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Postnatal development of thalamic synaptic events underlying evoked recruiting responses and electrocortical activation
Brain Research, 60 (1973) 21-34
© ElsevierScientificPublishingCompany, Amsterdam- Printed in The Netherlands
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POSTNATAL DEVELOPMENT OF THALAMIC SYNAPTIC EVENTS UNDERLYING EVOKED RECRUITING RESPONSES AND ELECTROCORTICAL ACTIVATION
ROBERT W. THATCHER* ANDDOMINICK P. PURPURA Department of Anatomy and the Rose F. Kenned), Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y. 10461 (U.S.A.)
(Accepted March 20th, 1973)
SUMMARY
Postsynaptic potentials were intracellularly recorded from thalamic neurons in kittens of different ages during stimulation of medial thalamic (MTh) regions at low and high frequencies. In neonatal and young kittens ( < 10 days old) low-frequency (3.3/sec) MTh stimulation elicited IPSPs in thalamic neurons with a mean duration of 50 m s e c . EPSPs at this age were weak and variable. In older kittens (2-3 weeks old) MTh-evoked IPSPs exhibited a mean duration of 125 msec and EPSPs were more prominent in EPSP-IPSP sequences. High-frequency (80/sec) MTh stimulation in young kittens produced sustained summation of IPSPs and suppression of cell discharge. IPSPs were also elicited by high-frequency MTh stimulation in 2-3-week-old kittens but these IPSPs were rapidly terminated by powerful and sustained EPSPs. The relationship of these observations to previous studie ~, of evoked synchronizing and desynchronizing processes in thalamic neuronal organizations in adult animals indicates that: (1) the development of evoked thalamic neuronal synchronization is associated with the functional maturation of interneuronal pathways involved in the production of EPSP-IPSP sequences in thalamic neurons; and (2) the development of electrocortical activation subsequent to high-frequency MTh stimulation occurs i pari passu with an increasing capacity of excitatory synaptic inputs to elicit powerful and sustained EPSPs in thalamic neurons. Thalamic synaptic mechanisms underlying evoked synchronization and desynchronization of electrocortical activity attain functional maturation by the end of th~~. third postnatal week in the kitten.
* Present address: Department of Physiology, New York Medical College, 106th St. and Fifth Avenue, New York, N.Y. 10029, U.S.A.
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R. W. THATCHER AND D. P. PURPURA
INTRODUCTION
Electrocortical activity of altricial newborn subprimate mammals is poorly organized with respect to rhythmically recurring spindle waves and deficient in the high-frequency low-voltage activity characteristic of wakefulness in mature animals7,10,2L Although these features of EEG ~immaturity' reflect the morphophysiological and biochemical immaturity of the brain as a whole, virtually nothing is known concerning the synaptic events that contribute to the physiological development of synchronizing and desynchronizing mechanisms controlling electrocortical activity. One approach to this complex problem is the subject of the present report. It is generally held that the thalamus is the major source of the afferent projection systems that powerfully influence electrocortical activity1,4,9,12. Intracellular studies of thalamic neurons during evoked electrocortical synchronizing and desynchronizing activities in adult animals have disclosed two fundamentally different patterns of thalamic synaptic events in association with these activities 15. During electrocortical synchronization elicited by low-frequency stimulation of medial thalamic (MTh) nonspecific nuclei a large proportion of thalamic neurons exhibit rhythmically recurring EPSP-IPSP sequencesS,14,~s,~9,22. In contrast, during electrocortical desynchronizafion elicited by high-frequency MTh stimulation thalamic neurons generally exhibit sustained excitatory synaptic activity23. A major factor contributing to the generalized synchronization of thalamic neuronal discharges is the inhibition of spontaneous activity effected by prominent and prolonged (100-200 msec) IPSPs ~s. During thalamically evoked reticulocorticai activation synchronizing IPSPs are attenuated or blocked and swamped by summating EPSPs 2a. Attenuation of synchronizing lPSPs is also observed following brain stem reticular stimulation 20. The foregoing summary of intracellular studies of thalamic neuronal operations and processes in adult animals provides a point of departure for assessing the maturational status of evoked synchronizing and desynchronizing synaptic events in the thalamus of the immature animal. The present study examines the synaptic events elicited in thalamic neurons during low- and high-frequency MTh stimulation in kittens of different postnatal ages. Data are provided on developmental differences in effects of MTh stimulation and on the factors which contribute to the synchronization and desynchronization of thalamic neuronal activity during postnatal ontogenesis. A preliminary report on some aspects of this study has appeared elsewhere ~. METHODS
Experiments were performed on kittens in 4 age groups: 1-3 days, 6-8 days, 13-15 days and 20-22 days. Ether anesthesia was employed during all operative procedures which included intratracheal intubation, exposure of the dorsolateral convexity of a cerebral hemisphere, cisternal drainage and suction ablation of cortex and structures overlying the thalamus. Following these procedures xylocaine was injected into all pressure points and applied in jelly form to exposed wound margins and skin surfaces. Thereafter the animals were paralyzed with intraperitoneal gal-
SYNAPTIC EVENTS IN IMMATURE THALAMUS
23
lamine triethiodide and artificially ventilated. Expired CO2 and heart rate were monitored periodically. Normal body temperature was maintained by wrapping the kittens in a flexible heating coil connected to a DC source. Concentrically bipolar stimulating electrodes were introduced into medial regions of the exposed thalamus under direct vision. Responses evoked by stimulating pulses of different frequency were recorded monopolarly from pericruciate cortex. The indifferent electrode consisted of an AgAgC! wire embedded in nuchal musculature, Intracellular recordings were obtained with 2 M potassium citrate filled micropipettes (20-40 M ~ resistances) which were inserted into the thalamus 1.0-3.0 mm lateral to the stimulating electrodes. Other details as to stimulating and recording procedures were similar to those described in comparable studies of adult cats 1s.22,23. RESULTS
General remarks The extraordinary 'fragility' of immature neocortical and hippocampal neurons has been noted previously13,21,24. The size of immature thalamic neurons and the necessity to probe through several millimeters of tissue undoubtedly contributed to the greater difficulty in obtaining satisfactory impalements of immature thalamic neurons than immature cortical neurons. Negative DC shifts indicative of transmembrane potenti~gls were frequently not obtained in the course of a complete excursion of a micropipette in a particular electrode track. This was not necessarily related to the age of the kitten under investigation although such DC shifts were generally more frequent in 2-3-week-old kittens than neonatal animals. A neuron was included in the present series of over 200 cells when a sudden negative DC shift was associated •,vith spike potentials irrespective of the amplitude or configuration of the discharges. In approximately 90% of these elements IPSPs were elicited by low-frequency MTh stimulation. 1PSPs survived the loss of spike potentials as in the case of immature neocortical and hippocampal neurons21, 24. A membrar~e potential of 30 mV was chosen as an arbitrary value in classifying cells with membrane potentials (MPs) in excess of and less than this value. Presence and absenze of spike potentials were additional criteria for classification as noted in Table I. In all age groups more cells had MPs less than 30 mV than exhibited MPs in excess c f 30 mV. This indicates that there was little difference in the traumatic effects of impalement in neonatal and 2-3week-old kittens. Rarely neurons were held with stable MPs and evoked PSPs for longer than 2-3 min. More commonly recordings were obtained for 20-40 sec. Despite this brief period of intracellular or 'quasi-~tracellular' registration it was p~ssible to determine latency and duration characteristics of evoked I PSPs. With slightly longer recording times the effects of varying the frequency of MTh stimulation on evoked PSPs were also examined. It is of importance to note in Table ? that for any particular age group latency and duration of evoked IPSPs were similar for cells that showed some degree of spike potential activity and those that exhibi,ted spike inactivation. In agreement with studies of evoked cortical responses to thalamic stimulation, in immature rabbits e it was not possible to elicit typical long-latency recruiting re-
24
R. W. THATCHER AND D. P. P U R P U R A
A
B
•
14d 21d Fig. 1. Examples of evoked responses (negative upwards) recorded from pericruciate cortex during 3.3/sec (A), and 10/sec (B) stimulation of medial thalamic (MTh) regions in kittens of different ages as indicated (d - days). Note that the initial response to the repetitive stimulus is large and decrements with successive stimuli in neonatal and very young kittens. In 2- and 3-week-old kittens recruiting responses are well organized and exhibit characteristics similar to those observed in adult animals. Time bars, 100 msec, throughout.
sponses in young kittens with stimulus frequencies employed in adult animals. Indeed the effect of 3-10/sec MTh stimulation in neonatal and young kittens was more commonly seen as a decrementing series of cortical evoked responses rather than typical recruiting potentials. Examples of responses recorded from pericruciate cortex in kittens of different ages are illustrated in Fig. !. It is evident in this figure that recruiting responses to 3.3/sec and 10/sec MTh stimulation were obtained in 2- and 3-week-old kittens but that in neonatal and I-week-old kittens decrementing responses were elicited by both stimulus frequencies. These data are entirely consistent with observations on the maturation of recruiting responses in the rabbit s .
Characteristics of IPSPs elicited by low-frequency MTh stimulation Neonatal and 6-8.day.old kittens. All the thalamic neurons impaled in l-3-dayold kittens exhibited effects of membrane trauma. This was seen as a rapid attenuation of injury discharges subsequent to spike inactivation, low-amplitude long-duration spikes, or spikes with multiple components21, 24 (Fig. 2). Despite these obvious signs of injury, low-frequency (3.3/sec) MTh stimulation elicited prominent IPSPs in approximately 90% of impaled neurons widely distributed in the rostral thalamus. In a small proportion of cells reduction in discharge frequency was observed in the absence of IPSPs. Examples of the characteristics of MTh-evoked IPSPs encountered in thalamic neurons of l-3-day-old kittens are illustrated in Fig. 2. Although IPSPs were the most clearly detectable synaptic events observed in thalamic neurons in the neonatal period, small EPSPs were occasionally noted preceding IPSPs. IPSPs with characteristics similar to those observed in 1-3-day-old animals were also noted in thalamic neurons from kittens 6-8 days old. Fig. 3 illustrates these
SYNAPTIC EVENTS IN IMMATURE THALAIVlUS
25
A B
A
I00
muK
Fig. 2. Examples of characteristics of IPSPs evoked in thalamic neurons from-2- and 3-day-old kittens in reponse to low-frequency (3.3/sec) medial thalamic (MTh) stimulation. A and C, from a 3-day-old kitten. B, from a 2-d~y-old kitten. In this and subsequent figures upper channel records are cortical surface responses (negativity upwards). MTh stimuli indicated by dots. A: first 3 decrementing series of responses. B: alternating characteristics of cortical evoked responses during continued MTh stimulation. C: recorded during stabilization phase of cortically evoked response. IPSPs in these 3 cells exhibit variable duration. Further description in text and Table I.
A B •
•
•
C
I 0 0 msec
Fig. 3. Examples of synaptic events evoked in 4 different thalamic neurons by low-frequency MTh stimulation during the course of experiments on 7-day-old kittens. A: a slight increase in the interval between discharges immediately after the MTh stimulus is the only effect detectable. B and D: short duration IPSPs are elicited by MTh stimulation in cells with partial spike potentials. C: cell unaffected by MTh stimulation.
26
R. W. THATCHER AND D. P. PURPURA
IPSPs in cells from different experiments. In Fig. 3A the inhibitory effect of MTh stimulation is seen largely as an increase in the interval between discharges following each stimulus. IPSPs were also prominent in injured neurons exhibiting attenuated and partial spikes (Fig. 3B and D). For each cell included in the present series 30-60 IPSPs elicited at 3.3/sec MTh stimulation were examined for latency and duration. Latencies ranged from 5 to 50 msec in both neonatal and 6-8-day-old kittens. IPSP durations ranged from 30 toj 150 msec in animals from both age groups. Table I shows that the mean latency of IPSPs in cells exhibiting spikes and membrane potentials in excess of 30 mV was 13.5 msec in l-3-day-old kittens and 14.8 msec in 6-8-day-old kittens. Slightly longer mean latencies were found in cells without spikes and lower membrane potentials. The mean duration of IPSPs was 50 msec in cells with spikes and > 30 mV membrane potentials. This was not significantly different in the two age groups although the mean duration of IPSPs was slightly less in cells without spikes and ~ 30 mV membrane potentials. hi 13-22-day-old kittens. Spike potentials were considerably shorter in duration and larger in amplitude during injury discharges in thalamic neurons from 13-to 22-day-old kittens. Such discharges were maintained at a relatively high frequency for a longer period in older animals than in neonatal and very young kittens. Examples of IPSP characteristics observed in 14- and 22-day-old kittens are shown in Figs. 4 and 5, respectively. The smoothly summating characteristics of the prolonged IPSPs A
B
C
L
,_,
_
I
--
~
~
~llbl~- -
1
I
D mY 1 O0 m ~
Fig. 4. Characteristics of long-duration (80--160 msec) IPSPs observed in 4 different thalamic neurons from a 2-week-old kitten. Note that at this developmen~*al stage injury discharges occur at a higher frequency and spike potentials are shorter in duration th,~n in younger kittens (of. Figs. 2 and 3). IPSPs in A and C have multiple components.
27
SYNAPTIC EVENTS IN IMMATURE THALAMUS
A.
•
C
.
D
,p ¥
•
•
• I00
msec
Fig. 5. Examples of IPSP characteristics observed in 4 different thalamic neurons from a 3-week-old kitten during low-frequency MTh stimulation. Spike potential amplitude and ~.uration are similar to spikes of thalamic neurons in adult animals xs,2a. This display emphasizes the synchronizing features of the long-duration IPSPs which limit spike discharges, to the brief periods after IPSPs and just prior to the succeeding stimulus of the repetitive train. Note in particular that IPSPs in B attain a duration of nearly 200 msec. In the other cells the IPSPs range from 100 to 150 msec. observed in the older kitten (Fig. 5) were similar to those observed in adult animalslS,20,2~,~3. Surprisingly the range or mean IPSP latencies observed in 13-22-day-old kittens did not differ significantly from the range or mean latencies observed in younger kittens (Table I). However, highly significant differences were observed in IPSP duraTABLE 1 CHARACTERISTICS OF
Days
No. of cells
IPSPs IN IMMATURE THALAMIC NEURONS
Mean duration (msec)
With Without With spikes IPSPs IPSPs S.D. 1-3
24 34 6- 8 18 20 13-15 17 22 20-22 21 24
4 11 2 1 2 1 3 2
55.8 45.1 54.7 43.5 125.4 81.7 128.6 118.0
6.6 4.4 7.1 3.4 4.1" 5.3* 4.6* 5.1"
Mean latency (msec)
Without spikes With spikes
Membrane potential Without spikes (mV)
•
S.D.
•
S.D.
•
S.D.
50.2 38.7 53.6 44.4 123.2 115.8 125.2 120.8
6.8 4.0 6.2 5.9 4.1" 4.9* 5.7* 4.1"
13.5 22.6 14.8 17.5 15.1 21.3 14.9 19.1
2.0 3.9 1.3 2.1 1.5 4.5 2.2 2.9
10.6 23.4 15.2 18.4 14.8 20.9 15.5 20.2
1.8 4.1 1.7 1.8 1.1 2.0 1.9 1.1
>30 < 30 > 30 < 30 > 30 < 30 > 30 < 30
* Significant differences (P < 0.001) between these means and the means of 1-3- and 6-8-day-old kittens.
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R. W. THATCHER AND D. P. P U R P U R A
tions. The mean duration of IPSPs in cells with spike potentials and > 30 mV membrane potentials was 125 msee in 13-15-day-old animals and 129 msec in 20-22-dayold animals. Slightly shorter durations were observed in cells with spike inactivation. The data indicate that during the second and third postnatal weeks MTh-evoked IPSPs in thalamic neurons undergo a 2- to 3-fold increase in duration. IPSP characteristics attain mature form by the third postnatal week.
Developmental differences in synaptic effects of high-frequency MTh stimulation Neurons exhibiting relatively stable membrane potentials during and immediately after low-frequency (3.3/see) M T h stimulation were also examined for synaptie effects of high-frequency (80/sec) M T h stimulation. Remarkably different effects were observed during and following such high-frequency stimulation in very young ( < 1
A
I00
mse¢
B
O
Fig. 6. Changing effects of high-frequency (80/sec) MTh stimulation on thalamic neurons as a function of postnatal age. A: recordings from 3 different neurons from 3-day-old kittens. Onset of highfrequency MTh stimulation indicated by dots. IPSP summation is prominent though variable in the 3 neurons. In the upper and lower sets ~" records spike discharges occur shortly after the last stimulus of the repetitive train. In the middle set the increase in membrane potential associated with IPSP summation persists after cessation of MTh stimulation. Electrocortical activation is not seen at this age during high-frequency MTh stimulation. B: neurons from 14-day-old kittens. High-frequency MTh stimulation initially evokes 60-80 msec duration IPSPs which are terminated by sustained depolarizing shifts in membrane potential. Recovery of membrane potential occurs slowly after cessation of stimulation. Note induction of high-frequency electrocortical activity during MTh stimulation.
SYNAPTIC EVENTS IN IMMATURETHALAMUS
29
week old) and older kittens (2-3 weeks of age). A comparison of the basic difference in the two age groups is illustrated in Fig. 6. Examples of findings from 3 different preparations of 2-3-day-old animals are shown in Fig. 6A. The first stimulus of the repetitive train initiated a long-latency IPSP as in the case of low-frequency stimulation. Subsequent stimuli prolonged the duration of the IPSP and suppressed discharges when these were present prior to the onset of the high-frequency stimulation (Fig. 6A upper and lower set of records). In other neurons the increase in membrane polarization initiated by high-frequency stimulation persisted beyond the period of stimulation (Fig. 6A, middle set of records). Entirely different effects of high-frequency M T h stimulation were observed in thalamic neurons of 2-3-week-old kittens. As in the case of the very young kittens the first stimulus of the repetitive train elicited an IPSP (Fig. 6B). However, this was terminated after 60-80 msec by a depolarizing shift in membrane potential that gradually subsided following cessation of the high-frequency MTh stimulation (Fig.
A
B
I
"J"-~J"~--
•
-
-
J"-
~
I,o __
-
~
-
-
•
•
,
"
100
,
[mV
mmc
Fig. 7. Postactivation facilitation of EPSPs and attenuation of IPSPs in thalamic neurons from a 2-week-old kitten following high-frequency MTh stimulation. A: upper set, low-frequency MTh stimulation evokes IPSPs wl~ich are not preceded by EPSPs. Middle set, onset of high-frequency (80/sec) MTh stimulation (at arrow). IPSP is terminated by depolarizing shift in membrane potential. Lower set, resumption of low-frequency MTh stimulation several seconds later• In the postactivation period each stimulus elicits an EPSP and spike discharge, but IPSPs are not observed. Spontaneous discharges occur at variable times after the evoked EPSP. Note persisting change in evoked cortical potential in the postactivation period. B: recordings from a traumatized neuron in the same animal. Sequence of changes in evoked PSPs, similar to that in A, occurs in the postactivation period. Towards the end of the lower set of records the EPSP induced by the prior period of high-frequency stimulation becomes attenuated along with 'reappearance' of the prolonged IPSP.
30
R. W. T H A T C H E R A N D D. P. P U R P U R A
6B). A similar sustained depolarization of thalamic neurons subsequent to highfrequency MTh stimulation in adult animals has been shown to be attributed to summation of EPSPs 2a. In 2-3-week-old kittens low-frequency MTh stimulation elicited prolonged IPSPs prior to the brief period of high-frequency MTh stimulation whereas such lowfrequency stimulation immediately after high-frequency stimulation failed to evoke prolonged IPSPs (Fig. 7A). Instead during the immediate postactivation period lowfrequency MTh stimulation elicited short-latency EPSPs which triggered spike discharges. In other neurons after loss of spike potentials a similar transformation in synaptic events evoked by low-frequency MTh stimulation was observed in the immediate postactivation period (Fig. 7B). Rapid attenuation of EPSPs and restitution of prolonged IPSPs occurred with continued low-frequency MTh stimulation (Fig. 7B). DISCUSSION
The foregoing results provide evi,,~ence that inhibitory synaptic activities are prominently displayed in intrathalamic internuclear interactions initiated by lowfrequency MTh stimulation in neonatal kittens. The same cannot be said for excitatory synaptic events which were generally not conspicuous in thalamic neurons from neonatal and young kittens under the present experimental conditions. These observations are consistent with previous findings in neocortical '~4 and hippocampal~a, '-'1 neurons of neonatal kittens with one exception. In the case of immature cortical neurons evoked IPSPs were found to be of long duration, comparable to, if not greater than IPSPs elicited in mature cortical neurons under similar conditions of examinationlL lPSPs in thalamic neurons of neonatal and young kittens have a mean duration that is significantly shorter than the mean duration of IPSPs in 2-3-week-old kittens. After the third week such IPSPs exhibit all the characteristics observed in adult animalslS-20, 2a. In view of the essential role of long-duration IPSPs in the local 2 and generalized synchronization 18 of thalamic neuronal activity it follows that the functional maturation of intrathalamic internuclear events associated with generalized synchronization of thalamic neuronal activity~4, x5 consists primarily in an increase in the mean duration of IPSPs. The progressive augmentation in the capacity of internuclear pathways to elicit EPSPs in immature thalamic neurons represents a second feature of the maturational process of thalamic neuronal synchronization. The relative functional immaturity of thalamic internuclear excitatory synaptic pathways in neonatal and young kittens is also reflected in the synaptic events observed in these preparations during high-frequency MTh stimulation. During the first postnatal week high-frequency MTh stimulation elicits sustained depression of thalamic neuron activity. Summation of IPSPs was evident in most of these cells. By 2 weeks of age a new synaptic event is added in the form of a powerful and sustained EPSP which is capable of rapidly inactivating spike electrogenesis in cells exhibiting spike activity. Evidently the augmented excitatory synaptic activity so initiated persists within the interneuronal networks constituting the internuclear pathways. For
SYNAPTIC EVENTS IN IMMATURE THALAMUS
31
immediately following termination of the high-frequency MTh stimulation in older kittens EPSPs may be observed in response to low-frequency MTh stimulation when such stimulation prior to high-frequency stimulation failed to evoke them. Moreover there is a striking attenuation of low-frequency evoked IPSPs in the postactivation period which is similar to that observed in adult animals following high-frequency MTh stimulation 23. Thus the present findings suggest that during the second postnatal week excitatory internuclear intrathalamic synaptic pathways undergo a rapid phase of functional maturation. Evidently this developmental event heralds the capacity of thalamic neurons to exhibit powerful excitatory drives during MTh-evoked reticulocortical activation. Intracellular studies of thalamic neuronal events in adult animals during the transition from low- to high-frequency MTh stimulation have emphasized the frequency specificity of the sustained excitatory action initiated by MTh stimulation during the thalamocortical desynchronization process 23. This frequency specificity has been confirmed recently in similar studies of Broggi and Margnelli 5. It has been inferred from observations on the attenuation of synchronizing IPSPs in EPSP-IPSP sequences during the transition from low- to high-frequency MTh stimulation that such attenuation results from an inhibition or blockade of inhibitory pathways generating the synchronizing IPSPs as well as augmentation of excitatory synaptic drivesaa. If the attenuation of synchronizing IPSPs in thalamic neurons during highfrequency MTh stimulation was a consequence of the activation of inhibitory pathways by high-frequency stimulation it might be anticipated that IPSP attenuation would occur irrespective of the capacity of such stimulation to elicit powerful depolarizing EPSPs. The fact is that prior to the functional development of excitatory synaptic pathways capable of producing sustained EPSPs during high-frequency MTh stimulation IPSP attenuation does not occur. Rather low-frequency as well as highfrequency MTh stimulation elicits IPSPs in thalamic neurons of neonatal and young kittens. This suggests that high-frequency MTh stimulation does not activate pathways which are inhibitory to the interneuronal elements generating synchronizing IPSPs in thalamic neurons, at least in very young kittens. The possibility still exists that the attenuation of synchronizing IPSPs observed in older animals during highfrequency MTh stimulation could be due to the late development of a parallel pathway, inhibitory to elements generating synchronizing IPSPs. However the most parsimonious interpretation of the present observations is that the attenuation or 'blockade' of the synchronizing IPSP during high-frequency MTh stimulation is more apparent than real and represents the addition of EPSPs that in effect override simultaneously elicited IPSPs. One feature of the events observed in thalamic neurons of enc6phale isol6 adult animals during low-frequency MTh stimulation or during spontaneous or evoked spindle waves is seen in the cluster of spike discharges that succeed prolonged rhythmically recurring IPSPs 14,1s,19. Similar bursts superimposed on membrane depolarizations have been seen in ventrobasal neurons of barbiturized adult animals 1-3. Andersen and Andersson 1 have emphasized a role of postanodal exaltation or postinhibitory rebound in the generation of burst discharges during spindle waves. In
32
R. W. THATCHER AND D. P. P U R P U R A
contrast, evidence has been presented that depolarizations succeeding IPSPs in thalamic neurons are EPSP components of EPSP-IPSP sequences11,14, a view that has now been championed by Andersson and Manson 4. Results of the present study are relevant to the origin of these postinhibitory burst discharges. If postanodal exaltation was a general property of thalamic neurons it might be expected that burst discharges would occur following prolonged IPSPs whenever the latter were elicited. On the other hand, if such discharges were dependent upon the development of EPSPs succeeding the IPSPs then in the absence of EPSPs postinhibitory bursts should not occur. None of the thalamic neurons examined in very young kittens exhibited postinhibitory burst discharges of the type encountered in adult animals. Neither did they exhibit prominent spontaneous or evoked EPSPs to low-frequency MTh stimulation until after the third postnatal week. This suggests that the major determinant of postinhibitory burst discharges is related to the development of excitatory synaptic pathways which interact in some as yet poorly understood fashion with inhibitory interneuronal elements to give the EPSP-IPSP sequences characteristic of synchronizing activities of thalamic neurons t4,15. The present study sheds little light on the possible mechanisms underlying the increase in mean duration of IPSPs in thalamic neurons during postnatal development. To the extent that repetitive activity of inhibitory interneurons la contributes to the prolonged conductance increase associated with synchronizing IPSPs in thalamic neurons s, it is reasonable to consider developmental changes in repetitive responsiveness of immature thalamic neurons as an important factor in the increase in mean duration of IPSPs. The present observations taken together with previous studies of immature neocortica124 and hippocampal neurons 'z-t provide no convincing data for the role of repetitively active inhibitory interneurons in the production of prolonged IPSPs in immature brain. However, if inhibitory interneurons are relatively small and fragile compared to the cells impaled in this and other ontogenetic studies it may well be that present failures to record from these elements may reflect largely operational difficulties. Alternatively it may be argued that high-frequency discharges of inhibitory interneurons may not be required to produce prolonged IPSPs in immature neurons if transmitter action is prolonged for various reasons t6. Apropos of this, it is of interest that preliminary electron microscope studies have suggested a remarkable development of complex synapses surrounded by glial processes in the thalamic neuropil during the first 2-3 weeks postnatally in the kitten 17 (Pappas and Purpura, unpublished). Conceivably such developmental events could contribute to the increase in duration of action of inhibitory transmitter at such synapses. Obviously this problem requires close pursuit in the further analysis of developmental changes in synaptic transactions in the immature brain 1~. Finally the developmental changes in patterns of synaptic activities in thalamic neurons described here call attention to the important role of intrathalamic interneuronal synaptic organizations in the electrographic events associated with the ontogenesis of sleep-wakefulness behavior.
SYNAPTIC EVENTS IN IMMATURE THALAMUS
33
ACKNOWLEDGEMENTS This s t u d y was supported in p a r t by a G r a n t f r o m the Alfred P. Sloan F o u n d a tion a n d the N a t i o n a l institute o f Neurological Diseases a n d Stroke (NS-07512). Dr. T h a t c h e r was s u p p o r t e d by N . I . M . H . G r a n t MH-6418.
REFERENCES 1 ANDERSEN,P., AND ANDERSSON,S. A., Physiological Basis of the Alpha Rhythm, Appleton-CenturyCrofts, New York, 1968. 2 ANDERSEN, P., AND ECCLES, J. C., Inhibitory phasing of neuronal discharge, Nature (Lond.), 196 (I 962) 645-647. 3 A~DERSEN,P., ECCLES,J. C., AND SEARS,T. A., The ventrobasal complex of the thalamus: Types of cells, their responses and their functional organization, J. Physiol. (Lond.), 174 (1964) 370-399. 4 ANDERSSON,S. A., AND MANSON,J. R., Rhythmic activity in the thalamus of the unanesthetized decorticate cat, Electroenceph. clin. Neurophysiol., 31 (1971)21-34. 5 BROGGI,G., AND MARGNELLI, M., Dynamic properties of synaptic input from intralaminar nuclei to ventrolateral neurons of the thalamus, Brain Research, 26 (1971) 192-194. 6 DOCARMO, R. J., Direct cortical and recruiting responses in postnatal rabbit, J. Neurophysiol., 23 (I 960) 496-504. 7 ELLINGSON,R. J., AND ROSE,G. H., Ontogenesis of the electroencephalogram. In W. A. HIMWlCH (Ed.), Developmental Neurobiology, Thomas, Springfield, 111., 1970, pp. 441-474. 8 FELDMAN, M. H., AND PURPURA, D. P., Prolonged conductance increase in thalamic neurons during synchronizing inhibition, Brain Research, 24 (1970) 329-332. 9 JASPER, H. H., Diffuse projection systems: the integrative action of the thalamic reticular system, Electroenceph. clin. Neurophysiol., 1 (1949) 405-420. 10 JOUVET-MOUNIER,D., ASTRI¢, L., AND LACOTE,D., Ontogenesis of the states of sleep in rat, cat and guinea pig during the first postnatal month, Develop. Psychobioi., 2 (1970) 216-239. 11 MAEKAWA,K., AND PURPURA, D. P., Properties of spontaneous and evoked sy'laptic activities of thalamic ventrobasal neurons, J. Neurophysiol., 30 (1967) 360-381. 12 PURPURA, D. P., Nature of electrocortical potentials and synaptic organizations in cerebral and cerebellar cortex, Int. Rev. Neurobiol., I (1959)47-163. 13 PURPURA, D. P., Stability and seizure susceptibility of immature brain. In H. H. JASPER, A. A. WARD AND A. POPE (Eds.), Basic Mechanisms of Epilepsy, Little, Brown, Boston, Mass., 1969, pp. 481-515. 14 PURPURA,D. P., Interneuronal mechanisms in synchronization and desynchronization of thalamic activity. In M. A. B. BRAZIER(Ed.), The Interneuron, UCLA Forum in Medical Sciences, Los Angeles, Calif., 1969, pp. 467-496. 15 PURPURA, D. P., Operations and processes in thalamic and synaptically related neural subsystems. In F. O. SCHMXTr(Ed.), The Neurosciences, Voi. 2, Rockefeller Univ. Press, New York, 1970, pp. 458-470. 16 PURPURA, D. P., Intracellular studies of synaptic organizations in the mammalian brain. In G. D. PAPPAS AND D. P. PURPURA (Eds.), Structure and Function of Synapses, Raven Press, New York, 1972, pp. 257-302. 17 PURPURA, D. P., Development of synaptic substrates for drug actions in immature brain. In A. VERNADAKIS(Ed.), Drugs and the Developing Brain, Plenum Press, New York, 1973, in press. 18 PURPURA,D. P., AND COHEN, B., Intracellular recording from thalamic neurons during recruiting responses, J. Neurophysiol., 25 (1962) 621-635. 19 PURPURA, D. P., FRIGYESI,T. L., MCMURTRY, J. G., AND SCARFF, T., Synaptic mechanisms in thalamic regulation of cerebello-cortical projection activity. In D. P. PURPURAAND M. D. YAHR (Eds.), The Thalamus, Columbia Univ. Press, New York, 1966, pp. 153-170. 20 PURPURA,D. P., MCMURTRY,J. G., AND MAEKAWA,K., Synaptic events in ventrolateral thalamic neurons during suppression of recruiting responses by brain stem reticular stimulation, Brain Research, I (1966) 63-76. 21 PURPURA,D. P., PRELEVI~,S., AND SANTINI, M., Postsynaptic potentials and spike variations in the feline hippocampus during postnatal ontogenesis, Exp. Neurol., 22 (1968) 408-422.
34
R. W. THATCHER AND D. P. PURPURA
22 PURPURA,D. P., SCARFF,T., ANO MCMURTRY, J. G., Intracellular study of internuclear inhibition in ventrolateral thalamic neurons, J. Neurophysioi., 28 (1965) 487-496. 23 PL'RPURA, D. P., AND SHOFER, R. J., Intracellular recording from thalamic neurons during reticulocortical activation, J. Neurophysiol., 26 (1963) 494-505. 24 PURPURA, D. P., SHOFER, R. J., AND SCARFF, T., Properties of synaptic activities and spike potentials of neurons in immature neocortex, J. Neurophysiol., 28 0965) 925-942. 25 SCHEIBEL, M., AND SCHEIBEL, A., Some structural and functional substrates of development in young cats. In W. A. HIMWICH AND H. E. H;MWXCH (Eds.), The Developing Brain, Progr. in Brain Research, Vol. 9, Elsevier, Amsterdam, 1964, pp. 6-25. 26 TIqATCHER,R. W., AND PURPtmA, D. P., Maturational status of inhibitory and excitatory synaptic activities of thalamic neurons in neonatal kitten, Brain Research, 44 (1972) 661-665.
© ElsevierScientificPublishingCompany, Amsterdam- Printed in The Netherlands
21
POSTNATAL DEVELOPMENT OF THALAMIC SYNAPTIC EVENTS UNDERLYING EVOKED RECRUITING RESPONSES AND ELECTROCORTICAL ACTIVATION
ROBERT W. THATCHER* ANDDOMINICK P. PURPURA Department of Anatomy and the Rose F. Kenned), Center for Research in Mental Retardation and Human Development, Albert Einstein College of Medicine, Yeshiva University, Bronx, N.Y. 10461 (U.S.A.)
(Accepted March 20th, 1973)
SUMMARY
Postsynaptic potentials were intracellularly recorded from thalamic neurons in kittens of different ages during stimulation of medial thalamic (MTh) regions at low and high frequencies. In neonatal and young kittens ( < 10 days old) low-frequency (3.3/sec) MTh stimulation elicited IPSPs in thalamic neurons with a mean duration of 50 m s e c . EPSPs at this age were weak and variable. In older kittens (2-3 weeks old) MTh-evoked IPSPs exhibited a mean duration of 125 msec and EPSPs were more prominent in EPSP-IPSP sequences. High-frequency (80/sec) MTh stimulation in young kittens produced sustained summation of IPSPs and suppression of cell discharge. IPSPs were also elicited by high-frequency MTh stimulation in 2-3-week-old kittens but these IPSPs were rapidly terminated by powerful and sustained EPSPs. The relationship of these observations to previous studie ~, of evoked synchronizing and desynchronizing processes in thalamic neuronal organizations in adult animals indicates that: (1) the development of evoked thalamic neuronal synchronization is associated with the functional maturation of interneuronal pathways involved in the production of EPSP-IPSP sequences in thalamic neurons; and (2) the development of electrocortical activation subsequent to high-frequency MTh stimulation occurs i pari passu with an increasing capacity of excitatory synaptic inputs to elicit powerful and sustained EPSPs in thalamic neurons. Thalamic synaptic mechanisms underlying evoked synchronization and desynchronization of electrocortical activity attain functional maturation by the end of th~~. third postnatal week in the kitten.
* Present address: Department of Physiology, New York Medical College, 106th St. and Fifth Avenue, New York, N.Y. 10029, U.S.A.
22
R. W. THATCHER AND D. P. PURPURA
INTRODUCTION
Electrocortical activity of altricial newborn subprimate mammals is poorly organized with respect to rhythmically recurring spindle waves and deficient in the high-frequency low-voltage activity characteristic of wakefulness in mature animals7,10,2L Although these features of EEG ~immaturity' reflect the morphophysiological and biochemical immaturity of the brain as a whole, virtually nothing is known concerning the synaptic events that contribute to the physiological development of synchronizing and desynchronizing mechanisms controlling electrocortical activity. One approach to this complex problem is the subject of the present report. It is generally held that the thalamus is the major source of the afferent projection systems that powerfully influence electrocortical activity1,4,9,12. Intracellular studies of thalamic neurons during evoked electrocortical synchronizing and desynchronizing activities in adult animals have disclosed two fundamentally different patterns of thalamic synaptic events in association with these activities 15. During electrocortical synchronization elicited by low-frequency stimulation of medial thalamic (MTh) nonspecific nuclei a large proportion of thalamic neurons exhibit rhythmically recurring EPSP-IPSP sequencesS,14,~s,~9,22. In contrast, during electrocortical desynchronizafion elicited by high-frequency MTh stimulation thalamic neurons generally exhibit sustained excitatory synaptic activity23. A major factor contributing to the generalized synchronization of thalamic neuronal discharges is the inhibition of spontaneous activity effected by prominent and prolonged (100-200 msec) IPSPs ~s. During thalamically evoked reticulocorticai activation synchronizing IPSPs are attenuated or blocked and swamped by summating EPSPs 2a. Attenuation of synchronizing lPSPs is also observed following brain stem reticular stimulation 20. The foregoing summary of intracellular studies of thalamic neuronal operations and processes in adult animals provides a point of departure for assessing the maturational status of evoked synchronizing and desynchronizing synaptic events in the thalamus of the immature animal. The present study examines the synaptic events elicited in thalamic neurons during low- and high-frequency MTh stimulation in kittens of different postnatal ages. Data are provided on developmental differences in effects of MTh stimulation and on the factors which contribute to the synchronization and desynchronization of thalamic neuronal activity during postnatal ontogenesis. A preliminary report on some aspects of this study has appeared elsewhere ~. METHODS
Experiments were performed on kittens in 4 age groups: 1-3 days, 6-8 days, 13-15 days and 20-22 days. Ether anesthesia was employed during all operative procedures which included intratracheal intubation, exposure of the dorsolateral convexity of a cerebral hemisphere, cisternal drainage and suction ablation of cortex and structures overlying the thalamus. Following these procedures xylocaine was injected into all pressure points and applied in jelly form to exposed wound margins and skin surfaces. Thereafter the animals were paralyzed with intraperitoneal gal-
SYNAPTIC EVENTS IN IMMATURE THALAMUS
23
lamine triethiodide and artificially ventilated. Expired CO2 and heart rate were monitored periodically. Normal body temperature was maintained by wrapping the kittens in a flexible heating coil connected to a DC source. Concentrically bipolar stimulating electrodes were introduced into medial regions of the exposed thalamus under direct vision. Responses evoked by stimulating pulses of different frequency were recorded monopolarly from pericruciate cortex. The indifferent electrode consisted of an AgAgC! wire embedded in nuchal musculature, Intracellular recordings were obtained with 2 M potassium citrate filled micropipettes (20-40 M ~ resistances) which were inserted into the thalamus 1.0-3.0 mm lateral to the stimulating electrodes. Other details as to stimulating and recording procedures were similar to those described in comparable studies of adult cats 1s.22,23. RESULTS
General remarks The extraordinary 'fragility' of immature neocortical and hippocampal neurons has been noted previously13,21,24. The size of immature thalamic neurons and the necessity to probe through several millimeters of tissue undoubtedly contributed to the greater difficulty in obtaining satisfactory impalements of immature thalamic neurons than immature cortical neurons. Negative DC shifts indicative of transmembrane potenti~gls were frequently not obtained in the course of a complete excursion of a micropipette in a particular electrode track. This was not necessarily related to the age of the kitten under investigation although such DC shifts were generally more frequent in 2-3-week-old kittens than neonatal animals. A neuron was included in the present series of over 200 cells when a sudden negative DC shift was associated •,vith spike potentials irrespective of the amplitude or configuration of the discharges. In approximately 90% of these elements IPSPs were elicited by low-frequency MTh stimulation. 1PSPs survived the loss of spike potentials as in the case of immature neocortical and hippocampal neurons21, 24. A membrar~e potential of 30 mV was chosen as an arbitrary value in classifying cells with membrane potentials (MPs) in excess of and less than this value. Presence and absenze of spike potentials were additional criteria for classification as noted in Table I. In all age groups more cells had MPs less than 30 mV than exhibited MPs in excess c f 30 mV. This indicates that there was little difference in the traumatic effects of impalement in neonatal and 2-3week-old kittens. Rarely neurons were held with stable MPs and evoked PSPs for longer than 2-3 min. More commonly recordings were obtained for 20-40 sec. Despite this brief period of intracellular or 'quasi-~tracellular' registration it was p~ssible to determine latency and duration characteristics of evoked I PSPs. With slightly longer recording times the effects of varying the frequency of MTh stimulation on evoked PSPs were also examined. It is of importance to note in Table ? that for any particular age group latency and duration of evoked IPSPs were similar for cells that showed some degree of spike potential activity and those that exhibi,ted spike inactivation. In agreement with studies of evoked cortical responses to thalamic stimulation, in immature rabbits e it was not possible to elicit typical long-latency recruiting re-
24
R. W. THATCHER AND D. P. P U R P U R A
A
B
•
14d 21d Fig. 1. Examples of evoked responses (negative upwards) recorded from pericruciate cortex during 3.3/sec (A), and 10/sec (B) stimulation of medial thalamic (MTh) regions in kittens of different ages as indicated (d - days). Note that the initial response to the repetitive stimulus is large and decrements with successive stimuli in neonatal and very young kittens. In 2- and 3-week-old kittens recruiting responses are well organized and exhibit characteristics similar to those observed in adult animals. Time bars, 100 msec, throughout.
sponses in young kittens with stimulus frequencies employed in adult animals. Indeed the effect of 3-10/sec MTh stimulation in neonatal and young kittens was more commonly seen as a decrementing series of cortical evoked responses rather than typical recruiting potentials. Examples of responses recorded from pericruciate cortex in kittens of different ages are illustrated in Fig. !. It is evident in this figure that recruiting responses to 3.3/sec and 10/sec MTh stimulation were obtained in 2- and 3-week-old kittens but that in neonatal and I-week-old kittens decrementing responses were elicited by both stimulus frequencies. These data are entirely consistent with observations on the maturation of recruiting responses in the rabbit s .
Characteristics of IPSPs elicited by low-frequency MTh stimulation Neonatal and 6-8.day.old kittens. All the thalamic neurons impaled in l-3-dayold kittens exhibited effects of membrane trauma. This was seen as a rapid attenuation of injury discharges subsequent to spike inactivation, low-amplitude long-duration spikes, or spikes with multiple components21, 24 (Fig. 2). Despite these obvious signs of injury, low-frequency (3.3/sec) MTh stimulation elicited prominent IPSPs in approximately 90% of impaled neurons widely distributed in the rostral thalamus. In a small proportion of cells reduction in discharge frequency was observed in the absence of IPSPs. Examples of the characteristics of MTh-evoked IPSPs encountered in thalamic neurons of l-3-day-old kittens are illustrated in Fig. 2. Although IPSPs were the most clearly detectable synaptic events observed in thalamic neurons in the neonatal period, small EPSPs were occasionally noted preceding IPSPs. IPSPs with characteristics similar to those observed in 1-3-day-old animals were also noted in thalamic neurons from kittens 6-8 days old. Fig. 3 illustrates these
SYNAPTIC EVENTS IN IMMATURE THALAIVlUS
25
A B
A
I00
muK
Fig. 2. Examples of characteristics of IPSPs evoked in thalamic neurons from-2- and 3-day-old kittens in reponse to low-frequency (3.3/sec) medial thalamic (MTh) stimulation. A and C, from a 3-day-old kitten. B, from a 2-d~y-old kitten. In this and subsequent figures upper channel records are cortical surface responses (negativity upwards). MTh stimuli indicated by dots. A: first 3 decrementing series of responses. B: alternating characteristics of cortical evoked responses during continued MTh stimulation. C: recorded during stabilization phase of cortically evoked response. IPSPs in these 3 cells exhibit variable duration. Further description in text and Table I.
A B •
•
•
C
I 0 0 msec
Fig. 3. Examples of synaptic events evoked in 4 different thalamic neurons by low-frequency MTh stimulation during the course of experiments on 7-day-old kittens. A: a slight increase in the interval between discharges immediately after the MTh stimulus is the only effect detectable. B and D: short duration IPSPs are elicited by MTh stimulation in cells with partial spike potentials. C: cell unaffected by MTh stimulation.
26
R. W. THATCHER AND D. P. PURPURA
IPSPs in cells from different experiments. In Fig. 3A the inhibitory effect of MTh stimulation is seen largely as an increase in the interval between discharges following each stimulus. IPSPs were also prominent in injured neurons exhibiting attenuated and partial spikes (Fig. 3B and D). For each cell included in the present series 30-60 IPSPs elicited at 3.3/sec MTh stimulation were examined for latency and duration. Latencies ranged from 5 to 50 msec in both neonatal and 6-8-day-old kittens. IPSP durations ranged from 30 toj 150 msec in animals from both age groups. Table I shows that the mean latency of IPSPs in cells exhibiting spikes and membrane potentials in excess of 30 mV was 13.5 msec in l-3-day-old kittens and 14.8 msec in 6-8-day-old kittens. Slightly longer mean latencies were found in cells without spikes and lower membrane potentials. The mean duration of IPSPs was 50 msec in cells with spikes and > 30 mV membrane potentials. This was not significantly different in the two age groups although the mean duration of IPSPs was slightly less in cells without spikes and ~ 30 mV membrane potentials. hi 13-22-day-old kittens. Spike potentials were considerably shorter in duration and larger in amplitude during injury discharges in thalamic neurons from 13-to 22-day-old kittens. Such discharges were maintained at a relatively high frequency for a longer period in older animals than in neonatal and very young kittens. Examples of IPSP characteristics observed in 14- and 22-day-old kittens are shown in Figs. 4 and 5, respectively. The smoothly summating characteristics of the prolonged IPSPs A
B
C
L
,_,
_
I
--
~
~
~llbl~- -
1
I
D mY 1 O0 m ~
Fig. 4. Characteristics of long-duration (80--160 msec) IPSPs observed in 4 different thalamic neurons from a 2-week-old kitten. Note that at this developmen~*al stage injury discharges occur at a higher frequency and spike potentials are shorter in duration th,~n in younger kittens (of. Figs. 2 and 3). IPSPs in A and C have multiple components.
27
SYNAPTIC EVENTS IN IMMATURE THALAMUS
A.
•
C
.
D
,p ¥
•
•
• I00
msec
Fig. 5. Examples of IPSP characteristics observed in 4 different thalamic neurons from a 3-week-old kitten during low-frequency MTh stimulation. Spike potential amplitude and ~.uration are similar to spikes of thalamic neurons in adult animals xs,2a. This display emphasizes the synchronizing features of the long-duration IPSPs which limit spike discharges, to the brief periods after IPSPs and just prior to the succeeding stimulus of the repetitive train. Note in particular that IPSPs in B attain a duration of nearly 200 msec. In the other cells the IPSPs range from 100 to 150 msec. observed in the older kitten (Fig. 5) were similar to those observed in adult animalslS,20,2~,~3. Surprisingly the range or mean IPSP latencies observed in 13-22-day-old kittens did not differ significantly from the range or mean latencies observed in younger kittens (Table I). However, highly significant differences were observed in IPSP duraTABLE 1 CHARACTERISTICS OF
Days
No. of cells
IPSPs IN IMMATURE THALAMIC NEURONS
Mean duration (msec)
With Without With spikes IPSPs IPSPs S.D. 1-3
24 34 6- 8 18 20 13-15 17 22 20-22 21 24
4 11 2 1 2 1 3 2
55.8 45.1 54.7 43.5 125.4 81.7 128.6 118.0
6.6 4.4 7.1 3.4 4.1" 5.3* 4.6* 5.1"
Mean latency (msec)
Without spikes With spikes
Membrane potential Without spikes (mV)
•
S.D.
•
S.D.
•
S.D.
50.2 38.7 53.6 44.4 123.2 115.8 125.2 120.8
6.8 4.0 6.2 5.9 4.1" 4.9* 5.7* 4.1"
13.5 22.6 14.8 17.5 15.1 21.3 14.9 19.1
2.0 3.9 1.3 2.1 1.5 4.5 2.2 2.9
10.6 23.4 15.2 18.4 14.8 20.9 15.5 20.2
1.8 4.1 1.7 1.8 1.1 2.0 1.9 1.1
>30 < 30 > 30 < 30 > 30 < 30 > 30 < 30
* Significant differences (P < 0.001) between these means and the means of 1-3- and 6-8-day-old kittens.
28
R. W. THATCHER AND D. P. P U R P U R A
tions. The mean duration of IPSPs in cells with spike potentials and > 30 mV membrane potentials was 125 msee in 13-15-day-old animals and 129 msec in 20-22-dayold animals. Slightly shorter durations were observed in cells with spike inactivation. The data indicate that during the second and third postnatal weeks MTh-evoked IPSPs in thalamic neurons undergo a 2- to 3-fold increase in duration. IPSP characteristics attain mature form by the third postnatal week.
Developmental differences in synaptic effects of high-frequency MTh stimulation Neurons exhibiting relatively stable membrane potentials during and immediately after low-frequency (3.3/see) M T h stimulation were also examined for synaptie effects of high-frequency (80/sec) M T h stimulation. Remarkably different effects were observed during and following such high-frequency stimulation in very young ( < 1
A
I00
mse¢
B
O
Fig. 6. Changing effects of high-frequency (80/sec) MTh stimulation on thalamic neurons as a function of postnatal age. A: recordings from 3 different neurons from 3-day-old kittens. Onset of highfrequency MTh stimulation indicated by dots. IPSP summation is prominent though variable in the 3 neurons. In the upper and lower sets ~" records spike discharges occur shortly after the last stimulus of the repetitive train. In the middle set the increase in membrane potential associated with IPSP summation persists after cessation of MTh stimulation. Electrocortical activation is not seen at this age during high-frequency MTh stimulation. B: neurons from 14-day-old kittens. High-frequency MTh stimulation initially evokes 60-80 msec duration IPSPs which are terminated by sustained depolarizing shifts in membrane potential. Recovery of membrane potential occurs slowly after cessation of stimulation. Note induction of high-frequency electrocortical activity during MTh stimulation.
SYNAPTIC EVENTS IN IMMATURETHALAMUS
29
week old) and older kittens (2-3 weeks of age). A comparison of the basic difference in the two age groups is illustrated in Fig. 6. Examples of findings from 3 different preparations of 2-3-day-old animals are shown in Fig. 6A. The first stimulus of the repetitive train initiated a long-latency IPSP as in the case of low-frequency stimulation. Subsequent stimuli prolonged the duration of the IPSP and suppressed discharges when these were present prior to the onset of the high-frequency stimulation (Fig. 6A upper and lower set of records). In other neurons the increase in membrane polarization initiated by high-frequency stimulation persisted beyond the period of stimulation (Fig. 6A, middle set of records). Entirely different effects of high-frequency M T h stimulation were observed in thalamic neurons of 2-3-week-old kittens. As in the case of the very young kittens the first stimulus of the repetitive train elicited an IPSP (Fig. 6B). However, this was terminated after 60-80 msec by a depolarizing shift in membrane potential that gradually subsided following cessation of the high-frequency MTh stimulation (Fig.
A
B
I
"J"-~J"~--
•
-
-
J"-
~
I,o __
-
~
-
-
•
•
,
"
100
,
[mV
mmc
Fig. 7. Postactivation facilitation of EPSPs and attenuation of IPSPs in thalamic neurons from a 2-week-old kitten following high-frequency MTh stimulation. A: upper set, low-frequency MTh stimulation evokes IPSPs wl~ich are not preceded by EPSPs. Middle set, onset of high-frequency (80/sec) MTh stimulation (at arrow). IPSP is terminated by depolarizing shift in membrane potential. Lower set, resumption of low-frequency MTh stimulation several seconds later• In the postactivation period each stimulus elicits an EPSP and spike discharge, but IPSPs are not observed. Spontaneous discharges occur at variable times after the evoked EPSP. Note persisting change in evoked cortical potential in the postactivation period. B: recordings from a traumatized neuron in the same animal. Sequence of changes in evoked PSPs, similar to that in A, occurs in the postactivation period. Towards the end of the lower set of records the EPSP induced by the prior period of high-frequency stimulation becomes attenuated along with 'reappearance' of the prolonged IPSP.
30
R. W. T H A T C H E R A N D D. P. P U R P U R A
6B). A similar sustained depolarization of thalamic neurons subsequent to highfrequency MTh stimulation in adult animals has been shown to be attributed to summation of EPSPs 2a. In 2-3-week-old kittens low-frequency MTh stimulation elicited prolonged IPSPs prior to the brief period of high-frequency MTh stimulation whereas such lowfrequency stimulation immediately after high-frequency stimulation failed to evoke prolonged IPSPs (Fig. 7A). Instead during the immediate postactivation period lowfrequency MTh stimulation elicited short-latency EPSPs which triggered spike discharges. In other neurons after loss of spike potentials a similar transformation in synaptic events evoked by low-frequency MTh stimulation was observed in the immediate postactivation period (Fig. 7B). Rapid attenuation of EPSPs and restitution of prolonged IPSPs occurred with continued low-frequency MTh stimulation (Fig. 7B). DISCUSSION
The foregoing results provide evi,,~ence that inhibitory synaptic activities are prominently displayed in intrathalamic internuclear interactions initiated by lowfrequency MTh stimulation in neonatal kittens. The same cannot be said for excitatory synaptic events which were generally not conspicuous in thalamic neurons from neonatal and young kittens under the present experimental conditions. These observations are consistent with previous findings in neocortical '~4 and hippocampal~a, '-'1 neurons of neonatal kittens with one exception. In the case of immature cortical neurons evoked IPSPs were found to be of long duration, comparable to, if not greater than IPSPs elicited in mature cortical neurons under similar conditions of examinationlL lPSPs in thalamic neurons of neonatal and young kittens have a mean duration that is significantly shorter than the mean duration of IPSPs in 2-3-week-old kittens. After the third week such IPSPs exhibit all the characteristics observed in adult animalslS-20, 2a. In view of the essential role of long-duration IPSPs in the local 2 and generalized synchronization 18 of thalamic neuronal activity it follows that the functional maturation of intrathalamic internuclear events associated with generalized synchronization of thalamic neuronal activity~4, x5 consists primarily in an increase in the mean duration of IPSPs. The progressive augmentation in the capacity of internuclear pathways to elicit EPSPs in immature thalamic neurons represents a second feature of the maturational process of thalamic neuronal synchronization. The relative functional immaturity of thalamic internuclear excitatory synaptic pathways in neonatal and young kittens is also reflected in the synaptic events observed in these preparations during high-frequency MTh stimulation. During the first postnatal week high-frequency MTh stimulation elicits sustained depression of thalamic neuron activity. Summation of IPSPs was evident in most of these cells. By 2 weeks of age a new synaptic event is added in the form of a powerful and sustained EPSP which is capable of rapidly inactivating spike electrogenesis in cells exhibiting spike activity. Evidently the augmented excitatory synaptic activity so initiated persists within the interneuronal networks constituting the internuclear pathways. For
SYNAPTIC EVENTS IN IMMATURE THALAMUS
31
immediately following termination of the high-frequency MTh stimulation in older kittens EPSPs may be observed in response to low-frequency MTh stimulation when such stimulation prior to high-frequency stimulation failed to evoke them. Moreover there is a striking attenuation of low-frequency evoked IPSPs in the postactivation period which is similar to that observed in adult animals following high-frequency MTh stimulation 23. Thus the present findings suggest that during the second postnatal week excitatory internuclear intrathalamic synaptic pathways undergo a rapid phase of functional maturation. Evidently this developmental event heralds the capacity of thalamic neurons to exhibit powerful excitatory drives during MTh-evoked reticulocortical activation. Intracellular studies of thalamic neuronal events in adult animals during the transition from low- to high-frequency MTh stimulation have emphasized the frequency specificity of the sustained excitatory action initiated by MTh stimulation during the thalamocortical desynchronization process 23. This frequency specificity has been confirmed recently in similar studies of Broggi and Margnelli 5. It has been inferred from observations on the attenuation of synchronizing IPSPs in EPSP-IPSP sequences during the transition from low- to high-frequency MTh stimulation that such attenuation results from an inhibition or blockade of inhibitory pathways generating the synchronizing IPSPs as well as augmentation of excitatory synaptic drivesaa. If the attenuation of synchronizing IPSPs in thalamic neurons during highfrequency MTh stimulation was a consequence of the activation of inhibitory pathways by high-frequency stimulation it might be anticipated that IPSP attenuation would occur irrespective of the capacity of such stimulation to elicit powerful depolarizing EPSPs. The fact is that prior to the functional development of excitatory synaptic pathways capable of producing sustained EPSPs during high-frequency MTh stimulation IPSP attenuation does not occur. Rather low-frequency as well as highfrequency MTh stimulation elicits IPSPs in thalamic neurons of neonatal and young kittens. This suggests that high-frequency MTh stimulation does not activate pathways which are inhibitory to the interneuronal elements generating synchronizing IPSPs in thalamic neurons, at least in very young kittens. The possibility still exists that the attenuation of synchronizing IPSPs observed in older animals during highfrequency MTh stimulation could be due to the late development of a parallel pathway, inhibitory to elements generating synchronizing IPSPs. However the most parsimonious interpretation of the present observations is that the attenuation or 'blockade' of the synchronizing IPSP during high-frequency MTh stimulation is more apparent than real and represents the addition of EPSPs that in effect override simultaneously elicited IPSPs. One feature of the events observed in thalamic neurons of enc6phale isol6 adult animals during low-frequency MTh stimulation or during spontaneous or evoked spindle waves is seen in the cluster of spike discharges that succeed prolonged rhythmically recurring IPSPs 14,1s,19. Similar bursts superimposed on membrane depolarizations have been seen in ventrobasal neurons of barbiturized adult animals 1-3. Andersen and Andersson 1 have emphasized a role of postanodal exaltation or postinhibitory rebound in the generation of burst discharges during spindle waves. In
32
R. W. THATCHER AND D. P. P U R P U R A
contrast, evidence has been presented that depolarizations succeeding IPSPs in thalamic neurons are EPSP components of EPSP-IPSP sequences11,14, a view that has now been championed by Andersson and Manson 4. Results of the present study are relevant to the origin of these postinhibitory burst discharges. If postanodal exaltation was a general property of thalamic neurons it might be expected that burst discharges would occur following prolonged IPSPs whenever the latter were elicited. On the other hand, if such discharges were dependent upon the development of EPSPs succeeding the IPSPs then in the absence of EPSPs postinhibitory bursts should not occur. None of the thalamic neurons examined in very young kittens exhibited postinhibitory burst discharges of the type encountered in adult animals. Neither did they exhibit prominent spontaneous or evoked EPSPs to low-frequency MTh stimulation until after the third postnatal week. This suggests that the major determinant of postinhibitory burst discharges is related to the development of excitatory synaptic pathways which interact in some as yet poorly understood fashion with inhibitory interneuronal elements to give the EPSP-IPSP sequences characteristic of synchronizing activities of thalamic neurons t4,15. The present study sheds little light on the possible mechanisms underlying the increase in mean duration of IPSPs in thalamic neurons during postnatal development. To the extent that repetitive activity of inhibitory interneurons la contributes to the prolonged conductance increase associated with synchronizing IPSPs in thalamic neurons s, it is reasonable to consider developmental changes in repetitive responsiveness of immature thalamic neurons as an important factor in the increase in mean duration of IPSPs. The present observations taken together with previous studies of immature neocortica124 and hippocampal neurons 'z-t provide no convincing data for the role of repetitively active inhibitory interneurons in the production of prolonged IPSPs in immature brain. However, if inhibitory interneurons are relatively small and fragile compared to the cells impaled in this and other ontogenetic studies it may well be that present failures to record from these elements may reflect largely operational difficulties. Alternatively it may be argued that high-frequency discharges of inhibitory interneurons may not be required to produce prolonged IPSPs in immature neurons if transmitter action is prolonged for various reasons t6. Apropos of this, it is of interest that preliminary electron microscope studies have suggested a remarkable development of complex synapses surrounded by glial processes in the thalamic neuropil during the first 2-3 weeks postnatally in the kitten 17 (Pappas and Purpura, unpublished). Conceivably such developmental events could contribute to the increase in duration of action of inhibitory transmitter at such synapses. Obviously this problem requires close pursuit in the further analysis of developmental changes in synaptic transactions in the immature brain 1~. Finally the developmental changes in patterns of synaptic activities in thalamic neurons described here call attention to the important role of intrathalamic interneuronal synaptic organizations in the electrographic events associated with the ontogenesis of sleep-wakefulness behavior.
SYNAPTIC EVENTS IN IMMATURE THALAMUS
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ACKNOWLEDGEMENTS This s t u d y was supported in p a r t by a G r a n t f r o m the Alfred P. Sloan F o u n d a tion a n d the N a t i o n a l institute o f Neurological Diseases a n d Stroke (NS-07512). Dr. T h a t c h e r was s u p p o r t e d by N . I . M . H . G r a n t MH-6418.
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