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Maturation of electrical activity of cerebral neocortex in tissue culture
EXPERIMENTAL
NEUROLOGY
Maturation
ARNOLD
48, 275-291
(1975)
of Electrical Activity of Cerebral in Tissue Culture L.
LEIMAN,
Dcpartmmt of Psychology, afld the Department and Statlford
FREDRICK
J.
SEIL
AND
M.
JAMES
University of California, Berkeley, Neurology, Veteraru Administration University School of Medicine, Palo Califorriia 94304
of
Rcccivcd
March
Neocortex
KELLY,
III 1
California 94720; Hospital Alto,
3, 1975
The neurophysiological development of cerebral neocortex cultures derived from 2 or 3 day old mice was studied using extracellular microelectrode recording techniques. The explants were examined during an ipa vitro growth period extending from 1 to 33 days. Spontaneous activity was rarely noted, although drugs such as penicillin and strychnine could provoke prolonged discharges. Electrical stimuli delivered to the dorsal cortical surfaces of the cultures provoked short-duration spike potentials during the initial 1 to 5 days of in vitro growth. These responses were attributed to axonal activity. After this early period, dorsal surface stimulation additionally elicited labile large-amplitude slow-wave activity which was of maximal amplitude in the middle third of the explants, a region often characterized by aggregates of pyramidal cell bodies and basal dendrites. Within the age range of 5 to 33 days of in vitro growth, the properties of the elicited slow waves failed to show any regular, progressive modifications such as increments in complexity of waveforms or prominence of oscillatory activity. These data were interpreted as reflecting the constraints on maximal functional development imposed by the isolation of a portion of an emerging nervous system. Although characteristic intrinsic circuitry may show extensive anatomical development in this state, more elaborate sequential functional development may depend upon the capacity of customary input pathways to provide triggers for additional structural and functional modifications.
INTRODUCTION Explants derived from prenatal or neonatal central nervous systems show many characteristic developmental features when grown in vitro. These include cell migration and differentiation (27, 29, 34)) synapse formation (5, 19, 21, 28), and myelinization (3, 4). Gross architectural 1 Veterans Administration Palo Alto, California.
Neurobiology
Fellow,
275 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
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features such as laminar forms of organization also become evident during in z&o growth and development (16, 17, 27-30, 34). The presence of such developmental phenomena in a preparation isolated from systemic and brain connection influences has led to suggestions that tissue cultures may provide a useful model for the study of neural ontogeny (9). These systems may be especially useful in the analysis of intrinsically guided processes related to the emergence of local circuit organization. However, there is evidence that some features of cerebral neocortical structural or functional developments (or both) are dependent on inputs derived from other brain regions, including corpus callosum and specific or nonspecific thalamic input pathways (2, 8, 13, 14). In the absence of these extrinsic influences it is possible that the extent and complexity of in vitro cerebral neocortical functional maturation may be appreciably constrained. The purpose of the present study was to systematically examine the extracellular microelectrode indicants of the potential functional changes related to the growth of cerebral neocortical tissue cultures. These cultures have been recorded from over an age range extending from 1 to 33 days in vitro. Across this age range in the in viva state, major changes occur in the rodent brain (1). Our perspective has been to determine the range of functional states that can be depicted during this period of isolated growth on functional development in cerebral and development. Observations neocortical tissue cultures have been reported by Crain and Bornstein (9-11). They have emphasized a continuous progression of increasing complexity in evoked activity which may be similar to in tivo functional developments. In the present study we have sought to determine the neurophysiological characteristics of cerebral neocortex in vitro, with particular emphasis on functional limitations which might reflect the simplification of circuitry in a developing system totally deprived of extrinsic afferent inputs. MATERIALS
AND
METHODS
Cultures derived from frontal and parietal cortex of Swiss-Webster mice 2 or 3 days old were prepared as described in a previous paper (29). Parasagittally oriented explants, approximately Z-Z.5 mm long from the rostra1 to the caudal end and l-l.5 mm deep from the cortical surface to the subcortical margin, were placed on collagen coated coverslips with a drop of nutrient medium and sealed in Maximow double coverslip assemblies. The cultures were incubated in the lying-drop position at 35.536 C, and were fed at twice weekly intervals. The nutrient medium consisted of two parts of 3 units/ml low-zinc insulin (supplied by the Squibb Institute for Medical Research), one part of 20% dextrose, four parts of bovine serum ultrafiltrate, four parts of Eagle’s minimum essential medium
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with Hanks’ base and added L-glutamine, seven parts of Simms’ X-7 balanced salt solution with sufficient added HEPES buffer to make its concentration 0.01 M in the fully constituted medium, and 12 parts of human placental serum. Recording techniques were similar to those described in our study of cerebellar explants (18). Cultures selected for recording varied in age from l-33 days in Z&V. Collagen coated coverslips containing the cultures formed the floor of a round 42 mm diameter Teflon coated steel chamber with 12 mm high sidewalls mounted on the mechanical stage of an inverted microscope. Electrode placements were made through an opening at the top of the chamber under direct visual control. Temperature was maintained by a d-c powered nichrome coil which surrounded the coverslip. A thermistor probe inserted into the medium covering the cultures enabled a continuous monitoring of temperature. Recordings were obtained at tem-
FIG.
The ment The plane cannot
1. Parasagittally oriented cerebral neocortex explant after 19 days in vitro. original cortical surface (CS) is evident near the top of the figure, and a segof subcortical tissue (SC) is present below the cortical portion of the explant. location of major horizontal bundles of neurites coursing in the rostra]-caudal of the culture (Ref. 29) is indicated by arrows. The rostra1 end of the explant be distinguished from the caudal end. Holmes stain. X60.
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peratures ranging from 2535 C. Within this range, temperature variations contributed only minor changes in amplitudes of evoked responses. No major effects attributable to temperature variations were noted. The medium during recording consisted of 5-7 drops of Simms’ X-7 balanced salt solution additionally buffered with 0.015 M HEPES. Extracellular potentials were recorded using either saline filled capillaries (0.9-2.0 M) or etched tungsten or platinum electrodes. The tip diameter of all electrodes was less than 1 pm. A chlorided silver reference electrode was placed in the medium bathing the explant. Electrical stimuli were delivered by a Grass S 88 stimulator through etched tungsten electrodes with the insulation partially removed from around the tip surface. Both constant current and constant voltage conditions were employed. Current levels ranged from 250 ernp to 1 mamp and pulse duration ranged from 50-80 psec. Electrical stimuli were delivered through closely spaced bipolar electrodes placed at various positions in the explant. Evoked activity was successively recorded at an array of positions whose location could be identified with the aid of a graticule placed in the microscope eyepiece. This enabled the precise mapping of the propagated effects of stimulation delivered to any position. Responses were recorded using a Grass P 15 preamplifier and stored on an Ampex SP 300 tape recorder or photographed from the oscilloscopic display with a Polaroid camera. Following recording, the explants were fixed as whole-mount preparations and stained either with thionine (28) or, in most cases, with the silver impregnation method of Holmes as modified for tissue culture by Wolf (33). Data described in this paper were obtained from 62 explants. RESUNLTS Light ikficroscopic Observations of Cerebral Neocortex Tissue Cultures. A low power view of a silver stained cerebral neocortex culture is shown in Fig. 1. This illustration shows a laminar organization of neurons and their processes in a culture after 19 days in vitro. Detailed microscopic observations of such preparations (29) have demonstrated that the dominant elements of this laminar organization are four horizontally coursing bundles of neurites, the positions of which are indicated by arrows in Fig. 1. In the present study, stimuli were usually delivered at the site of the dorsal bands of fibers. Pyramidal cell neurons are abundant in the lower two-thirds of the cortical portions of the explants, and are generally present in greatest concentration in the middle third of the explants. In almost half of the older cultures, the pyramidal cell basal dendrites in the middle third of the cortex appear interwoven, reminiscent of the basal dendritic bundles described in rodent and cat motor and visual
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cortex (24). Myelin is observed in these cultures after approximately 2 weeks in vitro. It is initially apparent in the deeper portions of the explants. Spontaneous Extracellular Electrical Activity. The display of spontaneous neural activity either in the form of spike discharges or slow potentials was uncommon in these studies. This observation was markedly dissimilar to the frequent occurrence of endogenous activity in cerebellar explants examined in our earlier study (18). On occasion, older explants eshibited slow-wave and unit discharge bursts which occurred once every 1-2 min. Observations of endogenous activity in cerebral neocortical explants were partially complicated by the unusual sensitivity of these explants to mechanical deformation imposed by the microelectrodes. In some instances, merely pressing a microelectrode against the top surface of a culture could provoke prolonged oscillatory activity which resembled electrically elicited activity. This observation might be related to speculations concerning the role of mechanical distortion of apical dendrites in the genesisof focal seizure activity (32). Maintained activity in these cultures could be produced readily under various pharmacological conditions. The application of penicillin (5000 units/ml, potassium penicillin G) to 12 to 19 day cultures provoked paroxysmal activity in which bursts were separated by long intervals of silence. These silent intervals lasted as long as 2-4 min. This activity persisted in one sessionfor a period of over 2 hr without any change in the distribution of burst intervals. Attempts to modify the temporal features of this penicillin-induced discharge pattern with l/set cortical surface stimulation were unsuccessful, i.e., a 2 min period of such stimulation did not modify the periodicity of discharge in a poststimulation observation period. Spontaneous discharges could be elicited in 19 day cultures by the application of strychnine (100 pg/ml) Rapidly reversible inhibitory or depressant effects have been produced by addition of glycine and human serums (unpublished observations) as well as by rodent serums (31). Extracellular Responses Elicited by Electrical Stinmlation. The range of types of extracellular responses elicited by stimulation of the dorsal cortical surface is illustrated in Fig. 2. Factors determining the form of evoked activity included developmental stage, recording position and stimulus intensity. Figure 2a shows a relatively rare instance of cell body discharge, presumably from a larger pyramidal cell. In these cultures, such large evoked extracellular spikes were quite rare and generally presented as single spike discharges. Figures 2b and c show two forms of smaller spike activity with 2b showing successive decrements in spike amplitude, and 2c showing a more tonic response pattern. These events could be recorded from many different positions in the culture and may have originated in fine neurite processes.The typical response of young cultures is shown in
h i FIG. 2. Varieties of evoked extracellular responses observed in cerebral neocortex cultures. In a, recorded from an 18 day culture at 26 C, a large amplitude cell body discharge was observed in a region of the explant in which pyramidal neurons were predominant. The time base marker at the bottom of the column equals 2 msec with reference to the response in a. Small amplitude, brief duration spike responses are illustrated in b, c, and d. The explants in b and c were both 19 days in vitro, and both responses were recorded at 26 C. The explant in d was 1 day, and the response was recorded at 32 C. The time base markers represent 5 msec with reference to b and d, and 100 msec with reference to c. The responses in e-i were recorded from a 23 day culture at 34.5 C at successively faster sweep speeds. The time base marker represents, in succession, 100, 50, 20 10, and 5 msec. Demonstrated are the characteristic slow wave responses which appear after the initial spike in older cultures.
Fig. 2d (described in more detail below) and Fig. Ze-i shows the slow potential at various time basesas seen in older cultures. On the day following explantation (day 1 in vibro), the only type of response that could be elicited by dorsal surface stimulation was a short duration event lasting 2-5 msec (Fig. Zd). These responses have been described as “spikes” (10, 11)) although they do not have the all-or-none property implied by the term. Indeed, this brief duration response appeared more analogous to a compound action potential. This was evident in observations of the effects of stimulus intensity changes. Figure 3 shows that this response was incremental with intensity changes over a wide range. In comparing cultures of three different ages the slope of this func-
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FIG. 3. Graph demonstrating the relationship between current of early, brief duration evoked responses in cerebral neocortical ranges. The stimulating electrodes in all cases were positioned surface, with similar interelectrode spacing.
FII elect1 with from vertic
:.
4 Evoke iode !S positi the -ecordi sue :essive :a1 1 laceme
zd activity recorded from an 8 day ioned at the dorsal cortical surface. ng electrode near the dorsal cortical vertically downward placements at nt patterns shown in Fig. 5.
level and amplitude cultures of three age at the dorsal cortical
culture at 34 C Pwith stin nulating The initial respons ;e x3ias (obtained surface, and subse quel nt l-f zsponses 120 pm intervals, sir nilar . to the
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tion varied markedly with age, being steepest in the oldest culture. Maximal amplitudes of this event were also more prominent in older cultures. These changes may reflect growth of neurites, increases of ason diameters and myelinization. The topography of this brief duration response in an 8 day explant is illustrated in Fig. 4, and Fig. 5 shows the distribution in an older culture
FIG. 5. Spatial distribution of activity evoked by placement of electrodes (,S) near the dorsal cortical surface (CS), and recording shown. The responses were obtained from a 27 day culture at 26 C.
the stimulating from points as
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(27 days in z&-o). This response could be elicited with stimulus and recording electrodes as widely separated as the entire length of the culture. This is in contrast to results with cerebellar explants, in which evoked activity could be obtained only when stimulus and recording electrodes were within 500 pm of each other (IS). Propagation in neocortical cultures was evident in both a horizontal and a vertical direction. A preferred direction of propagation of this response became apparent with aging. Comparisons amongst various ages suggest that horizontal propagation became more efficient with aging as opposed to the development of vertical transmission. This is in contrast to the situation in Z&JO, where vertical development of the neocortex predominates over horizontal development, both structurally and functionally, with progression towards maturity (25). During the first week in Z&V, the principal change in the brief duration response was an alteration in excitability cycle. This was evident in paired pulse stimulation experiments, as displayed in Fig. 6a and b which compares day 1 and day 8. At day 8, recovery of maximal amplitude was more rapid. Figure 6c and d shows that at day 23 there was a potentiation of this response, which was not seen in day 1 cultures. After 5-8 days the major functional change of in vitro development occurred. At this stage, dorsal surface stimulation elicited the spike observed at earlier stages followed by an extremely variable, longer duration slow wave. Unit discharge could be seen as part of the slow wave complex. Figure 2e-i shaws this response on various time bases. The response was characterized as a large positive-negative shift following an initial spike. On some occasions, oscillatory activity occurred with diminishing amplitude (Fig. Ze) . The characteristics of this slow potential included considerable variability which was not related to in z&o age. In some cultures, the ability to elicit the slow potential with surface cortical stimulation rapidly declined following an initial stimulus. Figure 7 shows some data of the slow recovery of such a response in a comparatively excitable culture. This illustration shows a minimal recovery period of at least 1 min, although recovery of rhythmic oscillations could take as long as lo-15 min. We have also noted that, unlike the early spike component, the amplitude of these responses was not simply related to stimulus intensity, although such observations were obviously complicated by the prolonged inexcitability of this response following a single stimulus. Topographic analyses which emphasized the slow wave component showed that the largest amplitude was invariably observed in the middle third of the explant, where pyramidal cell bodies and basal dendrite fields are present in greatest concentration. Successive recordings in a vertical direction from the dorsal cortical surface rarely demonstrated a phase reversal, Recordings in successive horizontal positions showed some in-
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-5msec
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SEIL
AND
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FIG. 6. Excitability cycles in cerebral neocortical explants at different ages. A and R are from 1 and 8 day cultures respectively, both recorded at 32 C. The comparatively less excitable property of the 1 day culture is evident. C and D were recorded at different time bases from a 23 day culture at 32.5 C. Note the hyperexcitability of the early component in C, and the prolonged inexcitability of the slower components.
creased complexity in waveform in addition to a decrease in amplitude. The paucity of phase reversals in the vertical plane is in contrast to observations of slow wave activity in cerebral neocortex explants described by Crain and Bornstein (10). Their finding of phase reversals in the vertical plane as a common event is consistent with an assumption that the basis of the slow-wave activity is synaptic inputs to vertically oriented apical dendrites. In the present study, however, maximal amplitudes of
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slow-wave responses were evident in the region of the basal dendrite synapses, not at the level of apical dendrites. A developmental question we have examined is whether the slow-wave response shows any regular, progressive changes with growth beyond the period when it is initially noted. At the level of extracellular analysis, we have been unable to detect any consistent functional differentiations with aging. Thus knowledge of stimulus parameters, recording position and form of slow wave response did not enable distinction of a 5-8 day old culture from a 33 day culture. This ambiguity partially derived from the fact that the variance of slow wave properties at any age of in vitro growth approximated the variance of the full time period of growth. Stimulation delivered to the deepest portion of the explant resulted in activity which was dissimilar to that provoked by cortical surface stimulation. Figure 8a shows a fast spike response recorded in the middle third of the explant by stimulation near the ventral margin. The complex large amplitude responses provoked by surface stimulation were never elicited by deep fiber stimulation. By contrast, when a rarely included group of subcortical nuclear neurons was stimulated, a long latency burst response
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7. Excitability characteristics of evoked activity elicited by cortical surface stimulation in an 8 day explant at 34 C. In A, the early fast component is emphasized; in B, the longer time base allows examination of the slower components. In both A and B, a-e represent successively recorded responses, with a being a “baseline” response, followed by b, recorded after a 1 min interval, and subsequently by c, d, and e, recorded after 30, 15 and 5 set intervals respectively. FIG.
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was recorded from the middle third of the explant (Fig. Sb) . This response could follow faster rates of stimulation and probably arose in an ascending fiber group. Oscillatory burst responses which could be elicited by stimulation of the superficial fiber bands were never elicited by deep filter stimulation. DISCUSSION The present experiments have shown that the functional development of neocortical explants is characterized by an early period where only short duration spike activity can be observed, followed by the appearance of more long duration slow wave evoked responses. The spike response is the only response observed during a period of l-5 days in vitro, and most likely indicates activity of axons. Changes in this response probably reflect developments in neurite growth, axon diameter and myelination. There is no evidence for a synaptic origin of this response, an assessment similar to that of Crain (9). The long-latency slow-wave response described in the present study differs in several features from a similar response descri.bed by Crain and Bornstein (10). For example, they noted phase reversals in a plane perpendicular to the cortical surface ; this phenomenon was rarely noted in our study. There are many intricacies of dipole analysis with laminar recording, including circumstances in which phase reversals may not be evident, although the implied organization of extracellular fields might lead one to expect phase reversals (26). In some cultures the absence of phase reversals may be related to the dispersion of pyramidal cells (29). Additionally, since many forms of characteristic input to cortical cells are eliminated in isolated explants, one might expect a rather sparse arrangement of synapses on apical dendrites, On the other hand, anatomical studies (29) show a rather dense meshwork of pyramidal cell basal dendrites in the middle third of some explants. If the primary source of synaptic electrogenesis is at the level of the basal dendrites, the absence of a vertically oriented dipole is not surprising. * The minimal examples of spontaneous activity noted in this study are in contrast to a recently reported study of endogenous activity in rat and cat cerebral neocortical explants. Calvet (7) noted spontaneous discharges occurring at intervals of 3 set to 1 min and suggested that they were the result of synaptic activity. From the description of the morphological organization of her cerebral neocortical cultures, it would seem that the synaptic density might be less than in thicker explants employed in the present study, in which there was a paucity of spontaneous activity. The reason for believing Calvet’s cultures to be more thinly dispersed, and therefore having fewer synapses, is based on her ability to resolve neurons
FIG. 8. A response recorded from tion delivered to the ventral margin deep band of fibers, as illustrated by With recording electrodes similarly group of subcortical nuclear neurons in b. Both responses were recorded at
the middle third of a 19 day explant to stimulaof the cortical portion of the explant (i.e., to the the lowermost arrow in Fig. 1) is presented in a. placed in a 12 day explant, stimulation of a burst response shown produced the lon g latency 26 C.
by phase contrast optics. Such resolution is not possible in thick explants. It would appear doubtful, therefore, that the spontaneous discharges described by Calvet are due to synaptic activation, unless there is a ‘selective depletion of inhibitory synapsesin her system. The absence of spontaneouscell discharge in explants described in this paper has also been described in immature rat (12, 20 )and neonatal cat cortex (15, 22) and in some reports of undercut adult cat cortex (6). The infrequent occurrence of pyramidal cell spike discharges in culture
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may be related to the late development of pyramidal cell spike activity, as described in various immature animals (12, 15, 22). On the other hand, the infrequent occurrence of pyramidal cell spikes in vitro may be a function of afferent deprivation rather than immaturity, for our earlier anatomical study ,(29) has indicated a considerable degree of morphological development after explantation. Crain in his review of functional development of explants of diverse origin (9) suggests that there is a general rule of development in cultures derived from fetal or neonatal nervous systems. He describes a sequence or progression proceeding from a simple spike response, which is assumed to be axonal, to complex slow wave electrogenesis based on synaptic activity. He further suggests that this slow-wave response shows an increasing complexity with development, including the emergence of oscillatory afterdischarge activity. Most of this progression was defined for spinal ganglion-spinal cord explants, although he briefly noted that a similar sequence was observed in extracellular studies of cerebral neocortical explants. The generality of this rule seems unusual across such disparately organized systems ; indeed, its existence might even be construed as evidence of the insensitivity of recording methods to structural differences. In our studies we consistently noted the priority of simple spike activity and the emergence of slow wave responses and synaptically driven activity in fine neurite processes. However, the most emphatic demonstration of a developmental progression would be the ability to depict approximate culture age, given knowledge of stimulus position and intensity, and recording position and characteristics of evoked activity. In our observations, elicited slow-wave activity does not provide the basis for such discriminations, although some features of the spike response provide a crude differentiation. Neuronal tissue cultures have been described as a model for the study of developmental processes. There is abundant evidence that characteristic architectural arrangements develop in vitro and many cellular developmental processes emerge within these systems (16, 17, 19, 21, 27-30, 34). Indeed, neural tissue cultures may show the maximum development of a brain subunit freed of extensive customary input pathways and target projection fields. What remains is the characteristic organization of comparatively small units of intrinsic circuitry. Such isolation in an emerging nervous system may strongly constrain the range of functional developments. In this paper we have noted a rather limited sequence of functional changes evident with cerebral neocortical growth in vitro. More elaborate sequential and progressive modifications with aging may depend on the capacity of customary input pathways to provide a set of triggers for both structural and functional developments. From this perspective, one might
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consider that each customary cortical input pathway provides both a capacity for transient signal communication and a trophic influence which are critical determinants of cortical ontogeny. A link between these factors is suggested by the work of Rutledge (23), who has shown that stimulation of isolated cortical slabs influences the maintenance of dendritic spines. One of the advantages of the tissue culture experiment is the ability to assess this conjecture under exacting conditions. For example, cerebrum explants which include thalamic tissue can be compared with those without such connections, both in terms of structural and functional developments. Contrasts between in vitro and in viva organizational developments are suggested from data discussed by the Scheibels (25). They have emphasized that structural studies and various neurophysiological investigations show the emerging dominance of vertical organization within tortes during the course of early development. Indeed, they note the disappearance of dorsal horizontal axonal fibers with aging and their replacement by apical dendrite branching. The dominance of horizontal activities in explants may reflect a persistence of early pathways in the absence of customary extrinsic inputs.
REFERENCES M. 1974. Development of the cerebral neocortex in the rat, pp. 7-67. In “Studies on the Development of Behavior and the Nervous System.” Vol. II. G. Gottlieb [Ed.]. Academic Press, New York. BLAKEMORE, C. 1974. Development of functional connexions in the mammalian visual system. Brit. Med. Bull. 30 : 152-157. BORNSTEIN, M. B. 1964. Morphological development of neonatal mouse cerebral cortex in tissue culture, pp. l-11. In “Neurological and Electrographic Correlative Studies in Infancy.” P. Kellaway and I. Peterson [Eds.]. Grune and ‘Stratton, New York. BORNSTEIN, M. B., and M. R. MURRAY. 1958. Serial observations on patterns of growth, myelin formation, maintenance and degeneration in cultures of newborn rat and kitten cerebellum. .I. Biophys. Biochwt. Cytol. 4: 499-504. BUNGE, R. P., M. B. BUNGE, and E. R. PETERSON. 1967. The onset of synapse formation in spinal cord cultures as studied by electron microscopy. Brain Rcs. 6 : 728-749. BURNS, B. D. 1958. “The Mammalian Cerebral Cortex,” pp. 52-60. Edward Arnold, London. CALVET, M.-C. 1974. Patterns of spontaneous electrical activity in tissues of mammalian cerebral cortex vs. cerebellum. Brain Res. 69: 281-295. CHOW, K. C., and A. L. LEIMAN. 1970. The structural and functional organization of the neocortex. Nrurosci. Res. Prog. Bull. 8: 153-220. CRAIN, S. M. 1974. Tissue culture models of developing brain functions, pp. 69-114. Ilz “Studies on the Development of Behavior and the Nervous System.” Vol. II. G. Gottlieb [Ed.]. Academic Press, New York.
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10. GRAIN, S. M., and M. B. BORNSTEIN. 1964. Bioelectric activity of mouse cerebral cortex during growth and differentiation in tissue culture. Exp. Ncurol. 10: 425-450. 11. CRAIN, S. M., and M. B. BORNSTEIN. 1974. Early onset in inhibitory functions during synaptogenesis in fetal mouse brain cultures. Brain Res. 68: 351-357. 12. DEZA, L., and E. EIDELBERG. 1967. Development of cortical electrical activity in the rat. Exp. Neurol. 17 : 425-438. 13. GLOBUS, A. 1971. Neural ontogeny: Its use in tracing connectivity, pp. 253-263. In “Brain Development and Behavior.” M. B. Sterman, D. J. McGinty and A. M. Adinolfi [Eds.]. Academic Press, New York. 14. GRUNER, J. E., J. C. HIRSCH, and C. SOTELO. 1974. Ultrastructural features of the isolated suprasylvian gyrus in the cat. J. Cornp. Neural. 154: l-28. 15. HUTTENLOCHER, P. R. 1967. Development of cortical neuronal activity in the neonatal cat. Exp. Neural. 17: 247-262. 16. KIM, S. U. 1972. Light and electron microscopic study of mouse cerebral neocortex in tissue culture. Exp. Newel. 35 : 305-321. 17. LAVAIL, J. H., and M. K. WOLF. 1973. Postnatal development of the mouse dentate gyrus in organotypic cultures of the hippocampal formation. Amer. J. Axat. 137 : 47-66. 18. LEIMAN, A. L., and F. J. SEIL. 1973. Spontaneous and evoked bioelectric activity in organized cerebellar tissue cultures. Exp. Nenrol. 40: 748-759. 19. MODEL, P. G., M. B. BORNSTEIN, S. M. CRAIN, and G. D. PAPPAS. 1971. An electron microscopic study of the development of synapses in cultured fetal mouse cerebrum continuously exposed to xylocaine. J. Cell Biol. 49: 362-371. 20. MYSLIVICEK, J. 1970. Electrophysiology of the developing brain-Central and Eastern European contributions, pp. 475-527. In “Developmental Neurobiology.” W. A. Himwich [Ed.]. Charles C Thomas, Springfield. 21. PAPPAS, G. D. 1966. Electron microscopy of neuronal junctions involved in transmission in the central nervous system, pp. 49-87. In “Nerve as a Tissue.” K. Rodahl and B. Issekutz [Eds.]. Harper and Row, New York. 22. PURPURA, D. P. 1972. Intracellular studies of synaptic organization in the mammalian brain, pp. 257-302. In “Structure and Function of Synapses.” G. D. Pappas and D. P. Purpura [Eds.]. Raven Press, New York. 23. RUTLEDGE, L. T. 1969. Effect of stimulation on isolated cortex, pp. 349-355. In “Basic Mechanisms of the Epilepsies.” H. H. Jasper, A. A. Ward and A. Pope [Eds.]. Little, Brown and Company, Boston. 24. SCHEIBEL, M. E., T. L. DAVIES, R. D. LINDSAY, and A. B. SCHEIBEL. 1974. Basilar dendrite bundles of giant pyramidal cells. Exp. Neural. 42: 307-319. 25. SCHEIBEL, M. E., and A. B. SCHEIBEL. 1975. Some thoughts on the ontogeny of memory and learning. In “Neural Mechanisms of Learning and Memory.” M. R. Rosenzweig and E. L. Bennett [Eds.]. MIT Press. (In press.) 26. SCHLAG, J. 1973. Generation of evoked potentials, pp. 273-317. In “Bioelectric ,Recording Techniques.” Part A. R. F. Thompson and M. M. Patterson [Eds.]. Academic Press, New York. 27. SEIL, F. J. 1972. Neuronal groups and fiber patterns in cerebellar tissue cultures. Brain Res. 42 : 33-51. 28. ,SEIL, F. J., and R. M. HERNDON. 1970. Cerebellar granule cells in vitro. A light and electron microscopic study. J. Cell Biol. 45 : 212-220. 29. SEIL, F. J., J. M. KELLY, III, and A. L. LEIMAN. 1974. Anatomical organization of cerebral neocortex in tissue culture. Exp. Nezcrol. 45: 435-450.
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30. SEIL, F. J., and A. L. LEIMAN. 1975. Neural subsystems and learning: Tissue culture approaches. 11%“Neural Mechanisms of Learning and Memory.” M. R. Rosenzweig and E. L. Bennett [Eds.]. MIT Press. (in press.) 31. SEIL, F. J., M. E. SMITH, A. I,. LEIMAN, and J. M. KELLY, III. 1975. Myelination inhibiting and neuroelectric blocking factors in experimental allergic encephalomyelitis. Scie?zce 187 : 951-953. 32. WARD, A. A., JR. 1961. Epilepsy. Int. XCV. Nc~~rol)iol. 3 : 137-186. 33. WOLF, M. K. 1964. Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum J. Cell. Biol. 22: 259-279. 34. WOLF, M. K. 1970. Anatomy of cultured mouse cerebellum. II. Organotypic migration of granule cells demonstrated by silver impregnation of normal and mutant cultures. J. Cowi). Ncwol. 140 : 281-297.
NEUROLOGY
Maturation
ARNOLD
48, 275-291
(1975)
of Electrical Activity of Cerebral in Tissue Culture L.
LEIMAN,
Dcpartmmt of Psychology, afld the Department and Statlford
FREDRICK
J.
SEIL
AND
M.
JAMES
University of California, Berkeley, Neurology, Veteraru Administration University School of Medicine, Palo Califorriia 94304
of
Rcccivcd
March
Neocortex
KELLY,
III 1
California 94720; Hospital Alto,
3, 1975
The neurophysiological development of cerebral neocortex cultures derived from 2 or 3 day old mice was studied using extracellular microelectrode recording techniques. The explants were examined during an ipa vitro growth period extending from 1 to 33 days. Spontaneous activity was rarely noted, although drugs such as penicillin and strychnine could provoke prolonged discharges. Electrical stimuli delivered to the dorsal cortical surfaces of the cultures provoked short-duration spike potentials during the initial 1 to 5 days of in vitro growth. These responses were attributed to axonal activity. After this early period, dorsal surface stimulation additionally elicited labile large-amplitude slow-wave activity which was of maximal amplitude in the middle third of the explants, a region often characterized by aggregates of pyramidal cell bodies and basal dendrites. Within the age range of 5 to 33 days of in vitro growth, the properties of the elicited slow waves failed to show any regular, progressive modifications such as increments in complexity of waveforms or prominence of oscillatory activity. These data were interpreted as reflecting the constraints on maximal functional development imposed by the isolation of a portion of an emerging nervous system. Although characteristic intrinsic circuitry may show extensive anatomical development in this state, more elaborate sequential functional development may depend upon the capacity of customary input pathways to provide triggers for additional structural and functional modifications.
INTRODUCTION Explants derived from prenatal or neonatal central nervous systems show many characteristic developmental features when grown in vitro. These include cell migration and differentiation (27, 29, 34)) synapse formation (5, 19, 21, 28), and myelinization (3, 4). Gross architectural 1 Veterans Administration Palo Alto, California.
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features such as laminar forms of organization also become evident during in z&o growth and development (16, 17, 27-30, 34). The presence of such developmental phenomena in a preparation isolated from systemic and brain connection influences has led to suggestions that tissue cultures may provide a useful model for the study of neural ontogeny (9). These systems may be especially useful in the analysis of intrinsically guided processes related to the emergence of local circuit organization. However, there is evidence that some features of cerebral neocortical structural or functional developments (or both) are dependent on inputs derived from other brain regions, including corpus callosum and specific or nonspecific thalamic input pathways (2, 8, 13, 14). In the absence of these extrinsic influences it is possible that the extent and complexity of in vitro cerebral neocortical functional maturation may be appreciably constrained. The purpose of the present study was to systematically examine the extracellular microelectrode indicants of the potential functional changes related to the growth of cerebral neocortical tissue cultures. These cultures have been recorded from over an age range extending from 1 to 33 days in vitro. Across this age range in the in viva state, major changes occur in the rodent brain (1). Our perspective has been to determine the range of functional states that can be depicted during this period of isolated growth on functional development in cerebral and development. Observations neocortical tissue cultures have been reported by Crain and Bornstein (9-11). They have emphasized a continuous progression of increasing complexity in evoked activity which may be similar to in tivo functional developments. In the present study we have sought to determine the neurophysiological characteristics of cerebral neocortex in vitro, with particular emphasis on functional limitations which might reflect the simplification of circuitry in a developing system totally deprived of extrinsic afferent inputs. MATERIALS
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Cultures derived from frontal and parietal cortex of Swiss-Webster mice 2 or 3 days old were prepared as described in a previous paper (29). Parasagittally oriented explants, approximately Z-Z.5 mm long from the rostra1 to the caudal end and l-l.5 mm deep from the cortical surface to the subcortical margin, were placed on collagen coated coverslips with a drop of nutrient medium and sealed in Maximow double coverslip assemblies. The cultures were incubated in the lying-drop position at 35.536 C, and were fed at twice weekly intervals. The nutrient medium consisted of two parts of 3 units/ml low-zinc insulin (supplied by the Squibb Institute for Medical Research), one part of 20% dextrose, four parts of bovine serum ultrafiltrate, four parts of Eagle’s minimum essential medium
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with Hanks’ base and added L-glutamine, seven parts of Simms’ X-7 balanced salt solution with sufficient added HEPES buffer to make its concentration 0.01 M in the fully constituted medium, and 12 parts of human placental serum. Recording techniques were similar to those described in our study of cerebellar explants (18). Cultures selected for recording varied in age from l-33 days in Z&V. Collagen coated coverslips containing the cultures formed the floor of a round 42 mm diameter Teflon coated steel chamber with 12 mm high sidewalls mounted on the mechanical stage of an inverted microscope. Electrode placements were made through an opening at the top of the chamber under direct visual control. Temperature was maintained by a d-c powered nichrome coil which surrounded the coverslip. A thermistor probe inserted into the medium covering the cultures enabled a continuous monitoring of temperature. Recordings were obtained at tem-
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1. Parasagittally oriented cerebral neocortex explant after 19 days in vitro. original cortical surface (CS) is evident near the top of the figure, and a segof subcortical tissue (SC) is present below the cortical portion of the explant. location of major horizontal bundles of neurites coursing in the rostra]-caudal of the culture (Ref. 29) is indicated by arrows. The rostra1 end of the explant be distinguished from the caudal end. Holmes stain. X60.
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peratures ranging from 2535 C. Within this range, temperature variations contributed only minor changes in amplitudes of evoked responses. No major effects attributable to temperature variations were noted. The medium during recording consisted of 5-7 drops of Simms’ X-7 balanced salt solution additionally buffered with 0.015 M HEPES. Extracellular potentials were recorded using either saline filled capillaries (0.9-2.0 M) or etched tungsten or platinum electrodes. The tip diameter of all electrodes was less than 1 pm. A chlorided silver reference electrode was placed in the medium bathing the explant. Electrical stimuli were delivered by a Grass S 88 stimulator through etched tungsten electrodes with the insulation partially removed from around the tip surface. Both constant current and constant voltage conditions were employed. Current levels ranged from 250 ernp to 1 mamp and pulse duration ranged from 50-80 psec. Electrical stimuli were delivered through closely spaced bipolar electrodes placed at various positions in the explant. Evoked activity was successively recorded at an array of positions whose location could be identified with the aid of a graticule placed in the microscope eyepiece. This enabled the precise mapping of the propagated effects of stimulation delivered to any position. Responses were recorded using a Grass P 15 preamplifier and stored on an Ampex SP 300 tape recorder or photographed from the oscilloscopic display with a Polaroid camera. Following recording, the explants were fixed as whole-mount preparations and stained either with thionine (28) or, in most cases, with the silver impregnation method of Holmes as modified for tissue culture by Wolf (33). Data described in this paper were obtained from 62 explants. RESUNLTS Light ikficroscopic Observations of Cerebral Neocortex Tissue Cultures. A low power view of a silver stained cerebral neocortex culture is shown in Fig. 1. This illustration shows a laminar organization of neurons and their processes in a culture after 19 days in vitro. Detailed microscopic observations of such preparations (29) have demonstrated that the dominant elements of this laminar organization are four horizontally coursing bundles of neurites, the positions of which are indicated by arrows in Fig. 1. In the present study, stimuli were usually delivered at the site of the dorsal bands of fibers. Pyramidal cell neurons are abundant in the lower two-thirds of the cortical portions of the explants, and are generally present in greatest concentration in the middle third of the explants. In almost half of the older cultures, the pyramidal cell basal dendrites in the middle third of the cortex appear interwoven, reminiscent of the basal dendritic bundles described in rodent and cat motor and visual
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cortex (24). Myelin is observed in these cultures after approximately 2 weeks in vitro. It is initially apparent in the deeper portions of the explants. Spontaneous Extracellular Electrical Activity. The display of spontaneous neural activity either in the form of spike discharges or slow potentials was uncommon in these studies. This observation was markedly dissimilar to the frequent occurrence of endogenous activity in cerebellar explants examined in our earlier study (18). On occasion, older explants eshibited slow-wave and unit discharge bursts which occurred once every 1-2 min. Observations of endogenous activity in cerebral neocortical explants were partially complicated by the unusual sensitivity of these explants to mechanical deformation imposed by the microelectrodes. In some instances, merely pressing a microelectrode against the top surface of a culture could provoke prolonged oscillatory activity which resembled electrically elicited activity. This observation might be related to speculations concerning the role of mechanical distortion of apical dendrites in the genesisof focal seizure activity (32). Maintained activity in these cultures could be produced readily under various pharmacological conditions. The application of penicillin (5000 units/ml, potassium penicillin G) to 12 to 19 day cultures provoked paroxysmal activity in which bursts were separated by long intervals of silence. These silent intervals lasted as long as 2-4 min. This activity persisted in one sessionfor a period of over 2 hr without any change in the distribution of burst intervals. Attempts to modify the temporal features of this penicillin-induced discharge pattern with l/set cortical surface stimulation were unsuccessful, i.e., a 2 min period of such stimulation did not modify the periodicity of discharge in a poststimulation observation period. Spontaneous discharges could be elicited in 19 day cultures by the application of strychnine (100 pg/ml) Rapidly reversible inhibitory or depressant effects have been produced by addition of glycine and human serums (unpublished observations) as well as by rodent serums (31). Extracellular Responses Elicited by Electrical Stinmlation. The range of types of extracellular responses elicited by stimulation of the dorsal cortical surface is illustrated in Fig. 2. Factors determining the form of evoked activity included developmental stage, recording position and stimulus intensity. Figure 2a shows a relatively rare instance of cell body discharge, presumably from a larger pyramidal cell. In these cultures, such large evoked extracellular spikes were quite rare and generally presented as single spike discharges. Figures 2b and c show two forms of smaller spike activity with 2b showing successive decrements in spike amplitude, and 2c showing a more tonic response pattern. These events could be recorded from many different positions in the culture and may have originated in fine neurite processes.The typical response of young cultures is shown in
h i FIG. 2. Varieties of evoked extracellular responses observed in cerebral neocortex cultures. In a, recorded from an 18 day culture at 26 C, a large amplitude cell body discharge was observed in a region of the explant in which pyramidal neurons were predominant. The time base marker at the bottom of the column equals 2 msec with reference to the response in a. Small amplitude, brief duration spike responses are illustrated in b, c, and d. The explants in b and c were both 19 days in vitro, and both responses were recorded at 26 C. The explant in d was 1 day, and the response was recorded at 32 C. The time base markers represent 5 msec with reference to b and d, and 100 msec with reference to c. The responses in e-i were recorded from a 23 day culture at 34.5 C at successively faster sweep speeds. The time base marker represents, in succession, 100, 50, 20 10, and 5 msec. Demonstrated are the characteristic slow wave responses which appear after the initial spike in older cultures.
Fig. 2d (described in more detail below) and Fig. Ze-i shows the slow potential at various time basesas seen in older cultures. On the day following explantation (day 1 in vibro), the only type of response that could be elicited by dorsal surface stimulation was a short duration event lasting 2-5 msec (Fig. Zd). These responses have been described as “spikes” (10, 11)) although they do not have the all-or-none property implied by the term. Indeed, this brief duration response appeared more analogous to a compound action potential. This was evident in observations of the effects of stimulus intensity changes. Figure 3 shows that this response was incremental with intensity changes over a wide range. In comparing cultures of three different ages the slope of this func-
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FIG. 3. Graph demonstrating the relationship between current of early, brief duration evoked responses in cerebral neocortical ranges. The stimulating electrodes in all cases were positioned surface, with similar interelectrode spacing.
FII elect1 with from vertic
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4 Evoke iode !S positi the -ecordi sue :essive :a1 1 laceme
zd activity recorded from an 8 day ioned at the dorsal cortical surface. ng electrode near the dorsal cortical vertically downward placements at nt patterns shown in Fig. 5.
level and amplitude cultures of three age at the dorsal cortical
culture at 34 C Pwith stin nulating The initial respons ;e x3ias (obtained surface, and subse quel nt l-f zsponses 120 pm intervals, sir nilar . to the
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tion varied markedly with age, being steepest in the oldest culture. Maximal amplitudes of this event were also more prominent in older cultures. These changes may reflect growth of neurites, increases of ason diameters and myelinization. The topography of this brief duration response in an 8 day explant is illustrated in Fig. 4, and Fig. 5 shows the distribution in an older culture
FIG. 5. Spatial distribution of activity evoked by placement of electrodes (,S) near the dorsal cortical surface (CS), and recording shown. The responses were obtained from a 27 day culture at 26 C.
the stimulating from points as
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(27 days in z&-o). This response could be elicited with stimulus and recording electrodes as widely separated as the entire length of the culture. This is in contrast to results with cerebellar explants, in which evoked activity could be obtained only when stimulus and recording electrodes were within 500 pm of each other (IS). Propagation in neocortical cultures was evident in both a horizontal and a vertical direction. A preferred direction of propagation of this response became apparent with aging. Comparisons amongst various ages suggest that horizontal propagation became more efficient with aging as opposed to the development of vertical transmission. This is in contrast to the situation in Z&JO, where vertical development of the neocortex predominates over horizontal development, both structurally and functionally, with progression towards maturity (25). During the first week in Z&V, the principal change in the brief duration response was an alteration in excitability cycle. This was evident in paired pulse stimulation experiments, as displayed in Fig. 6a and b which compares day 1 and day 8. At day 8, recovery of maximal amplitude was more rapid. Figure 6c and d shows that at day 23 there was a potentiation of this response, which was not seen in day 1 cultures. After 5-8 days the major functional change of in vitro development occurred. At this stage, dorsal surface stimulation elicited the spike observed at earlier stages followed by an extremely variable, longer duration slow wave. Unit discharge could be seen as part of the slow wave complex. Figure 2e-i shaws this response on various time bases. The response was characterized as a large positive-negative shift following an initial spike. On some occasions, oscillatory activity occurred with diminishing amplitude (Fig. Ze) . The characteristics of this slow potential included considerable variability which was not related to in z&o age. In some cultures, the ability to elicit the slow potential with surface cortical stimulation rapidly declined following an initial stimulus. Figure 7 shows some data of the slow recovery of such a response in a comparatively excitable culture. This illustration shows a minimal recovery period of at least 1 min, although recovery of rhythmic oscillations could take as long as lo-15 min. We have also noted that, unlike the early spike component, the amplitude of these responses was not simply related to stimulus intensity, although such observations were obviously complicated by the prolonged inexcitability of this response following a single stimulus. Topographic analyses which emphasized the slow wave component showed that the largest amplitude was invariably observed in the middle third of the explant, where pyramidal cell bodies and basal dendrite fields are present in greatest concentration. Successive recordings in a vertical direction from the dorsal cortical surface rarely demonstrated a phase reversal, Recordings in successive horizontal positions showed some in-
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FIG. 6. Excitability cycles in cerebral neocortical explants at different ages. A and R are from 1 and 8 day cultures respectively, both recorded at 32 C. The comparatively less excitable property of the 1 day culture is evident. C and D were recorded at different time bases from a 23 day culture at 32.5 C. Note the hyperexcitability of the early component in C, and the prolonged inexcitability of the slower components.
creased complexity in waveform in addition to a decrease in amplitude. The paucity of phase reversals in the vertical plane is in contrast to observations of slow wave activity in cerebral neocortex explants described by Crain and Bornstein (10). Their finding of phase reversals in the vertical plane as a common event is consistent with an assumption that the basis of the slow-wave activity is synaptic inputs to vertically oriented apical dendrites. In the present study, however, maximal amplitudes of
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slow-wave responses were evident in the region of the basal dendrite synapses, not at the level of apical dendrites. A developmental question we have examined is whether the slow-wave response shows any regular, progressive changes with growth beyond the period when it is initially noted. At the level of extracellular analysis, we have been unable to detect any consistent functional differentiations with aging. Thus knowledge of stimulus parameters, recording position and form of slow wave response did not enable distinction of a 5-8 day old culture from a 33 day culture. This ambiguity partially derived from the fact that the variance of slow wave properties at any age of in vitro growth approximated the variance of the full time period of growth. Stimulation delivered to the deepest portion of the explant resulted in activity which was dissimilar to that provoked by cortical surface stimulation. Figure 8a shows a fast spike response recorded in the middle third of the explant by stimulation near the ventral margin. The complex large amplitude responses provoked by surface stimulation were never elicited by deep fiber stimulation. By contrast, when a rarely included group of subcortical nuclear neurons was stimulated, a long latency burst response
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7. Excitability characteristics of evoked activity elicited by cortical surface stimulation in an 8 day explant at 34 C. In A, the early fast component is emphasized; in B, the longer time base allows examination of the slower components. In both A and B, a-e represent successively recorded responses, with a being a “baseline” response, followed by b, recorded after a 1 min interval, and subsequently by c, d, and e, recorded after 30, 15 and 5 set intervals respectively. FIG.
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was recorded from the middle third of the explant (Fig. Sb) . This response could follow faster rates of stimulation and probably arose in an ascending fiber group. Oscillatory burst responses which could be elicited by stimulation of the superficial fiber bands were never elicited by deep filter stimulation. DISCUSSION The present experiments have shown that the functional development of neocortical explants is characterized by an early period where only short duration spike activity can be observed, followed by the appearance of more long duration slow wave evoked responses. The spike response is the only response observed during a period of l-5 days in vitro, and most likely indicates activity of axons. Changes in this response probably reflect developments in neurite growth, axon diameter and myelination. There is no evidence for a synaptic origin of this response, an assessment similar to that of Crain (9). The long-latency slow-wave response described in the present study differs in several features from a similar response descri.bed by Crain and Bornstein (10). For example, they noted phase reversals in a plane perpendicular to the cortical surface ; this phenomenon was rarely noted in our study. There are many intricacies of dipole analysis with laminar recording, including circumstances in which phase reversals may not be evident, although the implied organization of extracellular fields might lead one to expect phase reversals (26). In some cultures the absence of phase reversals may be related to the dispersion of pyramidal cells (29). Additionally, since many forms of characteristic input to cortical cells are eliminated in isolated explants, one might expect a rather sparse arrangement of synapses on apical dendrites, On the other hand, anatomical studies (29) show a rather dense meshwork of pyramidal cell basal dendrites in the middle third of some explants. If the primary source of synaptic electrogenesis is at the level of the basal dendrites, the absence of a vertically oriented dipole is not surprising. * The minimal examples of spontaneous activity noted in this study are in contrast to a recently reported study of endogenous activity in rat and cat cerebral neocortical explants. Calvet (7) noted spontaneous discharges occurring at intervals of 3 set to 1 min and suggested that they were the result of synaptic activity. From the description of the morphological organization of her cerebral neocortical cultures, it would seem that the synaptic density might be less than in thicker explants employed in the present study, in which there was a paucity of spontaneous activity. The reason for believing Calvet’s cultures to be more thinly dispersed, and therefore having fewer synapses, is based on her ability to resolve neurons
FIG. 8. A response recorded from tion delivered to the ventral margin deep band of fibers, as illustrated by With recording electrodes similarly group of subcortical nuclear neurons in b. Both responses were recorded at
the middle third of a 19 day explant to stimulaof the cortical portion of the explant (i.e., to the the lowermost arrow in Fig. 1) is presented in a. placed in a 12 day explant, stimulation of a burst response shown produced the lon g latency 26 C.
by phase contrast optics. Such resolution is not possible in thick explants. It would appear doubtful, therefore, that the spontaneous discharges described by Calvet are due to synaptic activation, unless there is a ‘selective depletion of inhibitory synapsesin her system. The absence of spontaneouscell discharge in explants described in this paper has also been described in immature rat (12, 20 )and neonatal cat cortex (15, 22) and in some reports of undercut adult cat cortex (6). The infrequent occurrence of pyramidal cell spike discharges in culture
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may be related to the late development of pyramidal cell spike activity, as described in various immature animals (12, 15, 22). On the other hand, the infrequent occurrence of pyramidal cell spikes in vitro may be a function of afferent deprivation rather than immaturity, for our earlier anatomical study ,(29) has indicated a considerable degree of morphological development after explantation. Crain in his review of functional development of explants of diverse origin (9) suggests that there is a general rule of development in cultures derived from fetal or neonatal nervous systems. He describes a sequence or progression proceeding from a simple spike response, which is assumed to be axonal, to complex slow wave electrogenesis based on synaptic activity. He further suggests that this slow-wave response shows an increasing complexity with development, including the emergence of oscillatory afterdischarge activity. Most of this progression was defined for spinal ganglion-spinal cord explants, although he briefly noted that a similar sequence was observed in extracellular studies of cerebral neocortical explants. The generality of this rule seems unusual across such disparately organized systems ; indeed, its existence might even be construed as evidence of the insensitivity of recording methods to structural differences. In our studies we consistently noted the priority of simple spike activity and the emergence of slow wave responses and synaptically driven activity in fine neurite processes. However, the most emphatic demonstration of a developmental progression would be the ability to depict approximate culture age, given knowledge of stimulus position and intensity, and recording position and characteristics of evoked activity. In our observations, elicited slow-wave activity does not provide the basis for such discriminations, although some features of the spike response provide a crude differentiation. Neuronal tissue cultures have been described as a model for the study of developmental processes. There is abundant evidence that characteristic architectural arrangements develop in vitro and many cellular developmental processes emerge within these systems (16, 17, 19, 21, 27-30, 34). Indeed, neural tissue cultures may show the maximum development of a brain subunit freed of extensive customary input pathways and target projection fields. What remains is the characteristic organization of comparatively small units of intrinsic circuitry. Such isolation in an emerging nervous system may strongly constrain the range of functional developments. In this paper we have noted a rather limited sequence of functional changes evident with cerebral neocortical growth in vitro. More elaborate sequential and progressive modifications with aging may depend on the capacity of customary input pathways to provide a set of triggers for both structural and functional developments. From this perspective, one might
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consider that each customary cortical input pathway provides both a capacity for transient signal communication and a trophic influence which are critical determinants of cortical ontogeny. A link between these factors is suggested by the work of Rutledge (23), who has shown that stimulation of isolated cortical slabs influences the maintenance of dendritic spines. One of the advantages of the tissue culture experiment is the ability to assess this conjecture under exacting conditions. For example, cerebrum explants which include thalamic tissue can be compared with those without such connections, both in terms of structural and functional developments. Contrasts between in vitro and in viva organizational developments are suggested from data discussed by the Scheibels (25). They have emphasized that structural studies and various neurophysiological investigations show the emerging dominance of vertical organization within tortes during the course of early development. Indeed, they note the disappearance of dorsal horizontal axonal fibers with aging and their replacement by apical dendrite branching. The dominance of horizontal activities in explants may reflect a persistence of early pathways in the absence of customary extrinsic inputs.
REFERENCES M. 1974. Development of the cerebral neocortex in the rat, pp. 7-67. In “Studies on the Development of Behavior and the Nervous System.” Vol. II. G. Gottlieb [Ed.]. Academic Press, New York. BLAKEMORE, C. 1974. Development of functional connexions in the mammalian visual system. Brit. Med. Bull. 30 : 152-157. BORNSTEIN, M. B. 1964. Morphological development of neonatal mouse cerebral cortex in tissue culture, pp. l-11. In “Neurological and Electrographic Correlative Studies in Infancy.” P. Kellaway and I. Peterson [Eds.]. Grune and ‘Stratton, New York. BORNSTEIN, M. B., and M. R. MURRAY. 1958. Serial observations on patterns of growth, myelin formation, maintenance and degeneration in cultures of newborn rat and kitten cerebellum. .I. Biophys. Biochwt. Cytol. 4: 499-504. BUNGE, R. P., M. B. BUNGE, and E. R. PETERSON. 1967. The onset of synapse formation in spinal cord cultures as studied by electron microscopy. Brain Rcs. 6 : 728-749. BURNS, B. D. 1958. “The Mammalian Cerebral Cortex,” pp. 52-60. Edward Arnold, London. CALVET, M.-C. 1974. Patterns of spontaneous electrical activity in tissues of mammalian cerebral cortex vs. cerebellum. Brain Res. 69: 281-295. CHOW, K. C., and A. L. LEIMAN. 1970. The structural and functional organization of the neocortex. Nrurosci. Res. Prog. Bull. 8: 153-220. CRAIN, S. M. 1974. Tissue culture models of developing brain functions, pp. 69-114. Ilz “Studies on the Development of Behavior and the Nervous System.” Vol. II. G. Gottlieb [Ed.]. Academic Press, New York.
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30. SEIL, F. J., and A. L. LEIMAN. 1975. Neural subsystems and learning: Tissue culture approaches. 11%“Neural Mechanisms of Learning and Memory.” M. R. Rosenzweig and E. L. Bennett [Eds.]. MIT Press. (in press.) 31. SEIL, F. J., M. E. SMITH, A. I,. LEIMAN, and J. M. KELLY, III. 1975. Myelination inhibiting and neuroelectric blocking factors in experimental allergic encephalomyelitis. Scie?zce 187 : 951-953. 32. WARD, A. A., JR. 1961. Epilepsy. Int. XCV. Nc~~rol)iol. 3 : 137-186. 33. WOLF, M. K. 1964. Differentiation of neuronal types and synapses in myelinating cultures of mouse cerebellum J. Cell. Biol. 22: 259-279. 34. WOLF, M. K. 1970. Anatomy of cultured mouse cerebellum. II. Organotypic migration of granule cells demonstrated by silver impregnation of normal and mutant cultures. J. Cowi). Ncwol. 140 : 281-297.