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Myelination and the development of function in immature pyramidal tract
EXPERIMENTAL
NEUROLOGY
Myelination
29,
405-415
and
(1970)
the
Development
in Immature PETER Departwerkts
af
Neurology
of
Pyramidal R.
Tract
HUTTENLOCHER
arkd Pediatrics, ITale New Havelk. Co~knectimt Rccrivcd
July
Function
1 Uninrersity 06510
School
of
Medicilke,
23, lQ70
The functional properties of immature pyramidal tract neurons were investigated in kittens 3 days to 5 weeks of age. Pyramidal tract neurons were identified and activated by antidromic stimulation of their axons in the medullary pyramid. Antidromically evoked responses in neurons of sensorimotor cortex were found already in the youngest animals. However, these responses fatigued rapidly. Two effects were seen on repetitive stimulation. During the first postnatal week there was a progressive decrease in amplitude of extracellularly recorded unit spikes at stimulus rates of lO/sec or higher. In the older kittens, conduction block occurred in the axon during repetitive stimulation above ZO-30/set. This effect was seen consistently up to age 3 weeks, and it was present in some of the units recorded at age 5 weeks. Axons with conduction velocities below 2 m/set, which presumably were unmyelinated, showed conduction block on repetitive stimulation at low rates. Axons with conduction velocities above 3m/sec were able to conduct volleys at 40/set and higher. The results suggest that myelination increases the ability of central axons to fire repetitively. Introduction
For some time after the onset of electrical excitability the immature central nervous system continues to differ markedly from the adult in its functional properties. One such difference lies in the ability to maintain sustained activity. A rapid decrease in amplitude of evoked potentials to repetitive sensory or electrical stimuli has often been demonstrated in immature cerebral cortex (4, 5. 7). On the efferent side, movements elicited by electrical stimulation of immature motor cortex decrease in amplitude and then cease entirely when the stimulus is applied repeatedly (8, 22). The present study was undertaken to determine whether fulictional limitations
in
the
fiber
tracts
which
comprise
the
subcortical
white
matter
might contribute to the fatiguability of responses in the immature brain. Maturation of fibers in the subcortical white matter is a relatively late developmental event, In the kitten, stainable myelin does not appear in 1 Suprorte:!
by
Grant
NB
07105,
U.S.
Public 405
Health
Service.
406
HUTTENLOCHER
subcortical white matter until 2-3 weeks after the onset of neuronal activity in cerebral cortex (IO). The small, unmyelinated fibers found in the immature brain would be expected to conduct nerve impulses less efficiently than myelinated fibers of similar diameter (9 j . Observations have been carried out in single pyramidal tract axons of the kitten, prior to and during myelination. The pyramidal tract was chosen, since it forms a well-defined anatomic pathway of sufficient length for measurement of conduction velocities, and since its axons can be positively identified by the method of antidromic stimulation (17, 20). Methods
Pyramidal tract axons were activated by electrical stimulation of the medullary pyramid. Antidromically evoked single-cell spikes were recorded from sensorimotor cortex. Successful experiments were carried out in 18 animals, ages 3 days to 5 weeks. The kittens were anesthetized with ether, and ventilation was maintained with a pump respirator. The head was immobilized by use of a stereotaxic instrument with a rodent mouth bar attachment. The skull overlying the precruciate and postcruciate gyri on one side was removed, the dura mater being left intact. Bipolar stainlesssteel stimulating electrodes, insulated to the tip with enamel and 0.2 mm in diameter, were placed stereotactically into the medullary pyramid. This was achieved by flexing the head (mouth bar 15 mm below zero), and by inserting the electrodes through a burr hole at coordinates P 4-5, L 0.5. The medullary pyramid was reached 14-18 mm below the level of the dura mater, depending on the age of the animal. Most of the experiments were carried out with the animals under light ether anesthesia, supplemented with succinylcholine. In five kittens, a microelectrode pedestal and stimulating electrodes were implanted under ether anesthesia, and the observations were carried out after the animals had recovered from the anesthesia. Details of this method of implantation and recording are given in a previous report (10). Recordings were obtained from pyramidal tract neurons in precruciate and postcruciate gyri. Extracellular microelectrodes of the type described by Wolbarsht, MacNichol, and Wagner (24) were used. The microelectrodes were advanced into the brain by means of a hydraulic microdrive. Conventional amplifyin g equipment was used, and the single-unit activity was displayed on an oscillograph, and was recorded with a kymograph camera. Stimulation of pyramidal tract fibers in the medullary pyramid was achieved by means of a square-wave stimulator which provided single shocks 20-100 pv in duration. Correct placement of stimulating electrodes in the medullary pyramid was verified histologically. Stimulation of the medullary pyramid results in both direct (antidromic)
MYELINATION
AND
407
FUNCTION
and in synaptically conducted single cell responses in motor cortex. The latter may be secondary to synaptic activation of cortical cells via axon collaterals of pyramidal tract cells, or they may be due to spread of the stimulus current to afferent (lemniscal ) pathways in the medulla (3, 13). Several criteria for the distinction of direct antidromic from synaptically activated cortical units in adult cats have been laid down previously (17, 20). These include fixed latency of response, which should be less than 10 msec, invariable responses at stimulus rates of 50/set or higher, and activation by a small stimulating current (5v or less for 50 +ec or less). These criteria have to be modified for the immature animal ( 11) . Latencies are very much longer, and rather strong shocks to the medullary pyramid are needed to activate any cortical units. Filred latency of response and consistent following at low stimulus rates (up to lO/sec) remain as the most important criteria for identification of immature pyramidal tract units. Failure of evoked responses at low frequencies of stimulation is seen only when a spontaneous spike discharge precedes the stimulus by an interval equal to or less than the response latency (Fig. 1). A similar interaction between spontaneous and evoked spikes is not seen in synaptically activated
J-m--w
+----I
I
1 FIG. 1. A. Single unit record from a Z-day-old kitten. The unit is synaptically activated by the pyramidal tract stimulus. Multiple discharges are evoked by each pyramidal tract shock. There is no occlusion of evoked discharges by spontaneous discharges preceding the stimulus to the medullary pyramid (lower trace). B. Antidromically activated pyramidal tract neuron, same animal. Pyramidal tract stimuli are followed by single, fixed-latency spike discharges (upper and middle trace). The evoked spike is absent when the stimulus is preceded by a spontaneous spike discharge (lower trace). Calibration 500 pv, 50 msec. Positivity is indicated by a downward deflection.
408 units. The conducted occurring helpful in ducted.
HUTTENIACHER
effect appears to be explained by occlusion of the antidromically wave of depolarization by a simultaneous orthodromic discharge in the same axon. In the present study, this finding was often confirming that a given unit response was antidromically conResults
Response Latencies to Medulby Stimulation in Inwmture Pyrakdal Tract neurons. Thirty-six pyramidal tract neurons were identified and studied, in animals ranging from age 3 days to 5 weeks. Similar results were obtained in both unanesthetized and in lightly anesthetized, curarized animals. The data from both prepartions are, therefore, presented together. Latencies from medullary stimulus to cortical neuron spike in animals under age 2 weeks varied between 20 and 38 msec (Fig. 2). The measured distance between the stimulating and recording electrodes was about 30 mm, yielding conduction velocities of 0.8 - 1.5 m/set. After age 2 weeks units with response latencies below 10 msec, and with calculated conduction velocities up to 14 m/set were found. However, even at age 5 weeks some pyramidal tract fibers still had conduction times above 20 msec. Histologic sections of the pyramidal tracts in the medulla showed that stainable myelin appeared during the age period of the study. Faintly myelinated fibers were seen by age 14 days, and myelination progressed slowly over the next several weeks (Fig. 3). Response of Pyramidal Tract Neurons to Repetitive Stiwmla.tion. Responses of pyramidal tract neurons to repetitive stimuli were determined at stimulus rates of l-lm/sec for periods of 20 sec. All units showed con-
40 0
I
0 2 E -20
30
0 -
O
0
8
0 H
0 0 0
00
8
t Y P a-! lo-
ts 0
; 0
0 IO
20
AGE
FIG. 2. Latency from medullary 36 pyramidal tract neurons recorded between ages 3 and 40 days.
!j 30
40
(days)
pyramid shock from precruciate
to antidromic spike discharge in and postcruciate gyrus in kittens
MYELINATION
AND
FUNCTION
409
sistent responses at stimulus rates of 1-3/set, provided that prolonged repetitive stimulation had not occurred for at least 30 set prior to testing. However, a majority of the neurons were unable to follow consistently at stimulus rates above lo-20/set. In the youngest animals, especially in those studied prior to age 5 days, repetitive stimulation at lO/sec or higher resulted in a gradual decrease in amplitude and increase in duration of extracellularly recorded unit spikes. This effect was also seen during repetitive firing of spontaneously active cortical neurons in this age group. By age 1 week most cortical neurons were able to follow at rates of 10-20,’ set without marked decreases in spike amplitude. A second effect of repetitive stimulation was noticeable from birth, but became especially evident in the second and third postnatal weeks. after cortical spikes had become relatively stable. It consisted of a gradual increase in latency from pyramidal tract stimulus to cortical unit response during repetitive stimulation. Latency increases of up to 12 msec were seen. During continued repetitive stimulation lengthening in conduction time was followed by variable response of the pyramidal cell spike, and eventually by complete absence of evoked spikes. The failure of the pyramidal celI response was an all or none event, in contrast to the gradual failure of the cellular spike seen in the first postnatal week. Recovery was rather slow. After prolonged repetitive stimulation the latency did not return to resting levels until 30 set to 1 min after the last stimulus. Typical responses are illustrated in Figs. 4 and 5. Figure 4 shows the decrease in spike amplitude at lO/sec stimulation seen in the first postnatal week, Superimposed, there is gradual lengthening in conduction time. Slow recovery of conduction following repetitive stimulation is illustrated in ~olunm C. The first sweep in this column represents the unit response 30 set after a train of stimuli had been presented. The evoked spike has returned to full amplitude, but response latency is prolonged. The latency has returned to the prestimulation value 30 set later (middle trace). Figure 5 illustrates a unit recorded from a l4-day-old animal, The pyramidal cell spike is quite stable. ‘At lO/sec stimulation (middle trace) the unit spike at first follows very closely upon the subsequent stimulus artifact. After a brief train of stimuli there is a gradual increase in the distance between stimulus artifact and unit spike, indicative of increase in response latency. The unit responds only intermittently in the last trace. Fourteen pyramidal tract cell recordings were obtained in animals 3-5 weeks old. These neurons represented a heterogeneous group. A few cells were found to have characteristics of immature neurons, with Jong response latencies, and with development of conduction block on repetitive stimulation above 20-30/set. However, the majority of pyramidal tract cells in this age group had response latencies below 12 msec. These neurons were
410
HUTTENLOCHER
MYELINATION
t
AND
I------
411
FUNCTION
‘/r+----
1
4. Response to antidromic stimulation in a pyramidal tract neuron from a 3-dayold kitten. A. Unit spike, followed by evoked slow wave potential, at l/set stimulation. B. Rapid decrease in amplitude of the unit spike at lO/sec stimulation. The evoked slow wave response also fatigues rapidly. C. Recovery of unit response and of slow wave potential 30 set (top trace), 60 set (middle trace), and 90 set (lower trace) after cessation of repetitive stimulation. Calibration 500 pv, 50 msec. FIG.
able to maintain repetitive firing at stimulus rates of 40-60/set for at least 20 sec. No increase in response latency was seen during repetitive firing at these rates (Fig. 6). In general, units with long response latencies showed lengthening in conduction time and conduction block on repetitive stimulation. Units with response latencies below 12 msec were able to fire at stimulus rates of 40-60/set. The relationship between response latency and ability to maintain repetitive activity is illustrated in Fig. 7. Discussion
The present observations indicate that pyramidal tract neurons in kittens .differ markedly from the adult in their ability to undergo repetitive discharges. In the youngest animals, a decrease in amplitude and increase in duration of extracellularly recorded unit spikes is noted during repetitive activity. This finding confirms observations by Conway, Wright, and Bradley (4). who noted breakdown of extracellularly recorded unit spikes on repetitive pyramidal tract stimulation in the neonatal rabbit. The effect is FIG.
(top), x 30.
3. Myelination of the pyramidal 30-day-old kitten (middle), and
tract in the medulla of a 15-day-old kitten adult cat (lower). LVeil’s stain. Magnification
HUTTENLOCHER
412
C
B
A
3 FIG. 5. Response 14 days. A. ZO/sec progressive increase C. Recovery of the
of a pyramidal tract unit to repetitive antidromic stimulation, age stimulation. The unit follows wetl. B. 40/set stimulation. There is followed by conduction block (lower trace). in response latency, response after a period of rest. Calibration 200 pv, 50 msec.
&nilar to what is seen in mature neurons during injury discharges, during seizure discharges, or during very rapid repetitive stimulation. Phillips ( 17) observed breakdown of spikes at stimulus rates of about 50 to over 400 spikes/set in recordings from adult pyramidal tract cells. Breakdown of spikes at low rates of firing (4O/sec and less) appears to be characteristic of immature cortical neurons for the first few days after the onset of electrical excitability (4, 10, 11). After the first postnatal week in the kitten, repetitive firing in pyramidal tract units is no longer limited by inability of the soma to undergo repetitive activity. Rather, the limiting event now appears to be conduction along
1 FIG. 6. Response of a pyramidal 35 days. The unit shows persistent 50 msec.
I
1
tract unit to repetitive antidromic stimulation, age following at 60/set stimulation. Calibration 500 gv,
MYELINATION
AND
413
RJNCTION
ki 5 b u 5 % 3
20
-
9
c 10
c 20
LATENCY FIG.
sponse
7. Relationship in 33 pyramidal
0
0 30
0
I 40
(msec)
between maximum response rate tract neurons recorded between
and latency of antidromic ages 3 and 40 days.
re-
the axon. The response to repetitive stimulation in pyramidal tract cells between ages 1-3 weeks has features characteristic of conduction block in the axon, consisting of a progressive lengthening in conduction time, followed by abrupt and complete loss of evoked spikes recorded at cortical level. The positive correlation observed between conduction block and conduction velocity in pyramidal tract fibers suggests that myelination may be a factor in the ability of pyramidal tract axons to fire repetitively. One has to assume that fibers in immature pyramidal tract that conduct at velocities above 3 m/set have acquired functionally significant myelin along at least part of their course, since the pyramidal tract in the kitten does not contain unmyelinated fibers of a size which would allow such rapid conduction. Purpura et al. (18) did not find pyramidal tract fibers of greater than 2 11 diameter prior to age 4 weeks. Traces of stainable myelin become visible in the pyramidal tract at the time when units with rapid conduction velocity first appear (15, 18). Myelination would be expected to have a marked effect on the efficiency of conduction along the axon. Keynes and Ritchie (14) have estimated that the active membrane surface of a small, unmyelinated fiber is between 100 and 1000 times greater than is that of a myelinated fiber of equal length and diameter. The outflow of potassium per nerve impulse in a small, UWmyelinated fiber has been calculated to be about 300 times that of a myelinated fiber of the same size (9). As a result, the energy expenditure of unmyelinated fibers during repetitive activity has to be considerably greater
414
HUTTENLOCHER
than it is in fibers that are surrounded by a myelin sheath, provided that both types of membranes have to maintain similar ionic gradients. There is evidence which suggests that mature unmyelinated fibers in peripheral nerve have low resting membrane potentials, high internal sodium concentrations, and low potassium concentrations (14). However, it is doubtful that such special adaptation occurs in immature unmyelinated fibers in the central nervous system. While measurements of resting membrane potentials on immature central axons are unavailable, resting potentials of their soma membranes are similar to those in adults (19). Excessive outflow of potassium from immature axons during repetitive activity would provide an explanation both for the observed increase in conduction time and for conduction block. Both of these events occur when the internal potassium concentration of the axon is lowered (2). Depletion of internal potassium ion and accumulation of sodium ion may occur especially rapidly during repetitive activity of immature axons, since the sodium pump mechanism in immature brain appears to be as yet poorly developed (12). The inability of developing axons to maintain repetitive activity may be of functional importance in the immature brain. In the adult, rapid repetitive firing of cortical neurons is commonly seen during normal activity. As early as 1939 Adrian and Moruzzi (1) found that motor activity of an animal is correlated with high-frequency discharges in pyramidal tract fibers. Afferent stimuli or direct stimulation of motor cortex result in movement only when they produce rapid repetitive firing in pyramidal tract. More recent studies in the monkey by Evarts (6) have shown a correlation between bursts of rapid discharges in pyramidal tract units and voluntary contractions of small muscle groups. Firing rates of 50-lOO/sec were seen in pyramidal tract neurons during voluntary contraction of hand and forearm muscles. The inability of immature axons to maintain such rapid rates of activity may provide an explanation for the correlations that have been observed between myelination and the appearance of function in the immature brain (16, 21). On the other hand, it is clear that some functions, primarily those of simple reflex type, develop prior to and quite independent of myelination (23). References 1. ADRIAN, E.*lD., and G. MORUZZI. 1939. Impulses in the pyramidal tract, J. Physiol. Lortdon 97: 153-199. 2. BAKER, P. F., A. L. HODGKIN, and T. I. SHAW. 1%2. The effects of changes in internal ionic concentrations on the electrical properties of perfused giant axons. J. Physiol. LOPkdOlk. 164: 3.55-374. 3. CHANG, H. T. 195.5. Activation of internuncial neurons through collaterals of pyramidal fibers at cortical level. J. Neurophysiol. 18: 452-471.
MYELINATION
AND
FUNCTIOX
415
4. CONWAY, C. J., F. S. WRIGHT, and W. E. BRADLEY. 1969. Electrophysiological maturation of the pyramidal tract in the post-natal rabbit. Elcctvocncephalogr. Cl& Newophysiol. 26 : 565-577. 5. ELLINGSON, R. J., and R. C. WILCOTT. 1960. Development of evoked responses in visual and auditory cortices of kittens. J. Nc~~rophgsio.!. 23: 363-375 6. EVARTS, E. V. 1966. Pyramidal tract activity associated with a conditioned hand movement in the monkey. J. Nc~rophysiol. 29: 1011-1027. 7. GROSSMAN, C. 1955. Electra-ontogcnesis of cerebral activity. &4X.,4. Arch. Ncwol. Psych&. 74 : 18fXO2. 8. HENRY, E. W. 1943. Somatic motor responses produced by electrical stimulation of the cerebral cortex of new-born and young kittens. Fed. Proc. 2: 21. 9. HODGKIN, A. L. 1951. The ionic basis of electrical activity in nerve and muscle. Biol. Rezl. 26: 339409. 10. HUTTENLOCHER, P. R. 1967. Development of cortical neuronal activity in the neonatal cat. Errp. Ncwrol. 17: 247-262. 11. HUTTENLOCHER, P. R. 1969. Functional properties of pyramidal neurons during development of cerebral cortex in the kitten. Fed. PYOC. 28: 825. 12. HUTTENLOCHER, P. R., and M. D. RAWSON. 1968. Neuronal activity and adenosine triphosphatase in immature cerebral cortex. Exp. Nrurol. 22: 118-129. 13. JABBUR, S. J., and A. L. TOWE, 1961. Analysis of the antidromic cortical response following stimulation at the medullary pyramid. 1. Pizgsiol. Lotrdo~ 155: 14% 160. 14. KEYNES, R. D., and J. M. RITCHIE, 1967. The movement of labelled ions in mammalian non-myelinated nerve fibers. /. Physiol. London. 188: 309-327. 15. LANGWORTHY, 0. R. 1927. Histological development of cerebral motor areas in young kittens correlated with their physiological reaction to electrical stimulation. Co&rib. Enzbryol. 19: 177-208. 16. LANGWORTHY, 0. R. 1932. Development of behavior patterns and myelinization of tracts in the nervous system. A.M.A. drclt Nruvol. Psychiat. 28: 1365-1382. 17. PHILLIPS, C. G. 1959. Actions of pyramidal volleys on single Bertz cells in the cat. Quart. J. Enp. Physiol. 44: l-25. 18. PURPURA, D. P., R. J. SHOFER, E. M. HO~S~PIAN, and C. R. NOBACK. 1964, Comparative ontogenesis of structure-function relations in cerebral and cerebellar cortex. Progr. Braix Rcs. 4 : 187-221. 19. PURPURA, D. P., R. J. SHOFER, and T. SCARFF. 1965. Properties of synaptic activities and spike potentials of neurons in immature neocortex. J. Nnrrophysiol. 28 : 925-942. 20. ROSENTHAL, F., J. W. WOODBURY, and H. D. PATTON. 1966. Dipole characteristics of pyramidal cell activity in cat postcruciate cortex. J. N:c~~~ophysioZ. 28: 612625. 21. TILNEY, F., and J. CASAMAJOR. 1924. Myelinogeny as applied to the study of behavior. AMA Arch. Ncuvol. Psychiat. 12: l-66. 22. WEED, L. H., and 0. R. LANGWORTHY. 1926. Physiological study of cortical motor areas in young kittens and in adult cats. Coutrib. Embrgol. 17: 91-106. 23. WINDLE, W. F., M. W. FISH, and J. E. O’DONNELL. 1934. Myelogeny of the cat as related to development of fiber tracts and prenatal behavior patterns. J. Comp. Neural. 59: 139-165. 24. WOLBARSHT, M. L., E. F. MACNICHOL, and II. G. WAGNER. 1960. Glass insulated platinum microelectrodes. .I?ciettce 132 : 1309-1310.
NEUROLOGY
Myelination
29,
405-415
and
(1970)
the
Development
in Immature PETER Departwerkts
af
Neurology
of
Pyramidal R.
Tract
HUTTENLOCHER
arkd Pediatrics, ITale New Havelk. Co~knectimt Rccrivcd
July
Function
1 Uninrersity 06510
School
of
Medicilke,
23, lQ70
The functional properties of immature pyramidal tract neurons were investigated in kittens 3 days to 5 weeks of age. Pyramidal tract neurons were identified and activated by antidromic stimulation of their axons in the medullary pyramid. Antidromically evoked responses in neurons of sensorimotor cortex were found already in the youngest animals. However, these responses fatigued rapidly. Two effects were seen on repetitive stimulation. During the first postnatal week there was a progressive decrease in amplitude of extracellularly recorded unit spikes at stimulus rates of lO/sec or higher. In the older kittens, conduction block occurred in the axon during repetitive stimulation above ZO-30/set. This effect was seen consistently up to age 3 weeks, and it was present in some of the units recorded at age 5 weeks. Axons with conduction velocities below 2 m/set, which presumably were unmyelinated, showed conduction block on repetitive stimulation at low rates. Axons with conduction velocities above 3m/sec were able to conduct volleys at 40/set and higher. The results suggest that myelination increases the ability of central axons to fire repetitively. Introduction
For some time after the onset of electrical excitability the immature central nervous system continues to differ markedly from the adult in its functional properties. One such difference lies in the ability to maintain sustained activity. A rapid decrease in amplitude of evoked potentials to repetitive sensory or electrical stimuli has often been demonstrated in immature cerebral cortex (4, 5. 7). On the efferent side, movements elicited by electrical stimulation of immature motor cortex decrease in amplitude and then cease entirely when the stimulus is applied repeatedly (8, 22). The present study was undertaken to determine whether fulictional limitations
in
the
fiber
tracts
which
comprise
the
subcortical
white
matter
might contribute to the fatiguability of responses in the immature brain. Maturation of fibers in the subcortical white matter is a relatively late developmental event, In the kitten, stainable myelin does not appear in 1 Suprorte:!
by
Grant
NB
07105,
U.S.
Public 405
Health
Service.
406
HUTTENLOCHER
subcortical white matter until 2-3 weeks after the onset of neuronal activity in cerebral cortex (IO). The small, unmyelinated fibers found in the immature brain would be expected to conduct nerve impulses less efficiently than myelinated fibers of similar diameter (9 j . Observations have been carried out in single pyramidal tract axons of the kitten, prior to and during myelination. The pyramidal tract was chosen, since it forms a well-defined anatomic pathway of sufficient length for measurement of conduction velocities, and since its axons can be positively identified by the method of antidromic stimulation (17, 20). Methods
Pyramidal tract axons were activated by electrical stimulation of the medullary pyramid. Antidromically evoked single-cell spikes were recorded from sensorimotor cortex. Successful experiments were carried out in 18 animals, ages 3 days to 5 weeks. The kittens were anesthetized with ether, and ventilation was maintained with a pump respirator. The head was immobilized by use of a stereotaxic instrument with a rodent mouth bar attachment. The skull overlying the precruciate and postcruciate gyri on one side was removed, the dura mater being left intact. Bipolar stainlesssteel stimulating electrodes, insulated to the tip with enamel and 0.2 mm in diameter, were placed stereotactically into the medullary pyramid. This was achieved by flexing the head (mouth bar 15 mm below zero), and by inserting the electrodes through a burr hole at coordinates P 4-5, L 0.5. The medullary pyramid was reached 14-18 mm below the level of the dura mater, depending on the age of the animal. Most of the experiments were carried out with the animals under light ether anesthesia, supplemented with succinylcholine. In five kittens, a microelectrode pedestal and stimulating electrodes were implanted under ether anesthesia, and the observations were carried out after the animals had recovered from the anesthesia. Details of this method of implantation and recording are given in a previous report (10). Recordings were obtained from pyramidal tract neurons in precruciate and postcruciate gyri. Extracellular microelectrodes of the type described by Wolbarsht, MacNichol, and Wagner (24) were used. The microelectrodes were advanced into the brain by means of a hydraulic microdrive. Conventional amplifyin g equipment was used, and the single-unit activity was displayed on an oscillograph, and was recorded with a kymograph camera. Stimulation of pyramidal tract fibers in the medullary pyramid was achieved by means of a square-wave stimulator which provided single shocks 20-100 pv in duration. Correct placement of stimulating electrodes in the medullary pyramid was verified histologically. Stimulation of the medullary pyramid results in both direct (antidromic)
MYELINATION
AND
407
FUNCTION
and in synaptically conducted single cell responses in motor cortex. The latter may be secondary to synaptic activation of cortical cells via axon collaterals of pyramidal tract cells, or they may be due to spread of the stimulus current to afferent (lemniscal ) pathways in the medulla (3, 13). Several criteria for the distinction of direct antidromic from synaptically activated cortical units in adult cats have been laid down previously (17, 20). These include fixed latency of response, which should be less than 10 msec, invariable responses at stimulus rates of 50/set or higher, and activation by a small stimulating current (5v or less for 50 +ec or less). These criteria have to be modified for the immature animal ( 11) . Latencies are very much longer, and rather strong shocks to the medullary pyramid are needed to activate any cortical units. Filred latency of response and consistent following at low stimulus rates (up to lO/sec) remain as the most important criteria for identification of immature pyramidal tract units. Failure of evoked responses at low frequencies of stimulation is seen only when a spontaneous spike discharge precedes the stimulus by an interval equal to or less than the response latency (Fig. 1). A similar interaction between spontaneous and evoked spikes is not seen in synaptically activated
J-m--w
+----I
I
1 FIG. 1. A. Single unit record from a Z-day-old kitten. The unit is synaptically activated by the pyramidal tract stimulus. Multiple discharges are evoked by each pyramidal tract shock. There is no occlusion of evoked discharges by spontaneous discharges preceding the stimulus to the medullary pyramid (lower trace). B. Antidromically activated pyramidal tract neuron, same animal. Pyramidal tract stimuli are followed by single, fixed-latency spike discharges (upper and middle trace). The evoked spike is absent when the stimulus is preceded by a spontaneous spike discharge (lower trace). Calibration 500 pv, 50 msec. Positivity is indicated by a downward deflection.
408 units. The conducted occurring helpful in ducted.
HUTTENIACHER
effect appears to be explained by occlusion of the antidromically wave of depolarization by a simultaneous orthodromic discharge in the same axon. In the present study, this finding was often confirming that a given unit response was antidromically conResults
Response Latencies to Medulby Stimulation in Inwmture Pyrakdal Tract neurons. Thirty-six pyramidal tract neurons were identified and studied, in animals ranging from age 3 days to 5 weeks. Similar results were obtained in both unanesthetized and in lightly anesthetized, curarized animals. The data from both prepartions are, therefore, presented together. Latencies from medullary stimulus to cortical neuron spike in animals under age 2 weeks varied between 20 and 38 msec (Fig. 2). The measured distance between the stimulating and recording electrodes was about 30 mm, yielding conduction velocities of 0.8 - 1.5 m/set. After age 2 weeks units with response latencies below 10 msec, and with calculated conduction velocities up to 14 m/set were found. However, even at age 5 weeks some pyramidal tract fibers still had conduction times above 20 msec. Histologic sections of the pyramidal tracts in the medulla showed that stainable myelin appeared during the age period of the study. Faintly myelinated fibers were seen by age 14 days, and myelination progressed slowly over the next several weeks (Fig. 3). Response of Pyramidal Tract Neurons to Repetitive Stiwmla.tion. Responses of pyramidal tract neurons to repetitive stimuli were determined at stimulus rates of l-lm/sec for periods of 20 sec. All units showed con-
40 0
I
0 2 E -20
30
0 -
O
0
8
0 H
0 0 0
00
8
t Y P a-! lo-
ts 0
; 0
0 IO
20
AGE
FIG. 2. Latency from medullary 36 pyramidal tract neurons recorded between ages 3 and 40 days.
!j 30
40
(days)
pyramid shock from precruciate
to antidromic spike discharge in and postcruciate gyrus in kittens
MYELINATION
AND
FUNCTION
409
sistent responses at stimulus rates of 1-3/set, provided that prolonged repetitive stimulation had not occurred for at least 30 set prior to testing. However, a majority of the neurons were unable to follow consistently at stimulus rates above lo-20/set. In the youngest animals, especially in those studied prior to age 5 days, repetitive stimulation at lO/sec or higher resulted in a gradual decrease in amplitude and increase in duration of extracellularly recorded unit spikes. This effect was also seen during repetitive firing of spontaneously active cortical neurons in this age group. By age 1 week most cortical neurons were able to follow at rates of 10-20,’ set without marked decreases in spike amplitude. A second effect of repetitive stimulation was noticeable from birth, but became especially evident in the second and third postnatal weeks. after cortical spikes had become relatively stable. It consisted of a gradual increase in latency from pyramidal tract stimulus to cortical unit response during repetitive stimulation. Latency increases of up to 12 msec were seen. During continued repetitive stimulation lengthening in conduction time was followed by variable response of the pyramidal cell spike, and eventually by complete absence of evoked spikes. The failure of the pyramidal celI response was an all or none event, in contrast to the gradual failure of the cellular spike seen in the first postnatal week. Recovery was rather slow. After prolonged repetitive stimulation the latency did not return to resting levels until 30 set to 1 min after the last stimulus. Typical responses are illustrated in Figs. 4 and 5. Figure 4 shows the decrease in spike amplitude at lO/sec stimulation seen in the first postnatal week, Superimposed, there is gradual lengthening in conduction time. Slow recovery of conduction following repetitive stimulation is illustrated in ~olunm C. The first sweep in this column represents the unit response 30 set after a train of stimuli had been presented. The evoked spike has returned to full amplitude, but response latency is prolonged. The latency has returned to the prestimulation value 30 set later (middle trace). Figure 5 illustrates a unit recorded from a l4-day-old animal, The pyramidal cell spike is quite stable. ‘At lO/sec stimulation (middle trace) the unit spike at first follows very closely upon the subsequent stimulus artifact. After a brief train of stimuli there is a gradual increase in the distance between stimulus artifact and unit spike, indicative of increase in response latency. The unit responds only intermittently in the last trace. Fourteen pyramidal tract cell recordings were obtained in animals 3-5 weeks old. These neurons represented a heterogeneous group. A few cells were found to have characteristics of immature neurons, with Jong response latencies, and with development of conduction block on repetitive stimulation above 20-30/set. However, the majority of pyramidal tract cells in this age group had response latencies below 12 msec. These neurons were
410
HUTTENLOCHER
MYELINATION
t
AND
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411
FUNCTION
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1
4. Response to antidromic stimulation in a pyramidal tract neuron from a 3-dayold kitten. A. Unit spike, followed by evoked slow wave potential, at l/set stimulation. B. Rapid decrease in amplitude of the unit spike at lO/sec stimulation. The evoked slow wave response also fatigues rapidly. C. Recovery of unit response and of slow wave potential 30 set (top trace), 60 set (middle trace), and 90 set (lower trace) after cessation of repetitive stimulation. Calibration 500 pv, 50 msec. FIG.
able to maintain repetitive firing at stimulus rates of 40-60/set for at least 20 sec. No increase in response latency was seen during repetitive firing at these rates (Fig. 6). In general, units with long response latencies showed lengthening in conduction time and conduction block on repetitive stimulation. Units with response latencies below 12 msec were able to fire at stimulus rates of 40-60/set. The relationship between response latency and ability to maintain repetitive activity is illustrated in Fig. 7. Discussion
The present observations indicate that pyramidal tract neurons in kittens .differ markedly from the adult in their ability to undergo repetitive discharges. In the youngest animals, a decrease in amplitude and increase in duration of extracellularly recorded unit spikes is noted during repetitive activity. This finding confirms observations by Conway, Wright, and Bradley (4). who noted breakdown of extracellularly recorded unit spikes on repetitive pyramidal tract stimulation in the neonatal rabbit. The effect is FIG.
(top), x 30.
3. Myelination of the pyramidal 30-day-old kitten (middle), and
tract in the medulla of a 15-day-old kitten adult cat (lower). LVeil’s stain. Magnification
HUTTENLOCHER
412
C
B
A
3 FIG. 5. Response 14 days. A. ZO/sec progressive increase C. Recovery of the
of a pyramidal tract unit to repetitive antidromic stimulation, age stimulation. The unit follows wetl. B. 40/set stimulation. There is followed by conduction block (lower trace). in response latency, response after a period of rest. Calibration 200 pv, 50 msec.
&nilar to what is seen in mature neurons during injury discharges, during seizure discharges, or during very rapid repetitive stimulation. Phillips ( 17) observed breakdown of spikes at stimulus rates of about 50 to over 400 spikes/set in recordings from adult pyramidal tract cells. Breakdown of spikes at low rates of firing (4O/sec and less) appears to be characteristic of immature cortical neurons for the first few days after the onset of electrical excitability (4, 10, 11). After the first postnatal week in the kitten, repetitive firing in pyramidal tract units is no longer limited by inability of the soma to undergo repetitive activity. Rather, the limiting event now appears to be conduction along
1 FIG. 6. Response of a pyramidal 35 days. The unit shows persistent 50 msec.
I
1
tract unit to repetitive antidromic stimulation, age following at 60/set stimulation. Calibration 500 gv,
MYELINATION
AND
413
RJNCTION
ki 5 b u 5 % 3
20
-
9
c 10
c 20
LATENCY FIG.
sponse
7. Relationship in 33 pyramidal
0
0 30
0
I 40
(msec)
between maximum response rate tract neurons recorded between
and latency of antidromic ages 3 and 40 days.
re-
the axon. The response to repetitive stimulation in pyramidal tract cells between ages 1-3 weeks has features characteristic of conduction block in the axon, consisting of a progressive lengthening in conduction time, followed by abrupt and complete loss of evoked spikes recorded at cortical level. The positive correlation observed between conduction block and conduction velocity in pyramidal tract fibers suggests that myelination may be a factor in the ability of pyramidal tract axons to fire repetitively. One has to assume that fibers in immature pyramidal tract that conduct at velocities above 3 m/set have acquired functionally significant myelin along at least part of their course, since the pyramidal tract in the kitten does not contain unmyelinated fibers of a size which would allow such rapid conduction. Purpura et al. (18) did not find pyramidal tract fibers of greater than 2 11 diameter prior to age 4 weeks. Traces of stainable myelin become visible in the pyramidal tract at the time when units with rapid conduction velocity first appear (15, 18). Myelination would be expected to have a marked effect on the efficiency of conduction along the axon. Keynes and Ritchie (14) have estimated that the active membrane surface of a small, unmyelinated fiber is between 100 and 1000 times greater than is that of a myelinated fiber of equal length and diameter. The outflow of potassium per nerve impulse in a small, UWmyelinated fiber has been calculated to be about 300 times that of a myelinated fiber of the same size (9). As a result, the energy expenditure of unmyelinated fibers during repetitive activity has to be considerably greater
414
HUTTENLOCHER
than it is in fibers that are surrounded by a myelin sheath, provided that both types of membranes have to maintain similar ionic gradients. There is evidence which suggests that mature unmyelinated fibers in peripheral nerve have low resting membrane potentials, high internal sodium concentrations, and low potassium concentrations (14). However, it is doubtful that such special adaptation occurs in immature unmyelinated fibers in the central nervous system. While measurements of resting membrane potentials on immature central axons are unavailable, resting potentials of their soma membranes are similar to those in adults (19). Excessive outflow of potassium from immature axons during repetitive activity would provide an explanation both for the observed increase in conduction time and for conduction block. Both of these events occur when the internal potassium concentration of the axon is lowered (2). Depletion of internal potassium ion and accumulation of sodium ion may occur especially rapidly during repetitive activity of immature axons, since the sodium pump mechanism in immature brain appears to be as yet poorly developed (12). The inability of developing axons to maintain repetitive activity may be of functional importance in the immature brain. In the adult, rapid repetitive firing of cortical neurons is commonly seen during normal activity. As early as 1939 Adrian and Moruzzi (1) found that motor activity of an animal is correlated with high-frequency discharges in pyramidal tract fibers. Afferent stimuli or direct stimulation of motor cortex result in movement only when they produce rapid repetitive firing in pyramidal tract. More recent studies in the monkey by Evarts (6) have shown a correlation between bursts of rapid discharges in pyramidal tract units and voluntary contractions of small muscle groups. Firing rates of 50-lOO/sec were seen in pyramidal tract neurons during voluntary contraction of hand and forearm muscles. The inability of immature axons to maintain such rapid rates of activity may provide an explanation for the correlations that have been observed between myelination and the appearance of function in the immature brain (16, 21). On the other hand, it is clear that some functions, primarily those of simple reflex type, develop prior to and quite independent of myelination (23). References 1. ADRIAN, E.*lD., and G. MORUZZI. 1939. Impulses in the pyramidal tract, J. Physiol. Lortdon 97: 153-199. 2. BAKER, P. F., A. L. HODGKIN, and T. I. SHAW. 1%2. The effects of changes in internal ionic concentrations on the electrical properties of perfused giant axons. J. Physiol. LOPkdOlk. 164: 3.55-374. 3. CHANG, H. T. 195.5. Activation of internuncial neurons through collaterals of pyramidal fibers at cortical level. J. Neurophysiol. 18: 452-471.
MYELINATION
AND
FUNCTIOX
415
4. CONWAY, C. J., F. S. WRIGHT, and W. E. BRADLEY. 1969. Electrophysiological maturation of the pyramidal tract in the post-natal rabbit. Elcctvocncephalogr. Cl& Newophysiol. 26 : 565-577. 5. ELLINGSON, R. J., and R. C. WILCOTT. 1960. Development of evoked responses in visual and auditory cortices of kittens. J. Nc~~rophgsio.!. 23: 363-375 6. EVARTS, E. V. 1966. Pyramidal tract activity associated with a conditioned hand movement in the monkey. J. Nc~rophysiol. 29: 1011-1027. 7. GROSSMAN, C. 1955. Electra-ontogcnesis of cerebral activity. &4X.,4. Arch. Ncwol. Psych&. 74 : 18fXO2. 8. HENRY, E. W. 1943. Somatic motor responses produced by electrical stimulation of the cerebral cortex of new-born and young kittens. Fed. Proc. 2: 21. 9. HODGKIN, A. L. 1951. The ionic basis of electrical activity in nerve and muscle. Biol. Rezl. 26: 339409. 10. HUTTENLOCHER, P. R. 1967. Development of cortical neuronal activity in the neonatal cat. Errp. Ncwrol. 17: 247-262. 11. HUTTENLOCHER, P. R. 1969. Functional properties of pyramidal neurons during development of cerebral cortex in the kitten. Fed. PYOC. 28: 825. 12. HUTTENLOCHER, P. R., and M. D. RAWSON. 1968. Neuronal activity and adenosine triphosphatase in immature cerebral cortex. Exp. Nrurol. 22: 118-129. 13. JABBUR, S. J., and A. L. TOWE, 1961. Analysis of the antidromic cortical response following stimulation at the medullary pyramid. 1. Pizgsiol. Lotrdo~ 155: 14% 160. 14. KEYNES, R. D., and J. M. RITCHIE, 1967. The movement of labelled ions in mammalian non-myelinated nerve fibers. /. Physiol. London. 188: 309-327. 15. LANGWORTHY, 0. R. 1927. Histological development of cerebral motor areas in young kittens correlated with their physiological reaction to electrical stimulation. Co&rib. Enzbryol. 19: 177-208. 16. LANGWORTHY, 0. R. 1932. Development of behavior patterns and myelinization of tracts in the nervous system. A.M.A. drclt Nruvol. Psychiat. 28: 1365-1382. 17. PHILLIPS, C. G. 1959. Actions of pyramidal volleys on single Bertz cells in the cat. Quart. J. Enp. Physiol. 44: l-25. 18. PURPURA, D. P., R. J. SHOFER, E. M. HO~S~PIAN, and C. R. NOBACK. 1964, Comparative ontogenesis of structure-function relations in cerebral and cerebellar cortex. Progr. Braix Rcs. 4 : 187-221. 19. PURPURA, D. P., R. J. SHOFER, and T. SCARFF. 1965. Properties of synaptic activities and spike potentials of neurons in immature neocortex. J. Nnrrophysiol. 28 : 925-942. 20. ROSENTHAL, F., J. W. WOODBURY, and H. D. PATTON. 1966. Dipole characteristics of pyramidal cell activity in cat postcruciate cortex. J. N:c~~~ophysioZ. 28: 612625. 21. TILNEY, F., and J. CASAMAJOR. 1924. Myelinogeny as applied to the study of behavior. AMA Arch. Ncuvol. Psychiat. 12: l-66. 22. WEED, L. H., and 0. R. LANGWORTHY. 1926. Physiological study of cortical motor areas in young kittens and in adult cats. Coutrib. Embrgol. 17: 91-106. 23. WINDLE, W. F., M. W. FISH, and J. E. O’DONNELL. 1934. Myelogeny of the cat as related to development of fiber tracts and prenatal behavior patterns. J. Comp. Neural. 59: 139-165. 24. WOLBARSHT, M. L., E. F. MACNICHOL, and II. G. WAGNER. 1960. Glass insulated platinum microelectrodes. .I?ciettce 132 : 1309-1310.