to an.experiment per- formed in 1968 upon a sheep, hfith in its series. The third (0) response derived from the 15th puncture yielded four distinct units. 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See Appendices D and E following. 2 This unit was located in the border region between the cuneate-gracile nuclear complex and the external cuneate nucleus. As a consequence, it might be associated with either nucleus. See Appendix C, track 67132-7 3This animal was not processed through histology. .Animal 67133 was a pilot study for a nitrous oxide anesthesia project. The animal was disposed of before the recordings were found to be of value. Animal '-36 was utilized in a teaching experiment. An excessive number of electrode punctures made track tracing impossible. “The track containing this unit skins the hypoglossal nucleus. Locali- zation of puncture within the track was not precise enough to allow a definite attribution. 5Due to inability of the electronic apparatus used to discriminate spikes of differing amplitude imposed upon a wavering baseline, these histo- grams could not be plotted. 6This unit is placed in the NKPT) or periodic tailed histogram class be- cause of the extreme symmetry of the major peak - a characteristic peculiar to this type of interval histogram alone. Both units involved are ”somatic". 681hh-1A may not be in a physiologically “neutral" state. unit 681h5-3A was being ”stimulated" while the interval histogram was plotted because it was difficult to obtain data in the neutral position due to the presence of extraneous activity. 7These units could not be found. The microelectrode left no apparent track. Appendix C Tracings of the electrode tracks The tracings are presented by animal in the order experimented upon. The sections pertaining to a given animal are presented in the order in which they were sectioned. This may be in a rostro-caudal direction in one animal and the opposite in another. Thus: 671h3-58ht is the 58hth section (thionin) from animal 671h3. This section shows the hth electrode puncture which contains three single unit recording sites. 1&0 67141‘188-2001 E .C .N. .- U. . r. ' J 67132 265 7A Gr Q N \s. Al N QSe-sr 9.: 3.. N R F {$7 14A Gr.-Cu.N. l arm. .N. R F o N afi/\ ”N \p/ 67141 400! 67129-250! 1+1 Eovvv—mo Bmoévib “vow .0350 11-2? Boo—+9638 B—Nw.vv50 i—owézmo (o . .000 c» a: .960 . an. 9.8 a 5N.mv—mo wombvpmo 6: .> , «as . (\, .233 / 111.: .3 ll w 0 LL11 68145 -162t Py r. Dec. 68145-150t 1+5 . z. :2: 5 . 0.2 s 6303!: <' (Zak I '6. < QM 5.x Q Q0 68145 '198t 68145-190! 68145-162t Py r. Dec. 88145450t 1+5 s. R Q0 68145 '198t 68145-190t 68145-162t Py r. Dec. 88145-150t 1+5 u.’ . Z: ‘2 @2- <9 逥 9 < LY: ,._ p . vec- 3. Q Q0 88145 '1981 68145-190t «SN . name us Appendix D The Average Firing Rate Profiles #8 Figure D1: Notes on the average firing rate profiles. 67l29-2L: This unit responded in a steady-state fashion to alterations in the position of the lower Jaw. The "at rest" position is the free hanging or "neutral" position. The gap in the record at 500 plus seconds was caused by the time taken to change tape reels and not by a discontinuity in the spike sequence. To effect a "stimulated” condition a 2 by h inch block of wood was inserted between the Jews and left in position. The unit's re- sponse to this can.be seen in the "open" portion of the record. A slight adaptive trend was noted. The discontinuity before the on- set of this sequence was due to the shorting of the recording input during the time the block was being placed. To obtain the final portion of the record the block was re- moved without turning off the recording equipment. The Jaw was allowed to hang free as in the first "at rest" position. The grad- ual increase in firing rate from a level somewhat depressed from the first "at rest" state was noted. After stimulation approximately 1000 seconds elapsed before the unit attained its original resting level. No adaptation was Observed in the first resting sequence be- cause the recording of unit activity was not begun until the unit had been under Observation for over an hour. 67132-7A: This is a drivable unit whose response to the chosen stimulus, while readily apparent was not as "nice" as the response of the unit immediately preceeding. The free position response was re- corded from the unit while the leg lay undisturbed. The hoof was not in contact with any surface. The activity in the firing rate profile can be seen to alter firing rate entirely of its own accord, independently of any obvious stimulus. Upon propping the leg upward by placing a stool under the hoof, the response portrayed in "Pos. 1" was obtained. A fast transient response was noted initially which subsided to a very gradually di- minishing steady-state level subject to considerable minor irregular- ities in overall rate. A similar transient response was noted when a stronger stimulus was applied (forcing the leg even higher). The resulting activity on the part of the driven unit was even more irregular exhibiting much more variation in firing rate even to the point of exceeding the initial transient response for brief periods. Sudden removal of the stimulus resulted in approximately three minutes of silence." The unit was almost totally inhibited. This period of’activity was followed by a sudden rise to a steady- state level approximate eh, 3a free position, 13 me as that observed for the initial AVERAGE NUMBER OF SPIKES PER SECOND "Elli! IIIIII I"! I'llfllll Figure Dl. TIME IN SECONDS 1&9 h8 Figure Dl: Notes on the average firing rate profiles. 67129-2L: This unit responded in a steady-state fashion to alterations in the position of the lower Jew. The "at rest" position is the free hanging or "neutral" position. The gap in the record at 500 plus seconds was caused by the time taken to change tape reels and not by a discontinuity in the spike sequence. To effect a "stimulated” condition a 2 by h inch block of wood was inserted between the Jaws and left in position. The unit's re- sponse to this can be seen in the "open" portion of the record. A slight adaptive trend was noted. The discontinuity before the on- set of this sequence was due to the shorting of the recording input during the time the block was being placed. To obtain the final portion of the record the block was re— moved without turning off the recording equipment. The Jaw was allowed to hang free as in the first "at rest" position. The grad- ual increase in firing rate from a level somewhat depressed from the first "at rest" state was noted. After stimulation approximately 1000 seconds elapsed before the unit attained its original resting level. No adaptation was observed in the first resting sequence be- cause the recording of unit activity was not begun until the unit had been under observation for over an hour. 67l32-7A: This is a drivable unit whose response to the chosen stimulus, while readily apparent was not as "nice" as the response of the unit immediately preceeding. The free position response was re- corded from the unit while the leg lay undisturbed. The hoof was not in contact with any surface. The activity in the firing rate profile can be seen to alter firing rate entirely of its own accord, independently of any obvious stimulus. Upon propping the leg upward by placing a stool under the hoof, the response portrayed in "Pos. 1" was obtained. A fast transient response was noted initially which subsided to a very gradualxy di- minishing steady-state level subject to considerable minor irregular- ities in overall rate. A similar transient response was noted when a stronger stimulus was applied (forcing the leg even higher). The resulting activity on the part of the driven unit was even more irregular exhibiting much more variation in firing rate even to the point of exceeding the initial transient response for brief periods. Sudden removal of the stimulus resulted in approximately three minutes of "silence." The unit was almost totally inhibited. This period of activity was followed by a sudden rise to a steady- state level approximately the same as that observed for the initial free position. AVERAGE NUMBER OE SPIRES PER SECOND mum fllllli II" PIIIHIES Gila-2L (W) MM seam—Wis“ A 1:. A A U " ; 67132- 7A (LES’ TIME IN SECONDS Figure Dl . M9 #8 Figure Dl: Notes on the average firing rate profiles. 67129-2L: This unit responded in a steady-state fashion to alterations in the position of the lower Jaw. The "at rest" position is the free hanging or "neutral" position. The gap in the record at 500 plus seconds was caused by the time taken to change tape reels and not by a discontinuity in the spike sequence. To effect a "stimulated" condition a 2 by h inch block of wood was inserted between the Jews and left in position. The unit's re- sponse to this can be seen in the "open" portion of the record. A slight adaptive trend was noted. The discontinuity before the on- set of this sequence was due to the shorting of the recording input during the time the block was being placed. To obtain the final portion of the record the block was re- moved without turning off the recording equipment. The Jaw was allowed to hang free as in the first "at rest" position. The grad- ual increase in firing rate from a level somewhat depressed from the first "at rest" state was noted. After stimulation approximately 1000 seconds elapsed before the unit attained its original resting level. No adaptation was observed in the first resting sequence be- cause the recording of unit activity was not begun until the unit had been under observation for over an hour. 67132-7A: This is a drivable unit whose response to the chosen stimulus, while readily apparent was not as "nice" as the response of the unit immediately preceeding. The free position response was re- corded from the unit while the leg lay undisturbed. The hoof was not in contact with any surface. The activity in the firing rate profile can be seen to alter firing rate entirely of its own accord, independently of any obvious stimulus. Upon propping the leg upward by placing a stool under the hoof, the response portrayed in "Pos. 1" was obtained. A fast transient response was noted initially which subsided to a very gradualxy di- minishing steady-state level subJect to considerable minor irregular- ities in overall rate. A similar transient response was noted when a stronger stimulus was applied (forcing the leg even higher). The resulting activity on the part of the driven unit was even more irregular exhibiting much more variation in firing rate even to the point of exceeding the initial transient response for brief periods. Sudden removal of the stimulus resulted in approximately three minutes of "silence." The unit was almost totally inhibited. This period of activity was followed by a sudden rise to a steady- state level approximately the same as that Observed for the initial free position. AVERAGE NUMBER OE SPIKES PER SECOND "Ill“! flllli I"! I'llllllS Olin-2L (JUN) fiMbu Figure Dl. TIME IN SECONDS 1:9 50 Figure D2: Notes on the average firing rate profiles. 67lhl-3B: The discontinuity in the record Just before the 3000 second point was due to a tape reel change. The unit continued to fire during the change, but the spike activity was lost. 671hl-lOB-l: This unit was recorded continuously for over #000 seconds. The gap in the center of the record represents a period of absolute silence on the part of the unit. "IBIS! IIIIIIG II"! I'lllllllIS AVERAGE NUMBER OF SPIKES PER SECOND Figure D2 . 67141-35 we me he 07141-1084 W W 07“! JOB-2 s WW (i P T ‘ sooo TIME IN SECONDS 51 : n W”) 1 WU 52 Figure D3: Notes on the average firing rate profiles. 671hl-lhA: The break in the record is due to a tape change. The unit continued to fire. 671h3-33: Again the gaps in the record do not represent unit silence but are due to time lost while changing tape reels. 67lh3-hC: This unit's behavior is probably a good example of an irrita- bility reaponse followed by a "settling down" to a steady level. "III“! "III“ I“! Plllfllli ‘1 AVERAGE NUMBER OF SPIKES PER SECOND 67143-3A (ARM) A l 07141- 14A (NECK, 15 07143- 3D Figure D3 . (ARM) 67143 48 07143- 4C w. ‘45‘ "ME IN SECONDS 53 - ea Figure Dh: 681hh-11A: 5h Notes on the average firing rate profiles. The gap in the record does not indicate unit silence. AVERAGE NUMBER OF SPIKES PER SECOND "Ell“! flllllfl ll"! mum 67143-40 66144-1A (ARM) 66144-16 66144-3A 66144-6A 66144-66 a Figure DH. .[VN 66144-11A ( THUAX) TIME IN SECONDS 55 56 Figure D5: Notes on the average firing rate profiles. 681h5-3A-1: This unit was treated in much the same way as unit 67132-TA. Position 1. is the activity resulting from a free hanging arm. This is followed by position ii. which involved flexing the arm such that the elbow formed an acute angle. This was followed by a partial relaxation of the pressure which allowed the elbow to form an obtuse angle (position 111.). A return to the free position (iv.) was followed by an obtuse flexion (v.) which. in turn was followed by an acute flexion (vi.). Finally the limb was allowed to return to its resting state (vii.). It is apparent that the average rate profile does not afford a direct means of determining the limb position. The presumably equivalent free positions (1., iv., and vii.) display considerable variability in their effect upon the unit. The two acute positions (11. and vi.) show a higher overall rate than any of the other positions. The unit, in response to this degree of flaxion, would appear not to show the gradual rate in- crease exhibited by the obtuse positions (iii. and iv.). The gaps in the record are in all cases due to the cessation of recording during stimulus changes. AVERAGE NUMBER OF SPIKES PER SECOND "Elli! flllllfl III! I'IMIIES 66146 -1A 66145-16 UJW 66145-2A (LEG) -‘ fl. sens-3A4 ? ° N u u m u Iv u v n vi a m e '. ) Riki/1!“ #11 My”: (ARM) MA $ . r “I1 HAW RMJ’MMMWI I : 3 j I -‘ 1' H ‘ ‘ .a ll . l' ‘1 0 m I 1'. «lamb at said Ms TIME IN SECONDS Figure D5. 57 58 Figure D6: Notes on the average firing rate profiles. 68lh5-3Ar2: This unit was recorded simultaneously with unit 681h5-3A-l and, consequently, was subjected to identical external stimulus conditions. It would appear that this unit, though anatomically closely associated with unit 681u5-3A-1, is rather disinterested in the particular stimuli chosen to activate its neighbor. About the only obvious correlation is seen in position iv. where 681%5-3A-l takes a dip in firing rate and 681h5-3A-2 reactivates at the same time. There are then correlations which are (perhaps) related to the interactions of these two units but not necessarily directly coupling both units to the stimulus, but rather, possibly demonstrating a differential interest on the part of the two units to varying as- pects of the same stimulus. The record gaps are due to the halting of recording during stimulus changes. 681h5-8A: The gap at approximately 750 seconds is due to a tape change. The unit continued to fire. mum "ms nu mmn 66145-3A-2 N v N vi a wii 0 (ARM) WWW] Imj‘ M1 111111" 11% ' I I I I oddamuuemhiannnflx 66146 -66 AVERAGE NUMBER OF SPIKES PER SECOND 66146 -10A-2 (LEG) “$111K TIME IN SECONDS Figure D6. 59 58 Figure D6: Notes on the average firing rate profiles. 681h5‘3AP2: This unit was recorded simultaneously with unit 681h5-3A-l and, consequently, was subjected to identical external stimulus conditions. It would appear that this unit, though anatomically closely associated with unit 681115-3114, is rather disinterested in the particular stimuli chosen to activate its neighbor. About the only Obvious correlation is seen in position iv. where 681h5-3A-l takes a dip in firing rate and 681h5-3A-2 reectivates at the same time. There are then correlations which are (perhaps) related to the interactions of these two units but not necessarily directly coupling both units to the stimulus, but rather, possibly demonstrating a differential interest on the part of the two units to varying as- pects of the same stimulus. The record gaps are due to the halting of recording during stimulus changes. 681h5-8A: The gap at approximately 750 seconds is due to a tape change. The unit continued to fire. mum um um mmn 66145 ~3A-2 u v u vi as vii e (ARM) W MIIRI‘LIULH I‘LPJJLI‘LMMLW'NUI“ II I n - Mans d aid (1 WI 2 § ”“5.“ sens so u 3 6L n W as l[:::Jib & - o - W was I. O a sane -9A ““5'” = (LEG) E 3 z m 2 1 s O m m 0 L 5 1000 i TIME IN SECONDS Figure D6. 59 AVERAGE NUMBER OF SPIKES PER SECOND "(MEI Illlllli IIIIE PBIIIIIIS 66146-10A-3 I (LEG) WW 12 Figure D7. sens-us (new - ““5‘ .. seasons us . 9 J I sens-13A sens-13a 1000 TIME "6' SECONDS O 61 Figure D8: Estes on the average firing rate profiles. 681h5-lhB-2: The high peak at 900 seconds is due to an accidental stim- ulation of the unit's response area on the body surface. This unit was typical of the somatic responding units which responded to appropriate mechanical stimulation with a furious burst of activity that usually was very quick to adapt back to the maintained level. 681h5-15A: The gap is again due to a change of tape reels and not to unit shut down. AVERAGE NUMBER OF SPIKES PER SECOND "(MEI IIIIIE “If PIIIIIIS 66146-1” W was-14A 66146-146-1 (ARM) 66146-1464 (ARM) TIME IN SECONDS I i ure D8. 62 H CQ 63 Figure D9: Notes on the average firing rate profiles. 68lh5-15C—3: This unit was the lowest in amplitude of the h units ob- tained from this recording locus. It frequently was buried in rather large electrical artifacts (arising from a source external to the sheep) which made the presentation of a continuous record difficult. Hence, only a relatively clean sample is presented here. It should be noted that such artifacts were easily removed during the accumulation of the interspike interval histogram and, thus, do not constitute a serious source of error in the plotting of the spike interval distribution. Tbchnical vagaries, however, made ' suppression of these artifacts a difficult matter while the average firing rate profile was being computed. 681h5-18A: The dotted discontinuities represent electrical artifacts de- rived from an external source which, for reasons set forth in the discussion of unit 68115-1504. above, were not removable. These artifacts do not affect the accuracy of the interspike interval histogram calculated from these data, however. "ERIE! fllllli I"! Fllfllli AVERAGE NUMBER OF SPIKES PER SECOND amuse-s (MSWMTMV) v.51? e ' use See.- esus-tem 9 sens-teas L» yd {mfiVfi ”1 k I d o W ‘ ‘g 08145-17A (TM) TIME IN SECONDS 61L Figure D9. AVERAGE NUMBER OF SPIKES PER SECOND 1 hr 0 "III“! "MIG Ill“ Pllflfllls GEMS-NC TIME IN SECONDS 05 Q L ure DlO . Appendix E The Interspike Interval Histograms 07 Figure El: Notes on the interspike interval histograms. 67129-2L: See notes on this unit in.Appendix D, Figure D1. 67132-7A: See above. IIIIIISPIIE lIIIflWIl IIISIIIEIIAIS 67129-2L jaw. pos. 3 X 100 1 E- I); 67132-7A leg. 1s! {lesion NUMBER OF SPIKES PER INTERVAL _ _ 1 F 67132-7A lemretumsd to "as pos. W O 125 250 ems-2L jse.pos.2 125 25) s7132-7A leoJree nos. 0 125 2a) . 6N32JA leg. 20d flexion F D o :25 250 67133-1A «an 0 an) 500 INTERVAL IN MILLISECONDS Figure El . 68 Figure E2: Notes on the interspike interval histograms. 67lhl-lOB-2: The intervals are continuous in sections 1. through iii. The vertical scale is multiplied by a factor of 10: in section ii. and by.lOOx in section iii. 671hl-lhA: 10: vertical scale multiplication at 250 msec. NUMBER OF SPIKES PER INTERVAL IITEIISPIII IITIIWII IIISTIIBIIIIS 61133 .15 j 61133-6A O 125 A 2:50 0 250 . soc 5113M“ enemas 8 o 155 A 22.0 0 250 500 P x o . 137141-33 o 125 ‘ 3'55 A 375 57141-14A saw INTERVAL IN MILLISECONDS Figure 7272 . 7O 71 Figure E3: Notes in the interspike interval histograms. 67lh3-3A: 10: vertical scale multiplication at #00 msec. Note harmonic nature of marine. 68lhh-3A: See footnote h, Appendix B. The extreme symmetry of this unit's interval histogram may indicate that an unaccounted for stimulus was being applied during the recording session. NUMBER OF SPIKES PER INTERVAL IIITEIISPIKE IIIIHWII IIISTIIEIIMS ems-es 250 500 ems-40 I 0 125 250 05144-15 Figure E3. 4‘0 67143-35 arm 67143-4C 0 125 2 0 sane-1A arm 0 125 2.50 9;! 55144-1» Q h ' I 128 250 INTERVAL IN MILLISECONDS 72 73 Figure Eh: Notes on the interspike interval histograms. 681hh-5A: No scale expansion in inset. 681h4-8A: 2.5: scale expansion in inset. Note absence of tail in this region. NUMBER OF SPIKES PER INTERVAL IITEISPIKI llITllIfll IIISTIIEIIAIS cam-m 5 144. 8 BB thorax 8’ 125 250 ° '25 25° 0 125 250 ‘25 25° 5e145-2¢ I‘O #50 INTERVAL m MILLISECONDS Figure Eh. 7h 75 Figure E5: Notes on the interspike interval histograms. 681h5-3A-1: This unit was being driven while the interspike interval histogram accumulated. Note in particular the dimunition of the histogram tail and the increase in peak symmetry as the tension is increased. For further details concerning this unit and unit 681h5-3A-2 see Appendix D, fugures D5 and D6. 681h5-8A: 100x vertical scale expansion at 250 msec. Note harmonic nature of all but first maxima. 681h5-9A: 10: vertical scale expansion from hOO msec. on. NUMBER or 39111138 PER INTERVAL IIITEIISPIIE IITEIIVM IIISIIIBIIMIS 55145-5A-1 o . am1s1 "salon 55145- 3A- 1 arm. 2nd fission mum muse.) 55145 M 21. 155. 350 470. 550. 540 750 55145-10»: loo Figure E5 . 250 500 ° INTERVAL 1N MILLISECONDS 76 77 Figure E6: Notes on the interspike interval histograms. 681h5-l3B: 100x vertical scale expansion at 200 msec. Note harmonic nature of peaks. 681h5—l3C: 10: vertical scale expansion at #00 msec. IITEBSPIII llllflfll IIISTIIEIIMS 55145-11A 55145-115 arm o - 250 500 ‘ 125 250 .1 55145-12A 58145-13A g 1 11.1 '2 fi v r g g o O 125 250 u 125 J 250 )< E 05 3 53145-13c F 2 :3 :5 08145-130 BOMB-MA 0 250 500 0 125 - 250 INTERVAL IN MILLISECONDS Figure So. 78 79 Figure E7: Notes on the interspike interval histograms. 681h5-15A: 10: vertical scale expansion at 50 msec. 681h5-150-l: 10: vertical scale expansion at 100 msec. 68lh5-lfiC-3: 10: vertical scale expansion at 200 msec. 681h5-150-h: 10: vertical scale expansion at 50 msec. IIITEIISI'IK! IIITIIVM IIISTIIEIIMIS .. 58145-1484 I'm 0 | n A e Laa- 1.— 1 0 5'33 500 750 as 58145-145-2 2’ IN“ > N 0: 1.1.1 *- E e- D: 3': 0 250 500 (0 1.1.1 3 n 214 3‘ x g 5,? 55145-15c-1 55145-1502 8 redretory respiratory g 3000 4000 2 2H .3 Z 0 125 I 250 0 1000 2000 55145-15c-3 531454504 1 reflratory ;! respiratory INTERVAL IN MILLISECONDS Figure FY. 80 81 Figure E8: Notes on the interspike interval histograms 68lh5-l6A-2: 10x vertical scale expansion at 200 msec. 681h5-lBB: 10x vertical scale expansion at 1200 msec. 68lh5-180: Note the extreme regularity of firing and the harmonic nature of the multiple peaks. 10x scale expansion at 50 msec. IIITIIISPIIE IIITEIWAI IIISTIIBIIMIS 55145-15A-1 . ! 58145-16A-2 ! 1110 58145-17A 58145-18A trout 1......— NUMBER OF SPIKES PER INTERVAL X 100 9 55145-150 58145-155 9L 8| 8? 9. D J '0 :0 100 1 INTERVAL IN MILLISECONDS Figure E18 . 82 Appendix F Further Details of methods A. The Animal The subJects. The subjects were eight domestic sheep Ovis aries, of either Suffolk or a Suffolk-Dorset cross breed. All the subjects were female ranging in age from one to eight years and in weight from 100 to 225 pounds. Anesthesia. Food deprivation was in effect for 2h hours prior to each experiment. Approximately one hour before anesthetization, 100 to 250 milligrams of atropine sulfate were administered interperitoneally to prevent excess secretion. Seven of the animals in this series were anesthetized initially and maintained on a "dial Urethane" mixture. The anesthetic recipe will be found in Appendix G following. Anesthetic concentration was so arranged as to require approximately 1.0 milliliter of"1nai" per pound of animal when injected interperitoneally. One animal was initially anesthetized with Shrnylan (Parke, Davis and Co.) administered intervenouslyu This animmd.(67133) was later maintained on a nitrous-oxide and halothane gas mixture. Surgical Preparation. When the animal lost consciousness - from 15 to #5 minutes after injection for the'TJial" animals and almost instantly for the:5ernylan animal - it was transported quickly to the operating area and tracheotomized. Particular attention was paid to maintenance of the airway. Removal of all blood and mucous by aspiration followed by cauterization of all exposed wound surfaces proved beneficial in this regard. A glass cannula was inserted into the exposed trachea and su- tured in place. The neck wound was then clamped shut. From this point 83 SA on both oxygen and mechanical respiration were available in case of respiratory failure. Such failure was not uncommon.within the first few hours after anesthetization. Generally, a few minutes of mechanical assistance were all that were needed to bring the animal back to unaided breathing. Tracheotomy completed, the animal was hoisted onto supporting rails and the head positioned properly for eventual fastening into a head holder. A midline incision from the eyebrows to the mid-shoulder was made. Working caudally, the skull was exposed over a broad area later- ally'and back to the occipital ridge. At this point the head was posi- tioned in such a fashion as to allow maximum access to the as-yet-to-be- exposed medulla. Three-eighths inch brass rods were affixed to the skull with wood screws and dental acrylic. The head holder accepted the oppo- site ends of the rods, thus providing a firm support. The cranium caudal to the occiput and the first two cervical verte- brae were exposed from the dorsal aspect. Hide areas of muscle were retracted from the wound opening to provide maximum working area. Cau- terization and hemostatic clamps served to check excess bleeding and also provided good visualization. Additional brass rods were fixed to C2 and occasionally the lateral aspect of the occiput to provide the required rigidity. Medullary exposure was accomplished by removal of the portions of the skull case overlying the caudal portion of the cere- bellum and the dorsal portion of the first and the rostral half of the dorsal sapect of the second cervical vertebrae. Excessive bleeding was stemmed with gelatin sponges and bone wax. Care was taken to avoid dural puncture or the application of undue pressure to the neural tissue underlying the exposure. Exposure completed, a dam of dental acrylic 85 was built around the entire opening. The dura was carefully removed from both the medulla and the caudal portion of the cerebellum which was then aspirated away until the obex was exposed. The exposure was then flushed with 0.9% saline solution several times, drained and the dam filled with mineral 011. At this Juncture the preparation was ready for recording. Supportive measures. Normal supportive measures generally included periodic IP injections of normal saline solution to combat dehydration. Anesthetic and atropine were provided as needed during the course of the experiments which often lasted from 2h to 36 hours. Body tempera- ture was monitored and maintained between 3h and 37°C. by means of infra-red lamps. Periodic drainage of the trachea helped maintain the airway. Blood seepage occasioned the drainage, flushing and replacement of the mineral oil over the exposed medulla numerous times during the course of an experiment. B. The Recording Session Photography. Upon completion of the preparative procedures, a photograph was taken of the preparation and the exposure. Developed and printed before the recording session began, such a print served as a convenient means of locating topographical landmarks such as blood vessels, sulci and the like with respect to the electrode punctures. These punctures were marked on the photograph as they were made. Later they served as an aid in track tracing, giving the relative positions of the punctures with respect to one another. The receptive fields of those units which responded to mechanical stimulation of the body were mapped out and identified as to puncture on the whole body pictures. 86 The recording electrodes. The electrodes were either glass insu- lated tungsten microelectrodes similar to those used by Baldwin, gt 2;. (1965) or they were lacquered tungsten similar to the type used by Eubel (1957). In either case the best tip size was found to be on the order of 30 to ho microns of exposed metal beyond the insulation. Such a tip size typically gave a DC resistance of less than one megohm, there- by allowing a Tektronix type 122 AC preamplifier to be used without a cathode follower input. The recording equipment. Other recording equipment consisted of Tektronix types 502A or 565 oscilloscopes for visual monitoring. The preamplifier also drove a Grass type Ahkj audio monitor. The vertical output signal from the oscilloscope fed one channel of a high fidelity audio tape recorder (Magnecord type 1028); the second channel being re- served for voice commentary and time marking. The neural activity Obtained was always taped in this manner. Occasionally an interesting unit would be buried in considerable background activity or be plagued with a widely varying baseline. In these cases the signal was led through a 1.0 kHz filter which effectively removed most activity with risetimes less than that possessed by most nerve spikes. In general this was avoided where possible as it was felt that such signal proc- essing could be accomplished to better advantage upon playback of the signal. Recording procedures. The electrode punctures were ideally ar- ranged in rows running medic-laterally and from rostral to caudal. In such an arrangement prior punctures would interfere minimally with the neural inputs of the punctures yet to follow. The puncture rows and columns were generally spaced one-half millimeter to one millimeter 87 apart. In practice this seldom timed out to be the case. The pial sheath at the level oftheaxposed obex is suchthat allbutthehar- diest electrodes cone to grief when a puncture in this regim is attunpted. Hence, most punctures in this area were gladly accepted wherever they happened to fall. Elsewhere (:1 the medullery surface, blood vessels had to be avoided, as did the midline. Extreme lateral. partials were not available either as the electrodes usually will not enter the tissue unless the approach is at a near normal angle. The coverage obtained in these experiments was unquestimably weighted in favor of the regime of the dorsal column, the descending fibers of the fifth nerve and their respective nuclei. The rostral medulla was ignored conpletely. The more ventral portions of the medullary areas considered also suffered from lack of equal representation with respect to the dorsal half. Individual nmctm'es were conducted in the following manner. The medullary surfacehavingbeembroached -not always aneasymatter ashes been noted above - the surface position was noted as the vernier of the microdrive appuatus controlling the electrode movement. The tip was lowered until a single unit was discernable fro: the background. An attempt at amplitude maximization was made. This usually served as an indication as to whether the unit was responding primarily to the elec- trode or to, hopefully, sanething else. A wait of a mimte or two gave anideaastot‘nepermanencyoftheunit. Iftheamplitudeandfrequency remained reasonably constant over this period, it was seemed that the unit in question was not responding, as least primarily, to the pressure of the electrode. At this point mechanical stimlaticn of the bob sur- faces was begun to determine whether the unit oculd be so driven and thus attributed to the somatic sensory system. 88 The recording periods ranged from a minimum of 1.5 minutes to over an hour. The length of time that a unit was recorded depended upon a number of factors such as the frequency of the unit (that is, how rap- idly the spikes accumulated), the unit's viability and, to a certain extent, the durability of the experimenter. "Good" units would usually grow weaker or stronger in amplitude as the electrode was advanced or retracted slightly, but the unit's frequency would remain unaffected by electrode movement. Once a "good” unit was obtained, it was not bothered by further movement of the electrode nor was the animal touched at all until the desired data had been collected. However, certain units responded with varying discharge patterns to changes in limb and Jaw position. This positions could be varied to elicit a new firing rate or pattern in the unit being recorded. Once the somatic or non-somatic nature of a unit had been determined, the animal was left alone with the exception of units 67129-2L, 67132-7A, 681h5-3A—l and 681h5-3A-2. Further details on these "driven” units will be found in appendices D and E. C. Data Processing Playback procedures. Preliminary to the actual construction of either interspike interval histograms or average firing rate profiles, the taped data were played back through both the audio monitor and an oscilloscope. Qualitative Judgments were then made as to the retriev- ableness and subsequent reliability of the signals. Where needed, sharp low frequency filtering and/or rectification of the spike train under examination made the creation of standard pulses much easier. It was not infrequent that two or more spikes would be present simultaneously. Our equipment usually permitted the retrieval of the data when two spikes 89 were present. Three spikes usually resulted in retrieval of the average firing rate profiles but prevented an accurate construction of the interspike interval histogram of the smallest amplitude spike. For instance, two spikes of similar positive amplitude might have dis- similar negative amplitudes. Rectification of the positive portion of the signal and inversion of the negative portion would allow the ampli- tude gate to select one or the other of the signals. For these purposes both a voltage amplitude gate and a voltage "window” were used. The window was useful when the spike train of interest was of less amplitude than some interfering train. Both gating devices put out standard pulses which were then replayed into the oscilloscope and visually compared with the original neural signal. A one to one correspondence of shaped pulse to original spike was required before farther processing took place. A CAT hOOB computer was programmed to construct either interspike interval histograms or to count the spikes falling within a measured time interval. Close visual and aural monitoring of the signal while it was undergoing processing allowed some compensation for amplitude changes to he made. Large artifacts could sometimes be removed from the record by "riding" the input gate control if it was know when to expect these interfering signals. Construction of the average firing rate profiles. The data pre- sented in this thesis which is referred to as "average firing rate pro- files" were obtained from the CAT LOOB computer under the following conditions. An external square wave generator operating at 0.1 Hz/sec. advanced the CAT address every 10.0 seconds. Any pulse arriving at the CAT memory input during a given ten second interval would be stored 90 consecutively until the address was advanced to the next channel. In this way it was possible to obtain the number of spikes per ten seconds for periods extending to hOOO seconds. The ten second bin width was used throughout these experiments. Thus, all the profiles are directly comparable, though some plotted profiles are "doubled" into 20 second bins to conserve plotting Space. Computation of the interspike interval histograms. Interspike interval histograms could be computed directly with the CAT in its H or histogram mode. The entire #00 channel address bank would be swept at some predetermined rate such that the full scale sweep time was substantially longer than the majority of the interspike intervals. Each Spike would initiate a sweep; its successor would terminate it, entering that portion of the sweep which had been completed into the appropriate channel's memory, and starting the process over again. For most of the units in this study a sweep time of 1.0 second was used, giving a bin width of 2.5 milliseconds for each of the hOO channels. A few slower firing units required somewhat longer sweep periods. Data presentation. The processed data were printed out on a tele- type printer. Analogue output into an.X-Y'plotter was also possible and was used for on the spot checks of progress during computation. The data presented in graphic form in this study were plotted by hand from the numerical teletype data for reasons of accuracy and format. The plotted data are presented in appendices D and E. 91 D. Histology At the termination of the recording session, an electrode was driven into the medulla at a location sufficiently far from the recording sites so as not to be mistaken later for a true puncture. This electrode was released from the driving mechanism and its shaft clipped off Just above the point of entry into the medulla. The purpose of this was to provide a slicing plane reference for later microtoming so that the punctures would be in the plane of the cut. This accomplished, the animal was re- leased from the head holder given a fresh dose of anesthetic and low— ered to the floor, chest up. An intercardial perfusion of 0.9% saline followed by 10% formalin in 0.9% saline served to fix the tissue. The head was removed, partially skinned and placed in a bucket of fixative to await removal of the medulla. The carcass was incinerated. After the medulla was removed from the head, it was cleaned of ex- ‘cess dura and epipia and dehydrated in successive stages preparatory to celloidin embedding. The blocked medulla was sliced at 30 microns with alternate sections being Nissl (thicnin) stained.‘ Alternate sections of the remaining half of the slices received either a weil (hematoxylin) or a Sanidea Heidenhain (hematoxylin) stain for myelinated fibers. Generally the thionin sections were sufficient for electrode track localization. The tracks appeared as a greenish-yellow streak where glioais had occured in response to the presence of the electrode. Finer anatomical detail was often discernable with the other stains. Used conjointly, these stains made track localization and unit identification possible as a matter of routine. Appendix C gives the tracings of the electrode tracks with puncture locations of relevant units for the six animals in.which these techniques 92 were used. Individual unit locations within a puncture were roughly located on the basis of response to stimulation, if any, and relative depth as recorded from the microdrive verneir. Placing an electrolytic lesion at some known depth in the puncture sometimes allowed the precise location of a particular unit. Other units in the puncture could then be approximately located by using a simple proportional relationship. Differential tissue shrinkage and other factors make these methods rather imprecise, however. Appendix G The Recipe for Dia14Urethane Dissolve 29.5 grams of dial (5,5-diallybarbituric acid, Gene's Chemical works, 611 Broad $12., Carlstadt, NJ.) in 150 cc. of distilled water together with approximately 35 pellets of sodium hydroxide. Warm slightly until fully dissolved. Dissolve 9h.6 grams of urethane (Sargent,'USP grade) in 300 cc. of distilled water. Mix 1. and 2. Dosages: a. initially: 1.0 cc. per pound of animal (sheep) b. maintenance: 0.25 cc. per pound of animal as needed. 93