cJOUKNAL OF NEUROBIOLOGY, VOL. 7, NO. 6, PP. 551-566
A Comparative Statistical Study of Hippocampal Neuronal Spontaneous Spike Activity in Situ and in Vitro M. B. SHTARK, V. I. STRATIEVSKY, A. S. RATUSHNJAK, L. V. VOSKRESENSKAJA, a n d N. P. KARASEV Laboratory of Complex Research i n Neuron Systems, Institute of Automation and Electrometry, Siberian Branch o f Academy of Sciences, IJSSR, Novosibirsk, 1J.S.S.R
SUMMARY
The statistical characteristics of the spontaneous spike activity of rat hippocampal neurons in fields CAI-2 were compared in situ and in tissue culture. Statistical analyses have shown strong similarities in estimators of basic numerical characteristics of interspike interval (ISI) distributions. These similarities may serve as evidence of maintenance of normal functional properties and an “organotypic arrangement” of neurons in tissue culture, and they are also indicative of an intrahippocampal origin of the spontaneous impulse activity in the hippocampus. On the other hand, some differences are noted in the tests of firing patterns. Interpretation of these results leads t o some assumptions about mechanisms of the phenomenon under study.
INTRODUCTION
Nervous tissue culture, as a method combining morphological and functional pecularities of vertebrate CNS cellular elements with simplicity of networks unique for such neurons, is of interest for investigation of a number of insufficiently studied questions, closely associated with the mechanisms of development and the functional role of neuron impulse activity. In our view, the most significant results in this field of neurobiology have been achieved from analyses of development of central nervous “organotypic” cultures and dynamics of formation of synaptic electrogenesis and impulse activity (Crain, 1966; Corner and Crain, 1972, Shtark, Voskresenskaja, Ratushnjak, Olenev, and Popov, 1972; Zipser, Crain, and Bornstein, 1973; Nelson and Peacock, 1973). In conjunction with these types of research on nervous tissue culture, we think it interesting t o compare statistical properties of neuronal impulse firing in situ and in uitro. This may reflect relations between functions of a n individual neuron and the organization of the neural system of which i t is an element. 551 (c;
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Fig. 1. Morphological characteristics of newborn rat hippocampal tissue cultures. a-Frontal section of hippocampal formation of newborn rat (Nissl), showing CAI region used to prepare explants; F.d.-Fascia dentata. A-Str. pyramidale; section of newborn rat hippocampus (Bodian) (400X). R-neuritic outgrowth, 4 days in uitro (Holmes-Wolf) (400X). C-explant, 12 days in uitro (Bodian) (400X). D-pyramidal cells of field CAI 2, 18 days in uitro (living; phase-contrast). Calibration: 20 p. E-electron micrographs of pyramidal cell and axodendritic synapses (e),21 days i n uitro (araldit) (20,000X).
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METHODS Experiments were carried out on hippocampal neurons in situ in Wistar and random-bred albino rats of both sexes weighing 220-350 g. The animals were lightly anesthetized with ether. After tracheotomy and curarization, they were connected to a controlled breathing apparatus. Tubocurarine was used in doses of 0.02 mg/100 g body weight. Parts of the skin exposed to surgical manipulations and to squeezing by clamps of the head-holder were anesthetized with 2% novacaine solution. Body temperature was maintained automatically during all experiments within the range of 36-37°C with an automatic thermostatic table controlled by a rectal thermistor. Microelectrodes were introduced into the dorsal hippocampus (fields CAI -.), using coordinates of the stereotactic atlas of Marshall and Fifcova (Bures, Petran, and Zachar, 1967). The same region of hippocampus of newborn rats served for explantation in uitro (Shtark et al., 1972). After dissection of overlying neocortex, the hippocampus was displayed. A part of it was removed and cut into fragments about 0.5 X 1mm. These were placed on collagen-coated coverslips (Fig. 1). The coverslips were placed in culture vessels with nutrient medium (Eagle’s medium: 9G%; human blood serum: 10%; insulin: 0.1 units/ml; glucose: 600 mg%; antibiotics) and placed in a thermostatically controlled bath a t 37°C. After 3-30 days, following preliminary examination by phase-contrast microscopy, the culture coverslip was transferred to a thermostatically controlled chamber for electrophysiological investigations. The chamber was filled with the medium described above, and the pH was maintained a t 7.2-7.4 during the experiment. Micromanipulations were controlled by means of a microscope (MB1-3B) fitted with a phase-contrast system (KF-4). Visualization during advancement of the microelectrodes towards the cells was by means of closed-circuit TV (type PTH-23 M) incorporated into the microscope optics, with the TV screen facing the experimental chamber. Introduction of microelectrodes into various regions of the explant, and adjustments of their relations to neuronal cell bodies and dendrites, were carried out with a Zeiss micromanipulator (Jena). The microelectrodes were glass micropipettes with tip diameters of 0.5-1 Gm, filled with 3 M KCI solution, with a resistance of 4-6 mil and connected to the amplifier input through a cathode follower.
Fig. 2. Variants of spontaneous impulse activity of rat hippocampal pyramidal neurons (fields CAI-2) in tissue culture: A-randomly distributed spikes; B,C-cells firing in grouped bursts; D-a “bundle variant” of impulse activity. Time and amplitude calibration bars: 1mV and 2 sec.
SHTARK ET AL. Signals from the cells were displayed on an oscilloscope for visual control of the unit record. They were converted to standard square pulses by a triggering system. These standard pulses were recorded on magnetic tape. For monitoring mean discharge frequency in control records, and for selection of stationary epochs during an experiment, a “Didac-800” computer was used. Statistical analyses was carried out on spike trains of 4000 interspike intervals (ISI). The sequence of ISI’s was converted by the “Didac-800” into numerical form, and then introduced into a “Minsk-22’’ computer and processed according to a program described below. Distributions of ISI’s were evaluated by first-order interval histograms. Quantitative comparison of interval distributions was by the estimation of their basic statistical characteristics: mean firing rate, standard deviation, coefficients of variation, asymmetry and degree of “peakedness” (kurtosis) in the distribution.* Logarithmic plots of the probability functions were made from the first-order interval histogram for comparison of the actual interval distribution with a theoretical exponential one. Estimates were made of the distribution of long intervals. Curves were also made of postimpulse probability for the distribution function of ISI’s close to zero, in order to compare short and medium firing intervals with an exponential distribution.* For description of grouping of impulses and for estimation of correlative aspects of cell firing, we used interval histograms of the first to fourth order, sum of the interval histograms of the first to eighth order (autocorrelograms) and correlation fields of the first order (common-joint histograms
* We have calculated the so-called “moments” of a distribution. Just as the mean describes the location of the distribution on the abscissa, and the variance describes its dispersion, so do the higher moments describe other features. Thus the third moment describes skewness and the fourth moment indicates peakedness (kurtosis). The mathematical mean of impulse firing rate
where x , is the zth interspike interval. The standard deviation (the mean square error)
The asymmetry coefficient (skewness)
The coefficient of peakedness (kurtosis)
The variation coefficient
The reliability function logarithm (Cox and Lewis, 1969) In R ( x ) = In (1 - F ( x ) )
F ( x ) - distribution function. The postimpulse probability function
F ( x ) - density function.
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of adjacent intervals). Differences between the statistical distributions in the in situ and in uitro test series were evaluated with suitable goodness of fit tests. Explants were stained according to the Bodian, Holmes-Wolf, and Nissl methods (Kim, 1972, 1973; LaVail and Wolf, 1973) a t different periods of cultivation and microelectrode investigation (Fig. 1). Microelectrodes positioned in the CA1.2 region of the hippocampus in situ, and in the depths of the explant, were marked by passage of a marking current.
RESULTS
Morphological characteristics of hippocampal tissue culturest
As described above, oriented slices of newborn rat hippocampus from fields were explanted on collagen-coated coverslips (Fig. 1). T h e dominant feature of the explant was the dense layer of pyramidal cells lying a t a depth of not more than 100 pm from the alvear surface. This surface provided, therefore, a reliable reference for the microelectrodes in relation t o the cell bodies of the pyramids (Fig. 1A). During the first 5-6 days, a n intense growth of axonal processes was seen on the external edge of the explant extending out of the glial elements into an optically transparent zone (Fig. 1B). From 7-12 days, one may identify an “organotypic” pyrami$al layer in the depth of the explant, consisting of closely packed cells, 15-20 pm in diameter, with large pale nuclei (Figs. lC,D). These cellular elements may be considered characteristic of pyramids of Ammon’s horn (Kim, 1972). An “organotypic” arrangement of the pyramids was noted a t later stages in culture, around 18-21 days. With phase-contrast optics the pyramids are recognized as closely grouped cells (20-25 pm in diameter) with large central, or somewhat eccentric, nuclei, and clearly distinguishing nucleoli (Fig. I D ) . In agreement with previous electron-microscopy studies, our results show that in the pyramids of field CAl-2 most of the cell body is occupied by a relatively pale nucleus with evenly distributed chromatin (Fig. 1D). A narrow cytoplasmic rim contains all the characteristic organelles (clearly visible by light microscopy). A rough endoplasmic reticulum develops. The ribosomes and polysomes have a stellate form, and are clearly outlined. Mitochondria are polymorphous. Numerous axodendritic synapses are located on the large dendritic stem. They are characterized by comparatively wide (up to 300 A) synaptic clefts and electron-dense subsynaptic networks (Fig. 1E). Their appearance corresponds to type 1synapses of Gray (1959) or to asymmetrical synapses (Colonnier, 1968). The nuclear, cytoplasmic, and synaptic ultrastructures of hippocampal pyramidal neurons seen here in tissue culture are similar to those described by others (Bunge, Bunge, and Peterson, 1965; Schwartz, Pappas, and Purpura, 1968; Kim, 1972, 1973; LaVail and Wolf, 1973; Wenzel, Wenzel, Grosse, and Kirsche, 1973).
Qualitative description of t h e spontaneous spike activity of hippocampal neurons in vitro Stable spontaneous spike activity was recorded from neurons in hippocampal tissue cultures for periods of many minutes t o hours. Impulse activity was fre+
Electron-microscopic studies were carried out by E. G. Antonova.
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quently recorded only in neuronal groups (typical “nests”) and appears to be a function of density of the cell bodies a t that point. Neurons lying a t a distance from the cellular clusters were usually not spontaneously active. They were capable of generating spikes only by an artificial change in membrane polarization. Electrophysiological identification of neuronal elements in tissue culture may be based on this test (Cechner and Fleming, 1969; Crain, 1973). A qualitative description of the firing pattern of the background electrical activity in these hippocampal tissue cultures is consistent with the known types of unit firing, which are usually divided into three groups (Fig. 2)-randomly distributed spikes, cells firing in grouped bursts, and thirdly a “bundle variant“ of the background impulse activity similar to that seen in the hippocampus in situ (Green, 1964; Vinogradova, 1965; Noda, Manohar and Adey, 1969; see also Moore, Perkel and Segundo, 1966). In units with bundle activity, one may often observe impulse sequences with constantly decreasing amplitude of action potentials, consistent with development of inactivation of a spike-generating neuronal mechanism. This interpretation is supported by the finding that artificial change of membrane potential level by polarizing currents may stabilize the amplitude of the action potentials.
Quantitative description of the spontaneous spike activity in situ and in vitro Background firing activity of hippocampal neurons in vitro was recorded in explants after 7-30 days of culture. Until the seventh day in uitro, the explants rarely showed any background spike activity. We compared the statistical characteristics of background firing activity of 55 neurons of rat dorsal hippocampal fields CAI-2 in situ, and of 51 neurons of the same formation in tissue culture. T h e amplitude of the extracellular spike potentials and the occurrence of background activity primarily in compact clusters of cells which appear to be pyramidal neurons (Fig. lC), favor the view that almost all the firing in these studies was generated by pyramidal neurons in the CAI-2 regions. The distributions of the interspike intervals (ISI’s) of all the firing patterns studied were characterized by positive values of coefficients of asymmetry and excess. Distributions of mathematical means of the lengths of ISI’s and of the asymmetry and kurtosis coefficients did not show any significant differences, when checked by Student’s t-test. Differences in distributions of standard deviation by Fisher’s test were also nonsignificant. Further tests of differences were made by Kholmogorov’s test and Peerson’s test for all distributions and were similarly nonsignificant (Fig. 3). In view of the absence of evident differences in most statistical characteristics of the in situ and in vitro series, and the heterogeneity of the data in each of them, an attempt was made to classify the firing patterns by evaluating the distribution of all quantitative measures and by the character of a n “after-effect.” Aftereffect describes firing behavior different from that in a simple return to baseline
C d
Fig 3 Distributions of basic numerical characteristics of interspike intervals ( H I ) (solid line- zn 5ztu serieq) a-expectations, b-standard deviations, coefficients T h e distribution of interspike intervals (ISI) of all the firing patterns studied are characterized by positive L asymmetry coefficients, d-excess celues of coefficients of asymmetry and excess. Distributions of expectations of the lengths of ISI’s, asymmetry and exce5s coefficients do not show any significant differences
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Fig. 4. Characteristics of firing patterns of the first type: a-interval histogram (IH) of the first order; b -postimpulse probability function; c-correlation field; d-reliability function logarithm. The first type is characterized by near-exponential distributions of interspike intervals (1SI’s)unimodal interval histograms with exponential decay (a). The curves of postimpulse probability fluctuate close to the abscissa axis (b) and approach a straight line in the plot of the reliability function logarithm (d). The symmetrical correlation fields plotted with respect to the diagonal show an evenly condensing distribution toward the region of the shortest interval (c).
after a period of patterned firing. In this study, after-effect is defined as the appearance of firing behavior differing from the exponential distribution curve. Such cells thus show a dependence on prior firing intervals. The first type consisted of firing patterns in which the estimations we used did not show any signs of after-effect. Such firing patterns were characterized by nearly exponential distributions of interspike intervals. This was supported by unimodal interval histograms with exponential decay (Fig. 4a), curves of postimpulse probability which fluctuated close to the abscissa axis (Fig. 4b), and approximately linear plots of the reliability function logarithm (Fig. 4d). Their variation coefficients are close to unity. Aperiodic abruptly extinguishing autocorrelograms did not show any dependence of ISI’s on one another. The exponentiality of distribution of the ISI’s and the absence of a dependency between intervals are also indicated by the correlation fields (Fig. 4c), where the symmetrical plot around the diagonal shows the firing distribution condensing evenly in the region of the shortest intervals. Absence of an after-effect, in combination with the usual neuronal refractoriness, suggests that firing patterns
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Fig. 5. Characteristics of the second type. a-postimpulse probability function; b-correlation field. One may characterize the second type of firing pattern as a discharge with limited “after-effect” (see text). The first 10-20 msec of the postimpulse period is distinguished by a nonrandomly high probability of the spike occurring. The significant prevalence of short intervals with a stable duration for these discharges gives them a characteristic “firing” activity. The intervals here between firings are evenly distributed. The corresponding condensation lines of the correlation field points (b) are seen.
of this type are close to a Poisson distribution with a “standstill time.” The
modes of the ISI’s occur in most cases around 10-20 msec. One may characterize the second type of firing pattern as a discharge with limited after-effect. The after-effect appears as a sharp increase in the number of ISI’s with 10-20 msec duration in comparison with the exponentially distributed ones. The first 10-20 msec of the postimpulse period is distinguished by a nonrandomly high probability of the spike occurring, and then abruptly
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Fig. 6. Characteristics of third type: a-IH of the first-fourth orders; b-autocorrelograms; c-correlation field. The statistical parameters for the third type of firing pattern resemble those of pacemaker neurons. The interval histograms of different orders are almost symmetrical, separated by equal distances (a) and form autocorrelograms typical of regular firing (b). Points on the correlation field plot are located in a defined central zone (c). Firing patterns of this type have been detected only in uitro.
decreasing to a very low level (Fig. 5a). Since the autocorrelogram of the firing pattern of the second cell type also did not show any dependence between intervals, one may consider that the probability of a discharge arising is dependent only on the elapsed time since the immediately preceding spike, and is thus independent of its “prehistory.” The statistically significant occurrence of short intervals for these discharges gives these cells a characteristic firing pattern. In contrast to cells with bursting discharges, this second type of cell has a pattern with evenly distributed intervals. These firing patterns are characterized by (1)unimodal histograms with a sharp nonexponential recession and a long tail, TABLE 1 Distribution of T y p e s of Impulse Firing Patterns in Compared Series T y p e o f Firing Pattern Without after-effect With a limited after-effect Quasi-regular Quasi-periodic
N u m b e r in t h e In Situ Series
N u m b e r in t h e In Vitro Series
12 27
6 26 18 0
0
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Fig. 7. Characteristics of fourth type: a-IH of the first order; b-autocorrelogram; c,d-correlation fields. The fourth type of firing pattern typical for units in the cortex zn sztu, is characterized h y distributions, in which there are at least two and typically more modes of ISI’s (a). Summation of the interval-histograms of different orders does not smooth out the bimodality. It may be expressed in any tendency to alternation of the series of short intervals with definite pauses of sufficiently long duration (b). The same tendency is shown by the form of correlation fields which is characteristic of this type of firing pattern (c,d).
(2) by unimodal postimpulse probability curves with subsequent extension t o a plateau (Fig. 5a), ( 3 ) by the sharp recession of the probability function logarithms of the dominant intervals, and (4)by the corresponding condensation lines of the correlation field points (Fig. 5b). The statistical parameters for the third type of firing pattern resemble those of pacemaker neurons. The low values of the variation coefficients and the narrow, steep histograms of IS1 distribution support the relative stability of the length of the ISI’s. Interval histograms showing different orders (Fig. 6a) are almost symmetrical, separated by equal distances, and form autocorrelograms typical of regular firing (Fig. 6b). Points on the correlation field plot are located in a defined central zone and rarified towards the periphery (Fig. 6c). These features of discharges of units of the third type support the definition of quasiregular. The maxima of IS1 distributions are in the region of 60-70 msec. Firing patterns of this type have been detected only in uitro (Table 1). The fourth type of firing pattern typical for units in the cortex in situ is
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characterized by distributions in which there are a t least two and typically more modes in the ISI’s. Summation of the interval-histograms of different orders does not smooth out this bimodality, which may be evidence of the presence of connections between ISI’s. I t may be expressed in an‘y tendency to alternation of the series of short intervals with definite pauses of sufficiently long duration. The same tendency is shown by the form of correlation fields characteristic of this type of firing (Fig. 7). We refer t o this type of firing pattern as “quasiperiodical.” The distribution of impulse firing of different types in these series is presented by Table 1. DISCUSSION
A stable background of spontaneous spike activity of the neurons in uitro indicates that in situ such activity is apparently a phenomenon of “intrahippocampal” origin. The role of extrahippocampal influences seems to be restricted to modulation of the firing patterns generated in the hippocampal circuits. Of particular interest as origin of this modulation is the role of various sensory input stimuli, leading t o reverberation of impulses in closed neuronal networks, which are not controllable during the experiments in situ. It must be admitted, however, that under these conditions of prolonged deafferentation, the most likely sources of background spike activity in the cultures are the endogenous fluctuations of the excitation threshold of hippocampal pyramidal cells, and the spontaneous leakage of transmitter into the synaptic cleft. The absence of any significant differences between the basic statistical characteristics, considered with the well-known sensitivity of these parameters(e.g., mean frequency of discharge) to various nonspecific and injury influences, may be considered as evidence for maintenance of principal functional properties by the neurons in uitro. Furthermore, these results confirm conclusions about the endogenous sources of hippocampal background spike activity. Apparently, because of differences in age of the immature CNS tissues a t explantation, the reported time of onset of spontaneous impulse activity (in different studies) has ranged from 30 min t o 7-13 days after explantation (Crain, 1966; Schlapfer, 1969; Cechner and Fleming, 1969; Corner and Crain, 1972; Shtark et al., 1972). Also, and perhaps for similar reasons, changes in the firing patterns of spontaneously active neurons occurred during development in culture. Thus, there is not only a time sequence in the appearance of impulse activity in tissue culture, but even more significant, a progressive increase in numbers of spontaneously active cells with increasing age in uitro (Corner and Crain, 1972; Shtark et al., 1972). This is an important principle, since it suggests the progress in formation and elaboration of synaptic interaction in the cultures. I t also provides ample opportunity for study of this phenomenon under controlled conditions in culture as has been attempted in the present study. Prevalence of variation coefficients close t o unity in cells in situ is related t o the relatively large number of firings in this series approaching Poisson conditions (Fig. 8). Occurrence of such firing patterns may be related to overriding effects
I 84
Fig. 8. Distribution of variation coefficients (solid line--in situ series). The prevalence of variation coefficients close to unity in the in situ series is connected with relatively large number of firings, approaching the Poisson ones, in this series. The shift of the mode of distribution of variation coefficients towards lower values in the in uitro series is connected with the presence of firing patterns of the third type typical o f this series.
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of a large number of small nonorganized influences, which may predominate in the i n situ series, but may be partly reduced in tissue culture. Variation coefficients shift towards lower values in the in vitro series. The character of these firing patterns, and their clear predominance in the explants, suggests this form of activity as the closest to “true spontaneous activity” attributable to endogenous threshold fluctuations andlor spontaneous transmitter leakage into the synaptic cleft. A choice between these two possibilities by statistical analysis of firing patterns appears unfeasible because regularity of firing may be explained by periodicity in a pacemaker mechanism, or by regularity of the recovery process in the postimpulse period after spikes evoked by transmitter leakage. From these hypotheses, quasi-regular firing patterns may be considered as the primary ones in the absence of any manifest inhibitory or excitatory modulating influences. Therefore, firing patterns of the Poisson type result from many random influences upon the initial (regular) activity. In our view, i t should be possible t o settle this question by experiments with substances which selectively block synaptic mechanisms (e.g., Mg2+). The considerable number of distributions in both series with maxima in the region of 10-20 msec reflects the decrease in firing threshold during the corresponding postimpulse period, possibly related t o prolonged depolarization, lasting up to 20 msec in hippocampal pyramidal neurons (Kandel and Spencer, 1964). In particular, this factor is dominant for the most general case of firing patterns with limited after-effect. Formation of highly complex firing patterns characteristic of neurons in situ, and referred to as quasi-periodic, is influenced in part by organized synaptic activity initiated by extrahippocampal afferents. Analysis of the differences in statistical characteristics of hippocampal neurons in situ and in vitro is ambiguous due to the probable involvement of competing mechanisms. Thus, deafferentation of the hippocampus in uitro deprives its neurons of most excitatory and inhibitory extrahippocampal influences, and possibly changes its excitability. In addition, hippocampal pyramidal neurons may also be physiologically altered by the physicochemical environment in culture. To make these hypotheses more precise, and t o detect subtler differences by statistical analysis of background activity, it seem reasonable to simplify further the neural tissue culture experimental model, in addition t o perfecting analytical methods. Such simplification may be achieved by selective blocking of synaptic transmission in hippocampal explants, by utilizing cultures a t different stages of synaptogenesis, or by the use of cultures of dissociated and reaggregated cerebral neurons (Bornstein and Model, 1972; Crain and Bornstein, 1972,1974; Zipser, Crain, and Bornstein, 1973; Nelson and Peacock, 1973; Crain, Raine, and Bornstein, 1975). REFERENCES
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BURES,J., PETRAN, M., and ZACHAR, J. (1967). Electrophysiological Methods in Biological Research, 2nd Edit., Academia Publ. House, Czechoslovak Acad. Sciences, Prague. D. A. (1969). Modulation of spontaneous neuronal activity with CECHNER,P. L. and FLEMING, weak electrical current. Experiments in uitro. In: Systematic Organization of Physiological Functions. M. N. Liranov, Ed., State Publ. House, “Medicine,” Moscow. COLONNIER, M. (1968). Synaptic patterns of different cell types in the different laminae of the cat visual cortex. An electron microscope study. Brain Res. 9: 268-287. CORNER,M. A. and CRAIN,S. M. (1972). Patterns of spontaneous hioelectric activity during maturation in culture of fetal rodent medulla and spinal cord tissues. J. Neurobiol. 3: 25-46. COX,D. R. and LEWIS, P. A. (1969). The statistical analysis of series of events. “Mir” (“World”) Puhl. House, Moscow. CRAW,S. M. (1966). Development of “organotypic” bioelectric activities in central nervous tissue during maturation in culture. Internat. Reu. Neurobiol. 9: 1-43. CRAIN,S. M. (1973). Microelectrode recording in brain tissue cultures. In: Methods in Physiological Psychology, Vol. 1, Bioelectric Recording Techniques: Cellular Processes and Brain Potentials. R. F. Thompson and M. M. Patterson, Eds., Academic Press, New York, pp. 3975. CRAIN,S. M., and BORNSTEIN,M. B. (1972). Organotypic hioelectric activity in cultured reaggregates of dissociated rodent brain cells. Science 176: 182-184. CRAIN,S. M. and BORNSTEIN,M. B. (1974). Early onset in inhibitory functions during synaptogenesis in fetal mouse brain cultures. Brain Res. 68: 351-357. CHAIN,S. M., RAINE,C. S., and BORNSTEIN,M. B. (1975). Early formation of synaptic networks in cultures of fetal mouse cerebral neocortex and hippocampus. J. Neurobiol. 6: 329-336. GAHWLER,B. H., MAMOON,A. M., SCHLAPFER,W. T., and TOBIAS,C. A. (1972). Effects of temperature on spontaneous hioelectric activity of cultured nerve cells. Brain Res. 40: 527533. C. A. (1973). Spontaneous hioelectric activity GAHWILER,B. H., MAMOON,A. M., and TOBIAS, of cultured cerebellar Purkinje cells during exposure to agents which prevent synaptic transmission. Brain Res. 53,71-80. GRAY,E. G. (1959). Axosomatic and axodendritic synapses of the cerebral cortex: An electron microscope study. J . Anat. 93: 420-433. GREEN,J . D. (1964). The hippocampus. Physiol. Reviews 44: 561-608. W. A. (1961). Electrophysiology of hippocampal neurons. J . NeuKANDEL,E. R. and SPENCER, rophysiol. 24: 243-259. KIM, S. U. (1972). Light and electron microscope study of mouse cerebral neocortex in tissue culture. Exper. Neurol. 35: 305-321. KIM,S. IT.(1973). Morphological development of neonatal mouse hippocampus cultured in uitro. Exper. Neurol. 41: 150-162. LAVAII,,J. H. and WOLF, M. K. (1973). Postnatal development of the mouse dentate gyrus in organotypic cultures of the hippocampal formation. Amer. J . Anat. 137: 47-66. J. P. (1966). Statistical analysis and functional inMOORE.G. P., PERKEL,D. H., and SEGUNDO, terpretation of neuronal spike data. Ann. Reu. Physiol. 28: 439-522. J. H. (1973). Electrical activity in dissociated cell cultures from fetal NELSON,P. G. and PEACOCK, mouse cerebellum. Brain Res. 61: 163-170. S., and AL)EY,W. R. (1969). Spontaneous activity of cat hippocampal neurons NOIIA,H., MANOHAR, in sleep and wakefulness. Exper. Neurol. 24: 217-232. W. T. (1969). Bioelectric activity of neurons in tissue culture; synaptic interactions SCKLAPFER, and effects of environmental changes. Ph.D. Thesis, University of California, Berkeley. S(‘HWAKTZ, J. R., PAPPAS, G. D. and PURPURA, D. P . (1968). Fine structure of neurons and synapseqin the feline hippocampus during postnatal ontogenesis. Exper. Neurol. 22: 394-407. SHTARK,M. B. (1972). “The Brain of Hibernating Animals.” NASA, Washington, D.C. SHTARK,M. B., VOSKRESENSKAJA, L. W., RATUSHNJAK,A. S., OLENEV,S. N., and POPOV,J. W. (1972). A study of the impulse activity of the hippocampal neurons i n uitro. Rep. Ac. Sci. of lJSSR 202: 731-736. 0.S. (1965). Dinamicheskaja classificazija reaczii neironov gippocampa na sensornie VINOGRADOVA, rasdrasbenija. Shurn. uissh. nerun. deajt. 15: 500-512.
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A Comparative Statistical Study of Hippocampal Neuronal Spontaneous Spike Activity in Situ and in Vitro M. B. SHTARK, V. I. STRATIEVSKY, A. S. RATUSHNJAK, L. V. VOSKRESENSKAJA, a n d N. P. KARASEV Laboratory of Complex Research i n Neuron Systems, Institute of Automation and Electrometry, Siberian Branch o f Academy of Sciences, IJSSR, Novosibirsk, 1J.S.S.R
SUMMARY
The statistical characteristics of the spontaneous spike activity of rat hippocampal neurons in fields CAI-2 were compared in situ and in tissue culture. Statistical analyses have shown strong similarities in estimators of basic numerical characteristics of interspike interval (ISI) distributions. These similarities may serve as evidence of maintenance of normal functional properties and an “organotypic arrangement” of neurons in tissue culture, and they are also indicative of an intrahippocampal origin of the spontaneous impulse activity in the hippocampus. On the other hand, some differences are noted in the tests of firing patterns. Interpretation of these results leads t o some assumptions about mechanisms of the phenomenon under study.
INTRODUCTION
Nervous tissue culture, as a method combining morphological and functional pecularities of vertebrate CNS cellular elements with simplicity of networks unique for such neurons, is of interest for investigation of a number of insufficiently studied questions, closely associated with the mechanisms of development and the functional role of neuron impulse activity. In our view, the most significant results in this field of neurobiology have been achieved from analyses of development of central nervous “organotypic” cultures and dynamics of formation of synaptic electrogenesis and impulse activity (Crain, 1966; Corner and Crain, 1972, Shtark, Voskresenskaja, Ratushnjak, Olenev, and Popov, 1972; Zipser, Crain, and Bornstein, 1973; Nelson and Peacock, 1973). In conjunction with these types of research on nervous tissue culture, we think it interesting t o compare statistical properties of neuronal impulse firing in situ and in uitro. This may reflect relations between functions of a n individual neuron and the organization of the neural system of which i t is an element. 551 (c;
1976 by John Wiley & Sons, Inc.
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Fig. 1. Morphological characteristics of newborn rat hippocampal tissue cultures. a-Frontal section of hippocampal formation of newborn rat (Nissl), showing CAI region used to prepare explants; F.d.-Fascia dentata. A-Str. pyramidale; section of newborn rat hippocampus (Bodian) (400X). R-neuritic outgrowth, 4 days in uitro (Holmes-Wolf) (400X). C-explant, 12 days in uitro (Bodian) (400X). D-pyramidal cells of field CAI 2, 18 days in uitro (living; phase-contrast). Calibration: 20 p. E-electron micrographs of pyramidal cell and axodendritic synapses (e),21 days i n uitro (araldit) (20,000X).
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METHODS Experiments were carried out on hippocampal neurons in situ in Wistar and random-bred albino rats of both sexes weighing 220-350 g. The animals were lightly anesthetized with ether. After tracheotomy and curarization, they were connected to a controlled breathing apparatus. Tubocurarine was used in doses of 0.02 mg/100 g body weight. Parts of the skin exposed to surgical manipulations and to squeezing by clamps of the head-holder were anesthetized with 2% novacaine solution. Body temperature was maintained automatically during all experiments within the range of 36-37°C with an automatic thermostatic table controlled by a rectal thermistor. Microelectrodes were introduced into the dorsal hippocampus (fields CAI -.), using coordinates of the stereotactic atlas of Marshall and Fifcova (Bures, Petran, and Zachar, 1967). The same region of hippocampus of newborn rats served for explantation in uitro (Shtark et al., 1972). After dissection of overlying neocortex, the hippocampus was displayed. A part of it was removed and cut into fragments about 0.5 X 1mm. These were placed on collagen-coated coverslips (Fig. 1). The coverslips were placed in culture vessels with nutrient medium (Eagle’s medium: 9G%; human blood serum: 10%; insulin: 0.1 units/ml; glucose: 600 mg%; antibiotics) and placed in a thermostatically controlled bath a t 37°C. After 3-30 days, following preliminary examination by phase-contrast microscopy, the culture coverslip was transferred to a thermostatically controlled chamber for electrophysiological investigations. The chamber was filled with the medium described above, and the pH was maintained a t 7.2-7.4 during the experiment. Micromanipulations were controlled by means of a microscope (MB1-3B) fitted with a phase-contrast system (KF-4). Visualization during advancement of the microelectrodes towards the cells was by means of closed-circuit TV (type PTH-23 M) incorporated into the microscope optics, with the TV screen facing the experimental chamber. Introduction of microelectrodes into various regions of the explant, and adjustments of their relations to neuronal cell bodies and dendrites, were carried out with a Zeiss micromanipulator (Jena). The microelectrodes were glass micropipettes with tip diameters of 0.5-1 Gm, filled with 3 M KCI solution, with a resistance of 4-6 mil and connected to the amplifier input through a cathode follower.
Fig. 2. Variants of spontaneous impulse activity of rat hippocampal pyramidal neurons (fields CAI-2) in tissue culture: A-randomly distributed spikes; B,C-cells firing in grouped bursts; D-a “bundle variant” of impulse activity. Time and amplitude calibration bars: 1mV and 2 sec.
SHTARK ET AL. Signals from the cells were displayed on an oscilloscope for visual control of the unit record. They were converted to standard square pulses by a triggering system. These standard pulses were recorded on magnetic tape. For monitoring mean discharge frequency in control records, and for selection of stationary epochs during an experiment, a “Didac-800” computer was used. Statistical analyses was carried out on spike trains of 4000 interspike intervals (ISI). The sequence of ISI’s was converted by the “Didac-800” into numerical form, and then introduced into a “Minsk-22’’ computer and processed according to a program described below. Distributions of ISI’s were evaluated by first-order interval histograms. Quantitative comparison of interval distributions was by the estimation of their basic statistical characteristics: mean firing rate, standard deviation, coefficients of variation, asymmetry and degree of “peakedness” (kurtosis) in the distribution.* Logarithmic plots of the probability functions were made from the first-order interval histogram for comparison of the actual interval distribution with a theoretical exponential one. Estimates were made of the distribution of long intervals. Curves were also made of postimpulse probability for the distribution function of ISI’s close to zero, in order to compare short and medium firing intervals with an exponential distribution.* For description of grouping of impulses and for estimation of correlative aspects of cell firing, we used interval histograms of the first to fourth order, sum of the interval histograms of the first to eighth order (autocorrelograms) and correlation fields of the first order (common-joint histograms
* We have calculated the so-called “moments” of a distribution. Just as the mean describes the location of the distribution on the abscissa, and the variance describes its dispersion, so do the higher moments describe other features. Thus the third moment describes skewness and the fourth moment indicates peakedness (kurtosis). The mathematical mean of impulse firing rate
where x , is the zth interspike interval. The standard deviation (the mean square error)
The asymmetry coefficient (skewness)
The coefficient of peakedness (kurtosis)
The variation coefficient
The reliability function logarithm (Cox and Lewis, 1969) In R ( x ) = In (1 - F ( x ) )
F ( x ) - distribution function. The postimpulse probability function
F ( x ) - density function.
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of adjacent intervals). Differences between the statistical distributions in the in situ and in uitro test series were evaluated with suitable goodness of fit tests. Explants were stained according to the Bodian, Holmes-Wolf, and Nissl methods (Kim, 1972, 1973; LaVail and Wolf, 1973) a t different periods of cultivation and microelectrode investigation (Fig. 1). Microelectrodes positioned in the CA1.2 region of the hippocampus in situ, and in the depths of the explant, were marked by passage of a marking current.
RESULTS
Morphological characteristics of hippocampal tissue culturest
As described above, oriented slices of newborn rat hippocampus from fields were explanted on collagen-coated coverslips (Fig. 1). T h e dominant feature of the explant was the dense layer of pyramidal cells lying a t a depth of not more than 100 pm from the alvear surface. This surface provided, therefore, a reliable reference for the microelectrodes in relation t o the cell bodies of the pyramids (Fig. 1A). During the first 5-6 days, a n intense growth of axonal processes was seen on the external edge of the explant extending out of the glial elements into an optically transparent zone (Fig. 1B). From 7-12 days, one may identify an “organotypic” pyrami$al layer in the depth of the explant, consisting of closely packed cells, 15-20 pm in diameter, with large pale nuclei (Figs. lC,D). These cellular elements may be considered characteristic of pyramids of Ammon’s horn (Kim, 1972). An “organotypic” arrangement of the pyramids was noted a t later stages in culture, around 18-21 days. With phase-contrast optics the pyramids are recognized as closely grouped cells (20-25 pm in diameter) with large central, or somewhat eccentric, nuclei, and clearly distinguishing nucleoli (Fig. I D ) . In agreement with previous electron-microscopy studies, our results show that in the pyramids of field CAl-2 most of the cell body is occupied by a relatively pale nucleus with evenly distributed chromatin (Fig. 1D). A narrow cytoplasmic rim contains all the characteristic organelles (clearly visible by light microscopy). A rough endoplasmic reticulum develops. The ribosomes and polysomes have a stellate form, and are clearly outlined. Mitochondria are polymorphous. Numerous axodendritic synapses are located on the large dendritic stem. They are characterized by comparatively wide (up to 300 A) synaptic clefts and electron-dense subsynaptic networks (Fig. 1E). Their appearance corresponds to type 1synapses of Gray (1959) or to asymmetrical synapses (Colonnier, 1968). The nuclear, cytoplasmic, and synaptic ultrastructures of hippocampal pyramidal neurons seen here in tissue culture are similar to those described by others (Bunge, Bunge, and Peterson, 1965; Schwartz, Pappas, and Purpura, 1968; Kim, 1972, 1973; LaVail and Wolf, 1973; Wenzel, Wenzel, Grosse, and Kirsche, 1973).
Qualitative description of t h e spontaneous spike activity of hippocampal neurons in vitro Stable spontaneous spike activity was recorded from neurons in hippocampal tissue cultures for periods of many minutes t o hours. Impulse activity was fre+
Electron-microscopic studies were carried out by E. G. Antonova.
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quently recorded only in neuronal groups (typical “nests”) and appears to be a function of density of the cell bodies a t that point. Neurons lying a t a distance from the cellular clusters were usually not spontaneously active. They were capable of generating spikes only by an artificial change in membrane polarization. Electrophysiological identification of neuronal elements in tissue culture may be based on this test (Cechner and Fleming, 1969; Crain, 1973). A qualitative description of the firing pattern of the background electrical activity in these hippocampal tissue cultures is consistent with the known types of unit firing, which are usually divided into three groups (Fig. 2)-randomly distributed spikes, cells firing in grouped bursts, and thirdly a “bundle variant“ of the background impulse activity similar to that seen in the hippocampus in situ (Green, 1964; Vinogradova, 1965; Noda, Manohar and Adey, 1969; see also Moore, Perkel and Segundo, 1966). In units with bundle activity, one may often observe impulse sequences with constantly decreasing amplitude of action potentials, consistent with development of inactivation of a spike-generating neuronal mechanism. This interpretation is supported by the finding that artificial change of membrane potential level by polarizing currents may stabilize the amplitude of the action potentials.
Quantitative description of the spontaneous spike activity in situ and in vitro Background firing activity of hippocampal neurons in vitro was recorded in explants after 7-30 days of culture. Until the seventh day in uitro, the explants rarely showed any background spike activity. We compared the statistical characteristics of background firing activity of 55 neurons of rat dorsal hippocampal fields CAI-2 in situ, and of 51 neurons of the same formation in tissue culture. T h e amplitude of the extracellular spike potentials and the occurrence of background activity primarily in compact clusters of cells which appear to be pyramidal neurons (Fig. lC), favor the view that almost all the firing in these studies was generated by pyramidal neurons in the CAI-2 regions. The distributions of the interspike intervals (ISI’s) of all the firing patterns studied were characterized by positive values of coefficients of asymmetry and excess. Distributions of mathematical means of the lengths of ISI’s and of the asymmetry and kurtosis coefficients did not show any significant differences, when checked by Student’s t-test. Differences in distributions of standard deviation by Fisher’s test were also nonsignificant. Further tests of differences were made by Kholmogorov’s test and Peerson’s test for all distributions and were similarly nonsignificant (Fig. 3). In view of the absence of evident differences in most statistical characteristics of the in situ and in vitro series, and the heterogeneity of the data in each of them, an attempt was made to classify the firing patterns by evaluating the distribution of all quantitative measures and by the character of a n “after-effect.” Aftereffect describes firing behavior different from that in a simple return to baseline
C d
Fig 3 Distributions of basic numerical characteristics of interspike intervals ( H I ) (solid line- zn 5ztu serieq) a-expectations, b-standard deviations, coefficients T h e distribution of interspike intervals (ISI) of all the firing patterns studied are characterized by positive L asymmetry coefficients, d-excess celues of coefficients of asymmetry and excess. Distributions of expectations of the lengths of ISI’s, asymmetry and exce5s coefficients do not show any significant differences
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Fig. 4. Characteristics of firing patterns of the first type: a-interval histogram (IH) of the first order; b -postimpulse probability function; c-correlation field; d-reliability function logarithm. The first type is characterized by near-exponential distributions of interspike intervals (1SI’s)unimodal interval histograms with exponential decay (a). The curves of postimpulse probability fluctuate close to the abscissa axis (b) and approach a straight line in the plot of the reliability function logarithm (d). The symmetrical correlation fields plotted with respect to the diagonal show an evenly condensing distribution toward the region of the shortest interval (c).
after a period of patterned firing. In this study, after-effect is defined as the appearance of firing behavior differing from the exponential distribution curve. Such cells thus show a dependence on prior firing intervals. The first type consisted of firing patterns in which the estimations we used did not show any signs of after-effect. Such firing patterns were characterized by nearly exponential distributions of interspike intervals. This was supported by unimodal interval histograms with exponential decay (Fig. 4a), curves of postimpulse probability which fluctuated close to the abscissa axis (Fig. 4b), and approximately linear plots of the reliability function logarithm (Fig. 4d). Their variation coefficients are close to unity. Aperiodic abruptly extinguishing autocorrelograms did not show any dependence of ISI’s on one another. The exponentiality of distribution of the ISI’s and the absence of a dependency between intervals are also indicated by the correlation fields (Fig. 4c), where the symmetrical plot around the diagonal shows the firing distribution condensing evenly in the region of the shortest intervals. Absence of an after-effect, in combination with the usual neuronal refractoriness, suggests that firing patterns
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Fig. 5. Characteristics of the second type. a-postimpulse probability function; b-correlation field. One may characterize the second type of firing pattern as a discharge with limited “after-effect” (see text). The first 10-20 msec of the postimpulse period is distinguished by a nonrandomly high probability of the spike occurring. The significant prevalence of short intervals with a stable duration for these discharges gives them a characteristic “firing” activity. The intervals here between firings are evenly distributed. The corresponding condensation lines of the correlation field points (b) are seen.
of this type are close to a Poisson distribution with a “standstill time.” The
modes of the ISI’s occur in most cases around 10-20 msec. One may characterize the second type of firing pattern as a discharge with limited after-effect. The after-effect appears as a sharp increase in the number of ISI’s with 10-20 msec duration in comparison with the exponentially distributed ones. The first 10-20 msec of the postimpulse period is distinguished by a nonrandomly high probability of the spike occurring, and then abruptly
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Fig. 6. Characteristics of third type: a-IH of the first-fourth orders; b-autocorrelograms; c-correlation field. The statistical parameters for the third type of firing pattern resemble those of pacemaker neurons. The interval histograms of different orders are almost symmetrical, separated by equal distances (a) and form autocorrelograms typical of regular firing (b). Points on the correlation field plot are located in a defined central zone (c). Firing patterns of this type have been detected only in uitro.
decreasing to a very low level (Fig. 5a). Since the autocorrelogram of the firing pattern of the second cell type also did not show any dependence between intervals, one may consider that the probability of a discharge arising is dependent only on the elapsed time since the immediately preceding spike, and is thus independent of its “prehistory.” The statistically significant occurrence of short intervals for these discharges gives these cells a characteristic firing pattern. In contrast to cells with bursting discharges, this second type of cell has a pattern with evenly distributed intervals. These firing patterns are characterized by (1)unimodal histograms with a sharp nonexponential recession and a long tail, TABLE 1 Distribution of T y p e s of Impulse Firing Patterns in Compared Series T y p e o f Firing Pattern Without after-effect With a limited after-effect Quasi-regular Quasi-periodic
N u m b e r in t h e In Situ Series
N u m b e r in t h e In Vitro Series
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6 26 18 0
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Fig. 7. Characteristics of fourth type: a-IH of the first order; b-autocorrelogram; c,d-correlation fields. The fourth type of firing pattern typical for units in the cortex zn sztu, is characterized h y distributions, in which there are at least two and typically more modes of ISI’s (a). Summation of the interval-histograms of different orders does not smooth out the bimodality. It may be expressed in any tendency to alternation of the series of short intervals with definite pauses of sufficiently long duration (b). The same tendency is shown by the form of correlation fields which is characteristic of this type of firing pattern (c,d).
(2) by unimodal postimpulse probability curves with subsequent extension t o a plateau (Fig. 5a), ( 3 ) by the sharp recession of the probability function logarithms of the dominant intervals, and (4)by the corresponding condensation lines of the correlation field points (Fig. 5b). The statistical parameters for the third type of firing pattern resemble those of pacemaker neurons. The low values of the variation coefficients and the narrow, steep histograms of IS1 distribution support the relative stability of the length of the ISI’s. Interval histograms showing different orders (Fig. 6a) are almost symmetrical, separated by equal distances, and form autocorrelograms typical of regular firing (Fig. 6b). Points on the correlation field plot are located in a defined central zone and rarified towards the periphery (Fig. 6c). These features of discharges of units of the third type support the definition of quasiregular. The maxima of IS1 distributions are in the region of 60-70 msec. Firing patterns of this type have been detected only in uitro (Table 1). The fourth type of firing pattern typical for units in the cortex in situ is
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characterized by distributions in which there are a t least two and typically more modes in the ISI’s. Summation of the interval-histograms of different orders does not smooth out this bimodality, which may be evidence of the presence of connections between ISI’s. I t may be expressed in an‘y tendency to alternation of the series of short intervals with definite pauses of sufficiently long duration. The same tendency is shown by the form of correlation fields characteristic of this type of firing (Fig. 7). We refer t o this type of firing pattern as “quasiperiodical.” The distribution of impulse firing of different types in these series is presented by Table 1. DISCUSSION
A stable background of spontaneous spike activity of the neurons in uitro indicates that in situ such activity is apparently a phenomenon of “intrahippocampal” origin. The role of extrahippocampal influences seems to be restricted to modulation of the firing patterns generated in the hippocampal circuits. Of particular interest as origin of this modulation is the role of various sensory input stimuli, leading t o reverberation of impulses in closed neuronal networks, which are not controllable during the experiments in situ. It must be admitted, however, that under these conditions of prolonged deafferentation, the most likely sources of background spike activity in the cultures are the endogenous fluctuations of the excitation threshold of hippocampal pyramidal cells, and the spontaneous leakage of transmitter into the synaptic cleft. The absence of any significant differences between the basic statistical characteristics, considered with the well-known sensitivity of these parameters(e.g., mean frequency of discharge) to various nonspecific and injury influences, may be considered as evidence for maintenance of principal functional properties by the neurons in uitro. Furthermore, these results confirm conclusions about the endogenous sources of hippocampal background spike activity. Apparently, because of differences in age of the immature CNS tissues a t explantation, the reported time of onset of spontaneous impulse activity (in different studies) has ranged from 30 min t o 7-13 days after explantation (Crain, 1966; Schlapfer, 1969; Cechner and Fleming, 1969; Corner and Crain, 1972; Shtark et al., 1972). Also, and perhaps for similar reasons, changes in the firing patterns of spontaneously active neurons occurred during development in culture. Thus, there is not only a time sequence in the appearance of impulse activity in tissue culture, but even more significant, a progressive increase in numbers of spontaneously active cells with increasing age in uitro (Corner and Crain, 1972; Shtark et al., 1972). This is an important principle, since it suggests the progress in formation and elaboration of synaptic interaction in the cultures. I t also provides ample opportunity for study of this phenomenon under controlled conditions in culture as has been attempted in the present study. Prevalence of variation coefficients close t o unity in cells in situ is related t o the relatively large number of firings in this series approaching Poisson conditions (Fig. 8). Occurrence of such firing patterns may be related to overriding effects
I 84
Fig. 8. Distribution of variation coefficients (solid line--in situ series). The prevalence of variation coefficients close to unity in the in situ series is connected with relatively large number of firings, approaching the Poisson ones, in this series. The shift of the mode of distribution of variation coefficients towards lower values in the in uitro series is connected with the presence of firing patterns of the third type typical o f this series.
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of a large number of small nonorganized influences, which may predominate in the i n situ series, but may be partly reduced in tissue culture. Variation coefficients shift towards lower values in the in vitro series. The character of these firing patterns, and their clear predominance in the explants, suggests this form of activity as the closest to “true spontaneous activity” attributable to endogenous threshold fluctuations andlor spontaneous transmitter leakage into the synaptic cleft. A choice between these two possibilities by statistical analysis of firing patterns appears unfeasible because regularity of firing may be explained by periodicity in a pacemaker mechanism, or by regularity of the recovery process in the postimpulse period after spikes evoked by transmitter leakage. From these hypotheses, quasi-regular firing patterns may be considered as the primary ones in the absence of any manifest inhibitory or excitatory modulating influences. Therefore, firing patterns of the Poisson type result from many random influences upon the initial (regular) activity. In our view, i t should be possible t o settle this question by experiments with substances which selectively block synaptic mechanisms (e.g., Mg2+). The considerable number of distributions in both series with maxima in the region of 10-20 msec reflects the decrease in firing threshold during the corresponding postimpulse period, possibly related t o prolonged depolarization, lasting up to 20 msec in hippocampal pyramidal neurons (Kandel and Spencer, 1964). In particular, this factor is dominant for the most general case of firing patterns with limited after-effect. Formation of highly complex firing patterns characteristic of neurons in situ, and referred to as quasi-periodic, is influenced in part by organized synaptic activity initiated by extrahippocampal afferents. Analysis of the differences in statistical characteristics of hippocampal neurons in situ and in vitro is ambiguous due to the probable involvement of competing mechanisms. Thus, deafferentation of the hippocampus in uitro deprives its neurons of most excitatory and inhibitory extrahippocampal influences, and possibly changes its excitability. In addition, hippocampal pyramidal neurons may also be physiologically altered by the physicochemical environment in culture. To make these hypotheses more precise, and t o detect subtler differences by statistical analysis of background activity, it seem reasonable to simplify further the neural tissue culture experimental model, in addition t o perfecting analytical methods. Such simplification may be achieved by selective blocking of synaptic transmission in hippocampal explants, by utilizing cultures a t different stages of synaptogenesis, or by the use of cultures of dissociated and reaggregated cerebral neurons (Bornstein and Model, 1972; Crain and Bornstein, 1972,1974; Zipser, Crain, and Bornstein, 1973; Nelson and Peacock, 1973; Crain, Raine, and Bornstein, 1975). REFERENCES
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