Brain Research, 134 (1977) 429 444

429

© Elsevier/North-HollandBiomedicalPress

DEVELOPMENT OF KITTEN HIPPOCAMPAL NEURONS

PHILIP A. SCHWARTZKROIN and RICHARD J. ALTSCHULER Department of Neurology, Stanford University School of Medicine, Stanford, Calif. 94305 (U.S.A.)

(Accepted January 28th, 1977)

SUMMARY Cells in the CA1 region of the hippocampus of kittens were studied using an in vitro slice preparation. Good quality intracellular records were obtained from over 100 cells from kittens 2 days to 4 weeks of age. Cell resistance was high in 2-day-old animals and decreased over the following 4 weeks. Both excitatory and inhibitory synaptic potentials were seen in all animals. EPSPs were only weakly effective in triggering spikes in the youngest kittens, but were greatly potentiated by repetitive stimulation at 3-10/sec; IPSPs caused a potent blockade of cell discharge in even the youngest preparations. Stimulation of the orthodromic input pathway led to a complex series of excitatory and inhibitory synaptic events which was not seen in the adult. In the 2- and 4-week-old kittens, a cell type with physiological properties different from the predominant pyramidal cell began to appear in recordings from the CA1 region. Technical difficulties inherent in in vivo recordings from neonatal animals were considerably less with the in vitro technique. Careful developmental studies may now be pursued in the slice at the single cell synaptic level.

INTRODUCTION The developing mammalian central nervous system has been the subject of a large number of studies ~7,48which have employed physiological7,x1,17,21, anatomical 1,3°,41,45 and chemical 6,14,3a techniques of analysis to gain an understanding of normal maturation, of changes that can take place after perinatal manipulation, and consequently of the 'plasticity' inherent in developing nervous tissue 8,21,23. Most studies at the physiological level have dealt with EEGlZ,lv,~,a2, 40, evoked response 13,19,89,5°, and extracellular single unit dataea,25,49; relatively few reports are available which describe synaptic activities and other intracellular characteristics of cells in the CNS. The technical difficulties of maintaining very young animals in good physiological condition and of recording from small fragile cells in the developing nervous sys-

430 tem are considerable. Nonetheless, investigators have been able to obtain intracellular recordings at the spinal cord and cortical levels of kittens. Spinal cord studies suggested that the development of synaptic connection on to motoneurons is virtually complete by two days after birth16,2a,29, 50. The experiments of Purpura and associates on kitten neocortex, cerebellum, and hippocampus30,3a, 3~-3v indicated that synaptic connections in these higher CNS regions are somewhat slower to develop than in the spinal cord. In the young kitten, excitatory postsynaptic potentials (EPSPs) were less effective in triggering spikes than in the adult, there was little spontaneous activity, and there appeared to be active dendritic spike generation; inhibitory postsynaptic potentials (|PSPs) were well developed even in the youngest animals studied. These physiological results were presented together with histological and electron microscopic data from maturing kitten brain3a,34, 4z. Although the papers by Purpura and co-workers are important contributions to our understanding of the developing mammalian CNS, these reports illustrate the difficulty of interpreting results obtained from cells in the immature brain. The data from these studies suggested that cells in the immature brain had relatively low resting potentials and sluggish spike discharge. The authors concluded, however, that because of the fragility of these neurons, they were often injured by electrode penetration so that measures of membrane characteristics and synaptic potentials were not accurate. We have found that many of the technical difficulties encountered in recording from the in vivo kitten preparation - - proper maintenance of the animal over long periods, recording stability, good cell penetrations - - could be alleviated with in vitro methods, and have therefore undertaken a re-examination of the developing kitten hippocampus. High quality intracellular recordings have been obtained that show more clearly the course of development of cell characteristics and synaptic activity. METHODS Hippocampal slices were made from 14 kittens in the age range of 2 days to 4 weeks and from 2 adult cats; 4 animals were 2-3 days, 4 were 5-6 days, 4 were 2 weeks, and 2 were 4 weeks. The general procedure for preparing slices and maintaining them in vitro has been described previously 43. All animals were anesthetized with ether, decapitated, their brains quickly removed, and the hippocampus dissected free. Slices 400 #m thick were made transverse to the long axis of the hippocampus, and taken from the middle third of the structure. Time from animal death to placement of slices in the incubation chamber was less than 5 min in the kittens; a somewhat longer time (5-7 min) was required in dealing with the 2 adult cats. Slices in the in vitro chamber were maintained at 36.5 °C and bathed by a perfusion medium containing 124 m M NaC1; 5 m M KC1; 1.25 m M NaH2PO4 • H20; 2.0 m M MgSO4 • 7H20; 2.0 m M CaC~2 • 6H20; 26 m M NaHCO3; 10 m M dextrose. Slices were viable for periods of 4-8 h as determined by recording of single cell activity. Recordings were made from the CA1 region of hippocampal slices using micropipettes filled with 4 M potassium acetate (20-40 Mf~ measured at 60 Hz). Potentials were amplified and displayed using conventional electronics, and were stored on

431 magnetic tape for future examination and analysis. Stimulus pulses were delivered through monopolar tungsten electrodes to the stratum radiatum to activate fibers making synaptic contact with the CA1 pyramidal cells. Cell resistance was determined by passing current pulses through the recording electrode via a bridge circuit, and measuring the resultant voltage deflection when the bridge was balanced. Samples of hippocampus from animals of all ages were taken for electron microscopic examination. The tissue was fixed in 4 ~ paraformaldehyde - 1 ~ glutaraldehyde, post-fixed with 2 ~ osmium tetroxide, dehydrated, and embedded in Epon. Thin sections of the CA1 region were cut at 90-100 nm and stained with uranyl acetate and lead citrate. In addition, tissue from some animals was stained by a Golgi method using potassium chromate, potassium dichromate, and mercuric chloride. The tissue was embedded, and sections of hippocampus cut at 120 #m. RESULTS

Membrane and action potential characteristics In the youngest animals (2-3 days), considerable difficulty was experienced in

A 1

4 wk

B 2

200mseC

1

2

6 day ~. 20mY I 100msec

Fig. 1. A: two CA1 cells from 2-day-old kittens and one from a 4-week-old kitten. In A1, cells were clearly injured and generated broad oscillatory potentials in the young kittens. As the cells repaired, the oscillations disappeared and/or gave way to spike discharge (A2). In the injured cell from the 4-week-old kitten, broad oscillations were not present, but small, fast-rising potentials were seen, and functioned as prepotentials for full spikes. In all traces, stimulation to the orthodromic fiber pathway (stratum radiatum) (marked by the triangle) elicited a hyperpolarization. Early excitation was also seen when cells were relatively healthy (4-week-old example). B: fast prepotentials (FPPs) generated in cells of young kittens. In the first cell, from a 2-day-old kitten, traces are superimposed to show FPP relation to EPSP (1) and to spike discharge (2); note inflection on full spike rising phase which corresponds to FPP peak amplitude. Time calibration for this cell is given under trace 2. In another cell from a 2-day-old kitten, the FPP could be triggered at the peak of an EPSP (1) or on its falling phase; again, note inflection on spike rising phase (2). A bursting cell from a 6-day-old kitten is shown in the third row; FPPs were seen associated with stimulus-induced activity (1) and also spontaneously (2). In 2, the cell was slightly hyperpolarized to block full spike discharge, revealing isolated d-spikes. Bar above the signal trace is a current monitor; downward deflection, relative to its position in 1, signifies injection of hyperpolarizing current.

432 obtaining stable intracellular records with high membrane potential. Cells appeared to be widely separated, perhaps because of the large extracellular space characteristic of hippocampus at this age (see Anatomy section). In most penetrations, cells were damaged by the electrode, resulting in slow, low amplitude potentials and/or broad spikes (Fig. 1A1, top two rows). Such potentials could occur as rhythmic oscillations reminiscent of 'inactivation responses' seen during hippocampal theta activity18, 20. These potentials developed into cell action potentials as the penetration improved and the cell 'healed' (Fig. IAz). As the animals matured, it became somewhat easier to obtain good penetrations, although the slow rhythmic oscillations remained evident in preparations of kittens through 2 weeks of age. In 4-week-old kittens, these oscillations were apparently replaced by fast prepotential-like activity (Fig. IAa, third row), and were completely absent in adult cats. In cells from adult cats, too, spontaneous activity was somewhat lower than in kitten cells, although this observation is probably attributable to less cell damage and depolarization by electrode penetration in the slices from older animals. Eighteen intracellular recordings of good quality were obtained from the 4 kittens in the 2-3-day age group (Table I). These cells had average membrane resting level near 50 mV and overshooting action potentials. Sharp-rising, low amplitude potentials similar to previously described fast prepotentials 47 were seen in 7 of these penetrations (Fig. I B, top two rows). Many of the recordings showed very 'noisy' baselines, perhaps reflecting instabilities in the cell membrane. Cell resistance was high in these neurons (average value of 46 Mr2 in 9 cells), correlating with small cell size seen in histological preparations. It seemed likely, then, that the baseline 'noise' could also be due to spontaneous synaptic input onto dendritic processes which were close to the recording electrode, resulting in relatively large potentials. In 4 animals 5-6 days old, 31 good intracellular penetrations were obtained, and in many cases cells were held for several minutes. In 21 cells, an average cell resistance of 34 M~) was found (Table i). Fast prepotentials (FPPs) were observed in a lower percentage of the cells in the 5-6-day kitten (only 7 of the 31 healthy penetrations) than in younger animals (Fig. IB, third row). However, bursting activity (Fig. 1B, third row) was more obvious, both as a sign of cell injury and as a normal firing pattern. Cell bursting was seen in TABLE I Quantitative data obtained from cells in the CA 1 region o f slices o f kitten hippocampus maintained in vitro Age

Number o f animals

Cells' A verage spike (spike ~. 40 m V) amplitude (in V)

Average resting Average cell potential resistance (m II) (MD.)

2-3 days 5 6 days 2 weeks 4 weeks Adult

4 4 4 2 2

18 31 62 28 21

46 54 48 50 52

49 57 55 54 68

46 34 33 24 23

433 extracellular as well as intracellular recordings. I n t r a c e l l u l a r d e p o l a r i z i n g current pulses elicited trains o f spike discharge m u c h m o r e easily in cells in these slices t h a n in the 2 - 3 - d a y a n i m a l s ; at higher current levels, bursts o f spikes generated f r o m an active m e m b r a n e d e p o l a r i z a t i o n were sometimes triggered. W h e n d e p o l a r i z i n g current pulses e v o k e d high frequency discharge, a p e r i o d o f i n h i b i t i o n (50-200 msec) d u r i n g which s p o n t a n e o u s activity was blocked, often followed the spike train. This a f t e r h y p e r p o l a r i z a t i o n was seen consistently in cells o f all kittens after 5-6 days. Very little change was seen between the 5 - 6 - d a y a n d 2-week samples f r o m kitten h i p p o c a m p u s . A v e r a g e cell resistance, based on m e a s u r e m e n t s f r o m 23 o f 62 g o o d intracellular records, was 33 Mf~. F P P s were in evidence in only 6 healthy cells, a n d c o n t i n u e d to decrease in frequency o f occurrence in the o l d e r animals. Twenty-eight g o o d quality intracellular records were o b t a i n e d f r o m h i p p o c a m p i o f two 4-week-old kittens. In 14 cells, average resistance was 24 M ~ , much r e d u c e d f r o m that seen in y o u n g e r kittens. A c t i o n potential h y p e r p o l a r i z i n g u n d e r s h o o t s became obvious in cells in slices o f 4-week-old kittens (Fig. 2B1); some spike u n d e r s h o o t h a d been observed in cells in 2-week animals, but the u n d e r s h o o t at that age (and earlier) h a d been o b s c u r e d by d e p o l a r i z i n g a f t e r p o t e n t i a l s or underlying r h y t h m i c oscillations. T w e n t y - o n e intracellular records were o b t a i n e d f r o m h i p p o c a m p a l slices from two a d u l t cats. It p r o v e d s o m e w h a t m o r e difficult to remove, slice, a n d m a i n t a i n

I

2

A B

I

II IIIIII L

D

2

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4 wk

Z..__.___ re.t,n

200msec omvI V

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20 msec

Fig. 2. A: cell from 4-week-old kitten which showed a complex synaptic pattern - - short EPSP, short IPSP, long EPSP, long IPSP - - in response to orthodromic stimulation (A1). These potentials were exaggerated when the cell was depolarized (upward deflection of current monitor relative to 1) (As). Calibrations in B pertain also to A. B: another cell from a 4-week-old kitten, also showing the complex synaptic pattern; spike hyperpolarizing undershoots (marked by arrows) became apparent at this age. C: recording of field potential 'population spike' (top trace) elicited by stratum radiatum stimulation in slice of hippocampus of 4-week-old kitten. The negative peak of the population spike occurred at the same latency as orthodromically elicited spikes from single cells (bottom trace). D: cell from 5-day-old kitten in which effects of depolarizing and hyperpolarizing current on the pattern of EPSPs (early E at arrow, late E at open triangle) and IPSPs (first I at solid line, later I at dotted line) are shown. D1 and D2 show the same phenomena at different sweep speeds. Depolarizing current decreased EPSP amplitude and led to spike discharge from the later EPSP; IPSP amplitudes were exaggerated. Hyperpolarization decreased IPSP amplitude (particularly that of the first IPSP) and EPSPs were exaggerated and appeared to merge; inflection on the combined EPSPs corresponds to the early IPSP.

434 healthy tissue from the adult than fiom the kitten. This difficulty was primarily due to the larger amount of tissue and increased bone thickness in the adult which slowed the removal of the brain and impeded dissection of the hippocampus. Thus, the tissue examined from the two adult cats may have suffered more in removal than tissue from the kitten. Nevertheless, healthy cells and synaptic potentials were seen in slices from both adult cats. Average cell resistance measured in 11 cases was 23 Mg2.

Synaptic activity Extracellular recordings in slices from young kittens showed no field potentials evoked by stimulation in the orthodromic pathway. The absence of such signs of synchronous cell synaptic and spike activity was not surprising considering the small size of the EPSPs, the apparent loose packing of cells, and the variable latency of spikes when they were orthodromically triggered (see below). High amplitude field potential 'population spikes' were first recorded in the slices from 4-week-old kittens (Fig. 2C). At the single cell intracellular level, synaptic potentials and spike initiation were evoked, by stimulation in stratum radiatum, with increasing consistency as the animals matured. Even in recordings from apparently injured cells from 2-day-old kittens, slice stimulation in stratum radiatum produced hyperpolarizing potentials which caused a cessation of spontaneous injury discharge (Fig. 1A); inhibitory postsynaptic potentials were therefore judged functionally potent in the youngest animals studied. In cells apparently most 'healthy' (i.e., with highest resting level), stimulation elicited an initial short latency excitatory postsynaptic potential, which was immediately followed by a longer-lasting hyperpolarization (Fig. 2D1, resting). The early EPSP was lost upon cell injury and subsequent cell depolarization (Fig. 2D1, depol), and was not seen in poor penetrations; however, it was apparent in all cells in which the resting potential was high. Current-induced hyperpolarization was effective in unmasking the EPSP (Fig. 3A). This EPSP was not always sufficiently potent, even in healthy cells, to initiate action potentials. When action potentials were triggered, they sometimes occurred late, on the falling phase of the EPSP (Fig. 3B2). Cell spiking was more consistently triggered by orthodromic stimulation in the 5-6-day-old animals than the 2-3-day animals. When stratum radiatum was stimulated, the short latency, short duration EPSP was reliably evoked and often gave rise to an action potential. Action potential latency, however, could still be quite variable, with spikes sometimes arising from the falling phase of the EPSP. EPSP effectiveness in triggering spike discharge increased gradually as the animals matured until, in the adult, orthodromic stimulation could almost always be adjusted so that the EPSP reached spike initiation threshold. Stimulus-induced hyperpolarization, with associated blockade of cell spiking activity, was the most consistent synaptic response seen in preparations from all ages of kittens. Spontaneous activity could be blocked by this IPSP for several hundred msec, as was also the case in slices of hippocampus from the adult cat. Starting in the 2-3-day-old kittens, however, and becoming most apparent in the 2-4-week-old kitten, a series of synaptic events was found that complicated the adult synaptic pro-

435

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,

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c 20mY I |oo msec Fig. 3. Cells from 2-day-old kittens to show effects of cell hyperpolarization (A) and repetitive stimulation (B, C) on EPSP amplitude. In A1, orthodromic stimulation evoked no depolarizing EPSP; when the cell was hyperpolarized (A2), the EPSP became evident. Duration and magnitude of the current pulse is shown by the current monitor above the signal trace. B1 and C1 show two other cells from 2-day-old kittens in which no EPSP (B1) and a small EPSP (C]) were triggered by orthodromic stimulation at 0.3/sec; 3/sec stimulation (B2, C_~)resulted in large, long duration EPSPs which led to spike initiation.

file of an initial EPSP followed by a long-lasting IPSP (Fig. 2A, B, D). In the kitten, the IPSP was interrupted after about 50 msec by a small depolarizing hump which itself could trigger an action potential (triangle in Fig. 2D). The later excitatory potential, as well as the early EPSP (arrow in Fig. 2D), could initiate action potentials, so that a single stimulus often evoked pairs of spikes from a given cell, each triggered from separate depolarizing potentials. When subthreshold for spike initiation, the later hump could summate with a subthreshold depolarizing current pulse to trigger an action potential, Appearance of the second excitatory hump was not dependent on the initial excitation; spikes were triggered from the later potential even when the early EPSP was ineffective or absent. The two EPSPs were of very different time course; the initial EPSP was fast rising and brief ( < 5 msec) and more sensitive to cell depolarization than the later, longer (50-75 msec) depolarization. These two excitatory potentials appeared to merge as the cell was hyperpolarized (Fig. 2D, hyperpol), but an inflection remained as evidence of the underlying separation. The merged depolarizations could be extremely long and of high amplitude. The late excitatory potential was followed by an inhibitory period (dotted line in Fig. 2D), perhaps a continuation of the first IPSP (solid line in Fig. 2D). It appeared, however, that the two inhibitory periods were due to two separate processes, each of which resulted in blockade of cell spiking. The first inhibitory period lasted 50-100 msec and was associated with a large membrane hyperpolarization; the second, although effective in blocking Spike discharge for several hundred msec, was associated with little or no membrane hyperpolarization (Fig. 2A2). This complex EPSPIPSP sequence characteristic of kitten hippocampus was no longer obvious in the adult cat, although a hint of this potential sequence was seen in one or two cells.

436 Slice stimulation was normally carried out at a rate which produces no significant facilitation or potentiation effects in adult guinea pig and rabbit slices 44. With faster stimulation (3-10/sec), changes in the synaptic potentials were elicited f r o m cells in slices o f all aged kittens. Cell resting potential normally increased, with a subsequent increase in EPSP amplitude and duration, and decrease in IPSP amplitudes (Fig. 3B, C). This negative membrane drift could be long-lasting, suggesting that repetitive stimulation mobilized a metabolic p u m p mechanism 5. In cells where no initial EPSP was apparent (especially young animals), more rapid stimulation often brought out the EPSP (Fig. 3B). The initial and the later EPSPs tended to merge into one large long duration (60-80 msec) depolarizing potential, and spike initiation from the EPSP occurred more reliably. In older kittens, and particularly in adult cats, the degree of EPSP potentiation was less than in younger kittens. This finding appeared to be correlated with the absence o f the later EPSP in older animals.

Additional cell type Cells with very short spike duration (cf. Fig. 4A1 and B 1 ) , high spontaneous firing rate (cf. Fig. 4A4 and B4), and fast latency response to stimulation with a characteristic EPSP (cf. Fig. 4A2 and Bz) began to appear in recordings from hippocampi o f 2-week-old kittens. These cells often had smaller spike amplitudes than the rest of the population, but comparable resting membrane levels (Fig. 4A). The baseline noise in these recordings, too, seemed to be much 'busier' than in the majority o f cells,

A

1\

B

\ --

5 ~e

I00

500 m s e e

Fig. 4. Comparison of two types of cell recorded from stratum pyramidale of the CA1 region. A : more rare cell type. B: dominant pyramidal cell type. In 1, action potentials are presented at fast sweep speed, showing that the cell in A was much briefer and of slightly lower amplitude than the cell in B. Examples of spontaneous spiking are shown in the first traces of 2; note the hyperpolarizing undershoot following each spike in A, whereas in B, the cell spike is followed by a depolarizing afterpotential. Effects of orthodromic stimulation (triangle) in eliciting an initial excitatory response are illustrated in the second traces in 2. Row 3 shows responses of each cell to a depolarizing and hyperpolarizing current pulse (100 msec duration, 0.5 nA amplitude). Slow sweeps in 4 illustrate the relatively high spontaneous discharge rate of the cell in A compared to the cell in B (first trace) ; in both A and B, orthodromic stimulation (triangle) led to a long4asting IPSP during which spontaneous spiking was blocked.

437 and rheobase current was considerably lower (cf. Fig. 4Aa and Ba). In this newly recorded cell type, as well as in the predominant cell type, IPSPs were elicited by stimulation in stratum radiatum with consequent blockade of spontaneous spiking. Such cells were particularly obvious in 2- and 4-week-old animals, and were seen in smaller numbers in the adult cat.

Anatomy Samples from hippocampus of all the kittens that were studied in vitro were also examined in electron microscopic preparations. Cells in the young animals were small compared to cells in older kittens (compare somata of 2-day and 5-day animals in Fig. 5 with somata of 2- and 4-week kittens in Fig. 6), but already had elaborately branching dendrites and dendritic spines. There was a considerable amount of 'free' intercellular space; cells were not yet closely packed in stratum pyramidale. Non-pyramidal cells were also seen frequently (e.g., Fig. 5G), with dendritic branching patterns different from that of the pyramidal cells. Electron micrographs revealed only a small number of synapses in the youngest kittens, with synapses occurring both on the soma and on dendrites (Fig. 5A, B, D, E). Synaptic type, as judged by symmetry of membrane thickenings and vesicle shape criteria, indicated the presence of both inhibitory and excitatory synapses on somata and dendrites in the kittens. As kittens got older, there was a great proliferation of synaptic profiles both at the soma and on dendrites. The full adult profile was not reached, however, even in the 4-week-old animal. DISCUSSION The results of these experiments were gratifying from a technical point of view, since many good quality intracellular recordings were obtained from cells of kitten hippocampus maintained in vitro. Over 100 records were obtained from slices of only 14 kittens (Table I). Such success is important not only with regard to the prospects for future experiments involving intracellular recording from immature brain, but also in considering interpretation of the results. Three particular observations made in the slice preparation bear on earlier descriptions of developing synaptic and spiking activity in kitten hippocampus. Purpura et al. a5 were unable, in their recordings from the youngest neonatal animals, to consistently evoke EPSPs with fornix stimulation, but did observe IPSPs in almost all cells. It is probable that because of the low restling potentials of many of the cells recorded in the in vivo preparation, it was difficult to distinguish EPSPs. Given the small size of hippocampal cells in kittens of this age, it is to be expected that attempted electrode penetrations often cause cell injury and depolarization, as was also apparently the case in slice preparations of hippocampus from 2-3-day-old kittens. When recordings from healthy cells weie obtained in the slice, however, evoked EPSPs and cell spiking were found. The EPSP became particularly apparent when cells were hyperpolarized by current injection and could disappear completely if the cells were depolarized. As previously reported aS, we found that the IPSP was the synaptic event most usually associated with stimulation; this observation was probably due to the fact that most cells were depolarized by electrode-

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induced injury and thus IPSP-induced hyperpolarizations were exaggerated. In describing synaptic activity observed in hippocampal cells of neonatal kittens, Purpura et al. a5 also suggested that the IPSP was of relatively short duration (70-80 msec) compared to the IPSP in the adult. If the data on which this observation was based were derived from cells depolarized by impalement injury, a shortened IPSP might be observed; the driving electrochemical potential for return of the membrane potential to resting level from Eipsp would be greater than if the cell resting level were high. A longer duration IPSP would be consistent with findings in kitten neocortex37. In recordings from hippocampal slices of even the 2-3-day kittens, IPSP effects could be seen for several hundred msec, but the inhibitory potential was usually divided into two components: a relatively brief phase of the IPSP was associated with an especially large hyperpolarization; the inhibitory effect of the longer-lasting component occurred without a marked hyperpoladzation. Finally, it appeared to us, as it did to Purpura et al. 35, that spike duration was usually longer in cells of young kittens than in cells of the adult. Whether this was a real difference, or whether increased spike duration might simply have been a function of cell injury (which is much more likely to occur in the young animal) has not been determined. We have observed brief action potentials in cells of slices from the young kitten and have seen that cell depolarization could increase this spike duration. It therefore seems somewhat premature to conclude that broad spikes are characteristic of healthy neurons in the young animal. Field potential data, although not extensive in the present study, suggested that cell synchrony, as indicated in responses to afferent input, developed slowly over the first 2 postnatal weeks. Crain and associatesg, 10 have found that in hippocampal explants from fetal mice, slow field potential responses can be elicited, even though this tissue is at a very early stage in development. They have also evoked complex single cell potentials in fetal hippocampal explants 51. However, it should be noted that these investigators were not stimulating a selective orthodromic pathway, as was the case in experiments on the kitten hippocampal slice. Elicitation of synaptically induced field potentials in intact hippocampus 2 and in hippocampal slices4~ requires that stimulating and recording electrodes be aligned within a lamella and that a large number of cells receive and respond to the inputs simultaneously. It is possible that in the development of kitten hippocampus, there is a shift in lamellar alignment, so that slices were made at an appropriate orientation in the older kittens, but were cut across lamellae in the younger kittens. This explanation for the absence of synaptically evoked field potentials does not account for the presence of synaptic drive at a single Fig. 5. A - C : electron micrographs of hippocampal tissue of a 2-day-old kitten. A shows an example of a synapse in the dendrites, B a synapse on the soma, and C a lower power picture of the cell soma. In this and the following electron micrographs, the calibration bar represents 0.5/~m; the arrow on the cell soma gives the direction of the apical dendrite; arrows in the higher magnification photos indicate synapse location and direction. Abbreviations: D, dendrite; M, cell membrane; N, cell nucleus; V, synaptic vesicles. D - F : electron micrographs from 5-day-old kitten; D shows axodendritic synapse, E shows axosomatic synapse, and F shows the cell soma. G: example of pyramidal (open arrow) and non-pyramidal (solid arrow) cells in the 5-day-old kitten hippocampus, as seen in a Golgi-stained section. The brackets marks the extent of the CAI stratum pyramidale, approximately 40/zm wide.

440

Fig. 6. A - C : electron micrographs from 2-week-old kitten showing: A, cell soma; B, axosomatic synapse; C, axodendritic synapse. D - F : electron micrographs flora 4-week-old kitten showing: D, cell soma; E, axosomatic synapse; and F, axodendritic synapse. Note the decrease in extracellular space in the 2- and 4-week-old animals compared to the 2- and 5-day-old animals.

441 cell level. Our observations of large variability of synaptic latency, duration, amplitude, and efficacy in spike initiation in intracellular recordings from young animals seem to indicate simply a slow maturation in the circuitry necessary for generating population EPSPs and population spikes. Interesting new observations made in this in vitro study of kitten include the complex synaptic pattern elicited by stimulation in stratum radiatum. Although the synaptic morphology underlying the dual EPSPs and IPSPs is still a matter for conjecture, one may speculate that the two excitatory potentials and two inhibitory potentials were generated by different synaptic populations on the cell soma and dendrites. Our electron microscopic data indicated that, although synapses were few in number in the young animal, both excitatory and inhibitory synapses occurred on both somata and dendrites. Thus, the early EPSP might be attributed to excitatory input on the soma, and the late EPSP to input on the dendrites. The hypothesis that the two EPSPs were generated by geopgraphically distinct synaptic populations was supported by our observation that the early EPSP was more sensitive to somatic current injection than the late EPSP. These excitatory inputs were only weakly effective in triggering spike discharge, presumably because of their low density, and perhaps also because they were shunted by inhibitory synapses at respective somatic and dendritic sites. The slow rise and long duration of the late EPSP could be explained by asynchronous fiber volleys onto the dendrites, and by the dispersion of synaptic sites on the dendrites; as the animal matured, input synchrony increased, and the integrative capabilities of the dendrites improved as cell size and the dendritic length constant increased. A similar explanation may be proposed to describe the generation of the two IPSPs: the initial IPSP was produced by somatic synapses close to the site of electrode penetration, so that the electrode recorded a large hyperpolarization, especially when the soma was depolarized by electrode damage. The late IPSP was of longer duration and associated with little hyperpolarization since it was generated diffusely in the dendrites and was electrotonically conducted to the cell soma; inhibition, however, was still produced by the IPSP conductance shunting effect on excitatory dendritic inputs. The physiological profile of multiple synaptic inputs changed as the cat matured. Intracellular recordings from the adult hippocampus showed a single early excitatory potential which effectively triggered action potentials, and a single inhibitory potential which was associated with a large membrane hyperpolarization. In the adult, anatomical evidence indicates that the excitatory input is found largely on the dendrites and inhibitory input on the soma34,4z. The fine structure of the synapses, too, changes markedly over the first few postnatal weeks, with gradual thickening of postsynaptic densities and increase in number of vesicles in presynaptic terminals15. The in vitro recordings in kitten hippocampus also resulted in identification, based on physiological characteristics, of two distinct cell populations in stratum pyramidale of the CA1 region. The predominant cell type, presumably the pyramidal cell, was seen in all the animals studied; its firing properties, and membrane and response characteristics were similar to those described in intact adult animals26. At approximately two weeks of age, a different type of cell appeared in the kitten hippocampus. These cells were found less frequently than the presumed pyramidal cells,

442 and appeared to be small since they were easily damaged during electrode penetration. High spontaneous firing frequency, low spike height, and large hyperpolarizing afterpotentials seen in these cells could be due, at least in part, to cell injury. These characteristics were not substantially altered, however, when hyperpolarizing current was injected to stabilize and repair the neurons. With sufficient hyperpolarization, spontaneous spike discharge was blocked to reveal continuous membrane 'noise', suggesting a constant excitatory input onto these cells. The clarity of such 'noise' in recordings with a presumed somatically located electrode indicated that these cells were small (high input resistance resulting in large potentials), had large length constants, and/or had numerous effective excitatory synapses on the soma. Such a description o f these neurons, together with their very brief spike duration, makes it tempting to label these cells as interneurons. They do not, however, c o n f o r m completely to earlier descriptions o f hypothesized hippocampal interneurons3, 4 since they were often located in stratum pyramidale (not in stratum oriens) and could discharge at short latency with one or two spikes (not a burst) to orthodromic stimulation. Our anatomical data showed that there were a n u m b e r of non-pyramidal cell types in the stratum pyramidale of kitten hippocampus, but these cells were present much before the time at which they began to appear in physiological recordings. Such cells are now seen regularly in adult animals (unpublished observations) but their anatomical identification remains unclear. ACKNOWLEDGEMENTS This research was supported by Grants N u m b e r NS 12151 and NS 06477 from N I N C D S , N I H , a grant from the California C o m m u n i t y Foundation, and a gift from the Lucie Stern Trust. We are grateful to Dr. David A. Prince for helpful suggestions and to Ms. G. Chase for secretarial assistance.

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