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The preservation of nerve cells in rat neostriatal slices maintained in vitro: A morphological study
Brain Research, 197 (1980) 341-353 © Elsevier/North-Holland Biomedical Press
341
T H E PRESERVATION OF NERVE CELLS IN RAT N E O S T R I A T A L SLICES M A I N T A I N E D IN VITRO: A M O R P H O L O G I C A L STUDY
1L JIN BAK, ULRICH MISGELD*, MOLLY WEILER and ELLEN MORGAN ( I.J.B., M. VV. and E.M.) Department of Neurology, Reed Neurological Research Center and ( I.J.B. and M. 14/.) Department of Pharmacology, UCLA School of Medicine, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted March 27th, 1980) Key words: brain slice -- striatum -- functional and morphological preservation of neurons
SUMMARY The in vitro preservation of neurons in 300 #m thin neostriatal slices, which are routinely used for electrophysiological studies, was examined by light and electron microscopy and was compared to 700 #m thick neostriatal slices. The thin slices displayed well-preserved cells after up to 5 h of incubation. This finding correlated well with whether electrical activity could be recorded. In crosssection, the thin slices consisted of three layers: the inner layer contained many intact cells (80 ~ ) and was sandwiched between the outer layers where deteriorating cells predominated. In contrast to the thin slices, the thick slices (700 #m) displayed no layering of intact cells in cross-section. Instead, the majority of cells throughout these thick slices was swollen (98 ~), with only small patches of intact cells. Two types of deteriorating cells were apparent: swollen cells and dark (pycnotic) cells. The proportion of swollen cells increased with incubation time. In the thin slices this swelling occurred in the outer layers with the middle layer of intact cells remaining relatively unchanged over long incubation periods, whereas all cells in the thick slices were swollen after 2 h of incubation. Dark cells were localized to the outer portion of both slices and the number of such dark cells did not change with incubation time.
INTRODUCFION A variety of studies using intracellular recording techniques have demonstrated that brain slice preparations aa are valuable tools for in vitro electrophysiological * On leave of absence from: Max-Planck-Institut for Hirnforschung, Department of Neurobiology, Deutschordenstrasse 46, 6000 Frankfurt/M.-71, F.R.G.
342 studies. In the hippocampal slice preparation, tbr example, extensive studies of CA I neurons indicate that the neurons sustain their functional features in the in vitro environment26 zs. Many studies have been done on the neurochemical aspects of brain slices6,S,la, 16. Stemming from our long-standing interest in the ultrastructure of the neostriatumLa we decided to study the ultrastructure of neostriatal slices maintained in vitro in parallel with electrophysiological studies of these slices. We have l\~und neostriatal slices of rat to be suitable preparations in which to study intrinsic cholinergic neurotransmission in the neostriatum using electrophysiological, pharmacological and biochemical approaches20-23, ~°. The aim of the present investigation was to determine (1) whether the observed functional preservation of neostriatal neurons 2z finds its counterpart in ultrastructural integrity; (2) in which morphological characteristics neurons in neostriatal slices maintained in an artificial environment differ from 'normal' neurons (i.e. neurons as they appear in perfused neostriatal tissue) and (3) whether conditions optimal for electrophysiological recording are also optimal in terms of ultrastructural preservation. MATERIALS AND METHODS Sprague-Dawley albino rats, weighing an average of 200 g were used throughout the entire study. Rats were decapitated with a guillotine, the cranium opened and the whole brain removed. The brain tissue was prepared by cross-cutting at the level of the anterior commissure. The exposed cross-cut surface was further trimmed by removing the nucleus accumbens and the septal portion of the striatum. For acetylcholine measurement the adjacent cortex was also removed. The remaining striatal block was cross-sectioned using a glass guide. Slices were prepared at two different thicknesses, one at 300/~m and the other at 700/~m. The entire slice preparation takes less than 10 min. These slices were preincubated at 36 °C in oxygenated Krebs-Ringer bicarbonate solution (mM concentrations: NaCI 124; KC1 5.1 ; MgSO4 1.3; K H2PO4 1.22; NaHCO3 25.5; CaCle 2.3; and glucose 10.2). Electrical recording was done on the preincubated thick (700 btm) and thin (300 #m) slices after 0 min, 10 min, 1, 2, 4 or 5 h of incubation. All slices, after incubation and monitoring of electrical activities 19, were fixed in 1.25~ glutaraldehyde/1 ~ paraformaldehyde fixative for 3 h and post-fixed in 1 ~ OsO4 with 1.5 ~ potassium ferrocyanide or 1 ~ OsO4 followed by 2 ~ uranyl acetate en bloc staining. The fixed tissue samples were then dehydrated and embedded in an Araldite mixture and polymerized at 60 °C overnight. Polymerized blocks were semiand ultrathin sectioned for light and electron microscopy. Most blocks were cut so that the slice was cross-sectioned. Representative blocks from both 300 ~m and 700 /~m slices were cut horizontally. One/~m semithin serial sections were made horizontally. All sections were stained with Geimsa. In order to study the distribution of intact and morphologically changed neurons, every 25th section from the horizontal series was observed under the light microscope and the entire cell population in 1.2 sq.mm was counted and classified into intact neurons, swollen neurons and dark
343 neurons. Data is expressed in percentages of total number of cells in each section. The acetylcholine (ACh) concentration in the neostriatal slices after 1 h incubation was determined by combined gas chromatography/mass spectrometryt2,17. RESULTS
In order to determine the optimal conditions for slice preparation, two thicknesses of neostriatal slices, 300 #m and 700 #m, were compared morphologically and physiologically. Electrical activity as recorded extracellularly and intracellularly was correlated with the light and electron microscopic appearance of cells in the slices. For descriptive purposes we will refer to the 300 tzm slices as thin slices and the 700 #m slices as thick slices. The slices were examined for the presence of the typical field potential to judge the integrity of neuronal elements in the slices. The field potential has been shown to consist of two negative deflections, i.e. an antidromic and an orthodromic population spike 22. With physiological techniques a more detailed insight could be obtained by intracellular recording (Table I): the cells appeared to be well polarized according to their resting membrane potentials (RMP). Local stimulation triggered synaptically driven single action potentials and some antidromic spikes. The cells did not fire spontaneously. The membrane time constant measured in a few cells from small depolarizing and hyperpolarizing pulses was found to be almost equal to the decay time constant of the EPSP. We therefore consider the time constant of the EPSP as an approximate indication for the value of the passive membrane time constant. The mean time constant of 9.4 msec is comparable to the value reported from in vivo experiments2L Normal evoked field responses, extracellular spikes, and intracellular recording could be obtained from a central layer of the thin slices. This layer was about 100 #m thick. Normal electrophysiological responses of these three types were less easy and less common to find from the top and bottom layers of the thin slices. In a few thin slices, the normal physiological responses were few or absent. In these slices there were few normal-appearing nerve cells and much swelling (see below). TABLE I
Characteristics o f neurons in the neostriatal slices recorded intracelhdarly Values are the mean value and standard deviation obtained from 31 neurons. For the resting membrane potential, minimum was 40 and maximum 67 inV.
( A t resting membrane potential 50 m V) Action potential
Amplitude Orthodromic Antidromic Threshold voltage
53 =k 10.4 mV 100~ 26 ~ 7.9 d: 2.9 mV
EPSP
Latency Amplitude Duration Time to peak Time constant
2.0 7.3 17.7 3.8 9.4
i ± ± :k ±
0.6 msec 2.8 mV 5.9 msec 1 msec 3.3 msec
344
Fig. I. Light micrograph taken from horizontal section of the middle layer of a 300/~m thin slice incubated for 2 h, demonstrating well-preserved neostriatal cells. FB, fiber bundle in cross-section; arrows, dark cells. Fig. 2. Light micrograph taken from horizontal section of the middle area of a 700/~m thick slice incubated for 2 h, demonstrating all swollen cells with large cytoplasmic vacuoles and shrunken nuclei. Note swollen neuropile.
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Fig. 3. Distribution of intact, swollen, and dark cells throughout the entire thickness of thin (A) and thick (B) neostriatal slices. Data was expressed in percentage of the three cell types after cell counts of every 25th section from horizontal serial section of 300/zm and 700/~m slices. --, intact cells; . . . . , swollen cells; . . . . , dark cells. Normal physiological responses were infrequently obtained from the thick slices at any depth of recording. When one normal unitary response was found, it was not uncommon to find another neuron close by. Morphologically, the description of a neuron in a slice bathed in an artificial medium as being 'well-preserved' or 'intact' has to be delineated by comparison to cells in neostriatal tissue obtained from perfused animals described in earlier studies 1. Intact neurons in the slice can be defined morphologically by the following characteristics: (a) lack of swelling or shrinkage of their overall sizes (the average diameter of the cells in our preparations was 15-20 #m) (Fig. 1), (b) homogeneous nucleoplasm with distinct nucleoli, (c) mitochondria with parallel cristae and dense mitochondrial matrix, (d) manifest cell organelles (endoplasmic reticulum, Golgi apparatus) in the cytoplasmic matrix, and (e) smooth and uninterrupted cell and nuclear membranes (Fig. 5).
Thin slices The group of 300 # m slices demonstrated well-preserved cells, which in crosssections of the slice were localized in the middle layer (Fig. 4). A fewer number of intact cells was seen in both outer layers where more swollen cells and darkened cells were found. Neuropil in the inner layer was not swollen compared to the surface layers (Fig. 4). Neuropil swelling in the outer layers appeared as vacuoles which were identified as glial cell processes and less frequently as dendritic processes.
346 i
Fig. 4. Low magnification light micrograph prepared from a cross-section of a 300/tin thin neostrialal slice incubated for 2 h, demonstrating a middle layer of intact cells and well-preservedneuropile sandwiched by outer layers consisting of dark cells, swollen cells and swollen neuropile. Vertical arrow, dark cells; horizontal arrow, swollen cells. In serial horizontal sections more detailed information was obtained concerning the distribution of intact and abnormal cells. Of the total number of cells in the middle layer 88 ~ were intact (Fig. 3). This middle layer measures 100 # m in the center of the slice (Fig. 4). In both surface layers most of the cells were swollen. Besides the swollen cells, there were a number of dark, shrunken cells in the surface layers and a few such dark cells in the middle layer (Figs. 3 and 4). Electron microscopic examination of the middle layer revealed that the majority of cells had well-preserved cytoplasmic organelles such as mitochondria in the perikaryon (Fig. 5a). The nucleoplasm was also well preserved, showing the presence of a nucleolus and evenly distributed chromatin (Fig. 5a). In the neuropil there were well preserved neuronal elements and there were numerous nerve terminals synapsing on dendrites and more frequently on dendritic spines (Fig. 5b). Post-synaptic thickenings were apparent. On the pre-synaptic side there were synaptic vesicles, some closely associated, some fused to the membrane. The synaptic vesicles were mainly spherical and measured an average of 50 nm in diameter (Fig. 5c). The middle layer also had some swollen axonal, dendritic and glial cell processes, and in such elements we noted swollen mitochondria and various vacuolization. Such morphological changes were more frequent in the surface layer (Fig. 4).
Fig. 5. A: an electron micrograph taken from a 300 #m thin slice incubated for 2 h, demonstrating a well-preserved neostriatal medium-sized cell and surrounding neuropile. N, nucleus; No, nucleolus; G, golgi; M, mitochondria; D, dendrite. B: an electron micrograph taken from the middle layer of a 300/~m slice incubated for 2 h, demonstrating well-preserved neuropile with many axo-spinous synapses and clear post-synaptic thickenings. C: a high magnification electron micrograph prepared from a 300 /tm thin neostriatal slice incubated for 2 h, demonstrating well-preserved synapses. Post-synaptie thickening is apparent and numerous synaptic vesicles are present in the nerve terminal.
348
Thick slices A massive swelling effect was seen in the 700/~m slices. Most of the cells were swollen and only small patches of intact cells were observed. This was especially obvious in the horizontal serial sections (Fig. 2). Most of the intact cells seen in the thick slices were found at a depth of 50-150/~m and very few were localized in the middle portion of the slice (Fig. 3). This was about the same depth at which intact cells were found in the 3130/zm slices. This zone of patches of intact cells was often found in only one side of the 7130/zm slice rather than both sides. The rest of the slice contained 95-98 ~ swollen cells and some darkened cells (Fig. 3). Electron micrographs of the 700/~m slice further depicted swollen cells which were approximately twice as large as normal cells and had large cytoplasmic vacuoles. These cytoplasmic vacuoles were sometimes membrane bound (Fig. 6). Mitochondria and Golgi apparatuses were swollen with loss of their internal matrix (Fig. 6). The nuclei were markedly reduced in size with irregular nuclear membranes, and in the nucleoplasm chromatin was more aggregated making several patches. These characteristics were also clearly seen in low power light micrographs (Fig. 2). Alteration of the incubation temperature from 36 °C to room temperature (25 °C) did not improve the morphological appearance of the thick slices (700/zm). With extracellular recording, the thick slices showed very little electrical activity, and this was true for those maintained at room temperature or at 36 °C. Field potentials could be monitored only in very restricted portions within the tissue, the maximal amplitudes of the population spikes approached only 0.5 mV contrasting with the 1 2 mV amplitudes in thin slices. The incubation effects on the thick and thin slices were studied at varying incubation times: 0, 10, 60, 120 and 300 min. Each slice was checked for field potential just prior to fixation, except 0 and 10 rain 300 # m and 700 /zm slices. The 0 rain group, which was fixed immediately after removal from the brain, demonstrated no swollen cells. Both thick and thin slices, however, showed dark cells, mostly localized in the outer layers. These dark cells did not alter in number with prolonged incubation time. Most of the dark cells were pycnotic with electron-dense cytoplasm (Fig. 7). A second type of dark cell had moderately darkened cytoplasm without a change in cell contour and a third type was extremely shrunken and fragmented and often engulfed by glial cells. After 10 rain of incubation, both slices had minor swelling of some cell bodies and neuropil only in surface areas. In the 60 min incubation group, the 300/zm slices began to show a slightly swollen neuropil with darkened cells and swollen cells in both outer layers. Under low power microscopy, a reasonably normal central layer could be seen sandwiched between the pathological layers. Thin slices incubated for 120 min depicted few changes as compared to the 60 min group, except that the outer layers showed slightly more swollen neuropils (Fig. 4). With prolonged incubation, (4-5 h) the thin slice groups show very little change from the 2 h incubation group with a surprising number of intact neurons in the inner layer. The 700/~m thick slice group incubated for 60 min or 120 min showed swelling throughout the section and in the 120 min incubation group the slices showed an overall increase in swollen cells and neuropil relative to the 60 rain incubation.
349
Fig. 6. An electron micrograph prepared from a 700 ~ m thick slice incubated for 2 h, demonstrating a swollen cell with large cytoplasmic vacuoles. V, cytoplasmic vacuoles. Fig. 7. An electron micrograph prepared from a 300/~m thin neostriatal slice incubated for 2 h, demonstrating a dark cell with shrunken nucleus, wrinkled nuclear and cell membranes and darkened cytoplasm.
350 Thin and thick slices were also compared for their acetylcholine (ACh) levels since it had been found that neostriatal slices suitable for recording electrical activity can synthesize ACh to achieve concentration up to 10-fold higher than those in found in vivo 3o. For the thick slice at 36 °C after 1 h incubation, the mean ACh level was 3.85 :!: 0.25 retool/rag protein (n = 7), and at 25 :C the ACh level was 2.16 ~ 0.23 nmol/mg protein (n == 4). In the thin slices the ACh level after 1 h incubation was 8.34 ± 1.63 nmol/mg protein at 36 °C and at 25 °C the level was 3.16 :t: 0.35 nmol/mg protein (n = 4). Thus, the ACh level in the thick slices was lower than that in thin slices at either of the two temperatures (36 ~'C and 25 °C). DISCUSSION The present results indicate that neurons of neostriatal tissue can be well preserved in vitro if slices are appropriately prepared and maintained so that electrical potentials can be evoked by local stimulation. Two types of neuronal deterioration, however, were found, i.e. 'swollen' neurons and 'dark, pycnotic' neurons. Some cues for the understanding of the factors possibly responsible were seen in the distribution pattern of well preserved and deteriorated neurons in the slices, and in the different time course of appearance of these changes. The discussion will focus on the following two aspects: (a) significance of the morphological preservation of neostriatal elements for the interpretation of physiological investigations and (b) the significance of the types of neuronal disintegration observed to the preparation and maintenance of neostriatal slices. A substantial number of cells examined by the electron microscope were found to be well preserved, using the definition of well preserved cells in terms of their ultrastructure given in the Results section. This supports the conclusion drawn from the electrophy~iological recordings. Because of the data obtained by intracellular recording 22 (Table I) it was inferred that the neurons were functionally well preserved. Further, in slices eliciting no or only poor electrical activity following the local stimulation, none or only a few cells with well preserved ultrastructure could be found. There also is correlation in the evocation of synaptic potentials by local stimulation 2~ and in the preservation of synapses in terms of their ultrastructure. The parallel between the ultrastructural and functional preservation of neuronal elements in slices is in accordance with findings of Yamamoto et al. ~2 that olfactory slices appropriately maintained to show electrical activity also preserve their morphological integrity, and that ultrastructural abnormalities are associated with suppression of electrical activity. Electrophysiological analysis revealed that local stimulation in the neostriatum slice activates synapses of intrinsic neurons 22 which use ACh as a transmitter to excite their target neurons 2t-23. So far the target neurons have not been identified. Although this question cannot be settled by the present study, morphological analysis offers some hints. Considering the intact cells only, cells of various sizes were observed, including the rare large cells z. Based on the observation of varying cell sizes, it appears unlikely that the intact cells in the slice belong to only one group of neostriatal
351 neurons. Most probably they are representatives of various groups of the neuronal subgroups which have been classified in the neostriatum 1,1°,11,1s,24. Medium-sized cells, as defined with ordinary stains and with light microscopy, consist of several types, including at least one efferent type 7. Since about 50 ~ of neostriatal neurons are efferent neurons TM, and since in the middle layers of the thin slices most neurons are intact morphologically, there is a good chance that some neurons preserved in the slice are efferent neurons. This view is also supported by the following observations: the number of intact cells found in the inner layer had not been decreased when compared to the number of neurons found in a comparable field of normal perfused tissue, and the deteriorated cells were not distributed evenly but were predominantly in the outer layers. This implies that efferent neurons can survive, at least for a while even though their axons have been severed. Thin (300 ~m) slices displayed three distinct layers. In the middle layer the intact neurons prevailed, whereas in the outer layers deteriorated cells outnumbered the intact cells (Fig. 3). Two types of deteriorated cells were distinguished, the 'swollen' cells and the 'dark, pycnotic' cells. A considerable number of dark cells was already found in slices fixed at time 0, i.e. in slices which were not incubated before fixation, and their probability of occurrence did not increase with incubation time. Further, in thick (700 #m) slices they were found in relatively the same number. These 'dark, pycnotic' cells have also been observed in tissue fixed by perfusion, especially when the tissue was badly handled 5 (Bak, personal observation). We would therefore tend to consider these changes due to mechanical stress. On the other hand, the relative number of swollen neurons increased with time of incubation. In the thick slices only a few intact cells were found close to the surface. The middle of the thick slices was composed mainly of swollen cells (Fig. 3). Therefore, we believe that swelling of neurons occurs when maintenance conditions for the slices are inappropriate, the oxygen and glucose supply probably being the most important factors. In olfactory cortex slices it was found that by reducing the temperature of the surrounding medium and thereby reducing the oxygen consumption it was possible to employ sufficiently thick slices to incorporate more complete neural circuitries and thereby to preserve inhibitory elements 15. This approach appeared not to be suitable for neostriatal slices since thick slices maintained at room temperature had neurons more swollen than neurons in thick slices maintained at 36 °C. It follows that slice thickness, incubation temperature and mechanical stress are important considerations in the study of rat neostriatal slices, and probably in the application of the slice technique to other brain tissues as well. ACKNOWLEDGEMENTS The authors are grateful to Drs. C. H. Markham and D. J. Jenden for critical comments and revision of the manuscript. This study has received support from the Scott Trust.
352 REFERENCES 1 Bak, I. J., Choi, W. B., Hassler, R., Usunoff, K. G. and Wagner, A., Fine structural synaptic organization of the corpus striatum and substantia nigra in rat and cat. In D. B. Calne, T. N. Chase and A. Barbeau (Eds.), Advances in Neurology, Vol. 9, Raven Press, New York, 1975, pp. 2541. 2 Bak, I. J., Markham, C. H., Cook, M. L. and Stevens, J. G., lntraaxonal transport of herpes simplex virus in the rat central nervous system, Brain Research, 136 (1977) 415-429. 3 Bak, I. J., Markham, D. H., Cook, M. L. and Stevens, J. G., Ultrastructural and immunoperoxidase study of strionigral neurons by means of retrograde axonal transport of herpes simplex virus, Brain Research, 143 (1978) 361-368. 4 Buchwald, N. A., Price, D. D., Vernon, L. and Hull, C. C., Caudate intracellular response to thalamic and cortical inputs, Exp. Neurol., 38 (1973) 311-323. 5 Cammermeyer, J., Is the solitary dark neuron a manifestation of postmortem trauma to the brain inadequately fixed by perfusion? Histochemistry, 56 (1978) 97-115. 6 Cohen, M. M. and Hartmann, J. F., Biochemical and ultrastructural correlates of cerebral cortex slices metabolizing in vitro. In M. M. Cohen and R. S. Snider (Eds.), MorphologicalandBioehemical Correlates of Neural Activity, Harper and Row, New York, 1964, pp. 57-74. 7 DiFiglia, M., Pasik, P. and Pasik, T., A Golgi study of neuronal types in the neostriatum of monkeys, Brain Research, 114 (1976) 245-256. 8 Elliot, K. A. C., The use of brain slices. In A. Lajtha (Ed.), Handbook ofNeurochemistry, Vol. 2, Plenum Press, New York, 1969, pp. 103 114. 9 Feltz, P. and Albe-Fessard, D., A study of an ascending nigro-caudate pathway, Electroenceph. clin. Neurophysiol., 33 ( 1972) 179-193. 10 Fox, C. A., Andrade, A. N., Hillman, D. E. and Schwyn, R. C., The spiny neurons in the primate striatum: a Golgi and electron microscopic study, J. Hirnforsch., 13 (1971/1972) 181-201. 11 Fox, C. A., Andrade, A. N., Schwyn, R. C. and Rafols, J. S., The aspiny neurons and the glia in the primate striatum: a Golgi and electron microscopic study, J. Hirnforsch., 13 (1971/1972) 341-362. 12 Freeman, J. J., Choi, R. L. and Jenden, D. J., Plasma choline, its turnover and exchange with brain choline, J. Neurochem., 24 (1975) 729-734. 13 Garthwaite, J., Woodhams, P. L., Collins, J. J. and Balazs, R., On the preparation of brain slices: morphology and cyclic nucleotides, Brain Research, 173 (1979) 373 377. 14 Grofova, 1. and Rinvik, E., An experimental electron microscopic study on the striatonigral projection in the cat, Exp. Brain Res., 11 (1970) 249 262. 15 Harvey, J. A., Scholfield, C. N. and Brown, D. A., Evoked surface-positive potentials in isolated mammalian olfactory cortex, Brahz Research, 76 (1974) 235-245. 16 Ibata, Y., Piccoli, F., Pappas, G. D. and Lajtha, A., An electron microscopic and biochemical study on the effect of cyanide and low Na + on rat brain slices, Brain Research, 30 (1971) 137-158. 17 Jenden, D. J., Roch, M. and Booth, R., Simultaneous measurements of endogenous and deuterium labelled tracer variants of choline and acetylcholine in submicrogram quantities by gas chromatography/mass spectrometry, Analyt. Biochem., 55 (1973) 438-448. 18 Kemp, J. M. and Powell, T. P. S., The structure of the caudate nucleus of the cat: Light and electron microscopy, Phil. Trans. B, 262 (1971) 383-401. 19 Mcllwain, H. and Rodnight, R., Preparing neural tissues for metabolic study in vitro. In Practical Neurochemistry, Churchill, London, 1962, pp. 109-133. 20 Misgeld, U., Intra- and extracellular study on an intrinsic cholinergic excitation in the rat striatum slice, Appl. Neurophysiol., 42 (1979) 37-39. 21 Misgeld, U. and Bak, 1. J., Intrinsic excitation in the rat neostriatum mediated by acetytcholine, Neurosci. Lett., 12 (1979) 277-282. 22 Misgeld, U., Okada, Y. and Hassler, R., Locally evoked potentials in slices of rat neostriatum : A tool for the investigation of intrinsic excitatory processes, Exp. Brain Res., 34 (1979) 575-590. 23 Misgeld, U., Weiler, M. H., Bak, I. J. and Jenden, D. J., Receptors involved in acetylcholine mediated excitation in the rat neostriatum, Pfliigers Arch. ges. Physiol., R45 (1979) 379. 24 Pasik, P., Pasik, T. and DiFiglia, M., Quantitative aspects of neuronal organization in the neostriaturn of the Macaque monkey. In M. D. Yahr (Ed.), The Basal Ganglia, Raven Press, New York, 1976, pp. 57-90. 25 Richards, C. D. and Sercombe, R., Electrical activity observed in guinea pig olfactory cortex maintained in vitro, J. Physiol. (Lond.), 197 (1968) 667-683. 26 Schwartzkroin, P. A., Characteristics of CA1 neurons recorded intracellularly in the hippocampal in vitro slice preparation, Brain Research, 85 (1975) 423-436.
353 27 Schwartzkroin, P. A., Further characteristics of hippocampal CA1 cells in vitro, Brain Research, 128 (1977) 53-68. 28 Schwartzkroin, P. A. and Mathers, L. H., Physiological and morphological identification of a nonpyramidal hippocampal cell type, Brain Research, 5 (1978) 1-10. 29 Sugimori, M., Preston, R. J. and Kitai, S. T., Response properties and electrical constants of caudate nucleus neurons in the cat, J. Neurophysiol., 41 (1978) 1662-1675. 30 Weiler, M., Misgeld, U., Bak, I. J. and Jenden, D. J., Acetylcholine synthesis in rat neostriatal slices, Brain Research, 176 (1979) 401-406. 31 Yamamoto, C., Activation of hippocampal neurons by mossy fiber stimulationin thin brain sections in vitro, Exp. Brain Res., 14 (1972) 423-435. 32 Yamamoto, C., Bak, I. J. and Kurokawa, M., Ultrastructural changes associated with reversible and irreversible suppression of electrical activity in olfactory cortex slices, Exp. Brain Res., 11 (1970) 360-372. 33 Yarnamoto, C. and Mcllwain, H., Electrical activities in thin sections from the mammalian brain maintained in chemically defined media in vitro, J. Neurochem., 13 (1966) 1333-1343.
341
T H E PRESERVATION OF NERVE CELLS IN RAT N E O S T R I A T A L SLICES M A I N T A I N E D IN VITRO: A M O R P H O L O G I C A L STUDY
1L JIN BAK, ULRICH MISGELD*, MOLLY WEILER and ELLEN MORGAN ( I.J.B., M. VV. and E.M.) Department of Neurology, Reed Neurological Research Center and ( I.J.B. and M. 14/.) Department of Pharmacology, UCLA School of Medicine, Los Angeles, Calif. 90024 (U.S.A.)
(Accepted March 27th, 1980) Key words: brain slice -- striatum -- functional and morphological preservation of neurons
SUMMARY The in vitro preservation of neurons in 300 #m thin neostriatal slices, which are routinely used for electrophysiological studies, was examined by light and electron microscopy and was compared to 700 #m thick neostriatal slices. The thin slices displayed well-preserved cells after up to 5 h of incubation. This finding correlated well with whether electrical activity could be recorded. In crosssection, the thin slices consisted of three layers: the inner layer contained many intact cells (80 ~ ) and was sandwiched between the outer layers where deteriorating cells predominated. In contrast to the thin slices, the thick slices (700 #m) displayed no layering of intact cells in cross-section. Instead, the majority of cells throughout these thick slices was swollen (98 ~), with only small patches of intact cells. Two types of deteriorating cells were apparent: swollen cells and dark (pycnotic) cells. The proportion of swollen cells increased with incubation time. In the thin slices this swelling occurred in the outer layers with the middle layer of intact cells remaining relatively unchanged over long incubation periods, whereas all cells in the thick slices were swollen after 2 h of incubation. Dark cells were localized to the outer portion of both slices and the number of such dark cells did not change with incubation time.
INTRODUCFION A variety of studies using intracellular recording techniques have demonstrated that brain slice preparations aa are valuable tools for in vitro electrophysiological * On leave of absence from: Max-Planck-Institut for Hirnforschung, Department of Neurobiology, Deutschordenstrasse 46, 6000 Frankfurt/M.-71, F.R.G.
342 studies. In the hippocampal slice preparation, tbr example, extensive studies of CA I neurons indicate that the neurons sustain their functional features in the in vitro environment26 zs. Many studies have been done on the neurochemical aspects of brain slices6,S,la, 16. Stemming from our long-standing interest in the ultrastructure of the neostriatumLa we decided to study the ultrastructure of neostriatal slices maintained in vitro in parallel with electrophysiological studies of these slices. We have l\~und neostriatal slices of rat to be suitable preparations in which to study intrinsic cholinergic neurotransmission in the neostriatum using electrophysiological, pharmacological and biochemical approaches20-23, ~°. The aim of the present investigation was to determine (1) whether the observed functional preservation of neostriatal neurons 2z finds its counterpart in ultrastructural integrity; (2) in which morphological characteristics neurons in neostriatal slices maintained in an artificial environment differ from 'normal' neurons (i.e. neurons as they appear in perfused neostriatal tissue) and (3) whether conditions optimal for electrophysiological recording are also optimal in terms of ultrastructural preservation. MATERIALS AND METHODS Sprague-Dawley albino rats, weighing an average of 200 g were used throughout the entire study. Rats were decapitated with a guillotine, the cranium opened and the whole brain removed. The brain tissue was prepared by cross-cutting at the level of the anterior commissure. The exposed cross-cut surface was further trimmed by removing the nucleus accumbens and the septal portion of the striatum. For acetylcholine measurement the adjacent cortex was also removed. The remaining striatal block was cross-sectioned using a glass guide. Slices were prepared at two different thicknesses, one at 300/~m and the other at 700/~m. The entire slice preparation takes less than 10 min. These slices were preincubated at 36 °C in oxygenated Krebs-Ringer bicarbonate solution (mM concentrations: NaCI 124; KC1 5.1 ; MgSO4 1.3; K H2PO4 1.22; NaHCO3 25.5; CaCle 2.3; and glucose 10.2). Electrical recording was done on the preincubated thick (700 btm) and thin (300 #m) slices after 0 min, 10 min, 1, 2, 4 or 5 h of incubation. All slices, after incubation and monitoring of electrical activities 19, were fixed in 1.25~ glutaraldehyde/1 ~ paraformaldehyde fixative for 3 h and post-fixed in 1 ~ OsO4 with 1.5 ~ potassium ferrocyanide or 1 ~ OsO4 followed by 2 ~ uranyl acetate en bloc staining. The fixed tissue samples were then dehydrated and embedded in an Araldite mixture and polymerized at 60 °C overnight. Polymerized blocks were semiand ultrathin sectioned for light and electron microscopy. Most blocks were cut so that the slice was cross-sectioned. Representative blocks from both 300 ~m and 700 /~m slices were cut horizontally. One/~m semithin serial sections were made horizontally. All sections were stained with Geimsa. In order to study the distribution of intact and morphologically changed neurons, every 25th section from the horizontal series was observed under the light microscope and the entire cell population in 1.2 sq.mm was counted and classified into intact neurons, swollen neurons and dark
343 neurons. Data is expressed in percentages of total number of cells in each section. The acetylcholine (ACh) concentration in the neostriatal slices after 1 h incubation was determined by combined gas chromatography/mass spectrometryt2,17. RESULTS
In order to determine the optimal conditions for slice preparation, two thicknesses of neostriatal slices, 300 #m and 700 #m, were compared morphologically and physiologically. Electrical activity as recorded extracellularly and intracellularly was correlated with the light and electron microscopic appearance of cells in the slices. For descriptive purposes we will refer to the 300 tzm slices as thin slices and the 700 #m slices as thick slices. The slices were examined for the presence of the typical field potential to judge the integrity of neuronal elements in the slices. The field potential has been shown to consist of two negative deflections, i.e. an antidromic and an orthodromic population spike 22. With physiological techniques a more detailed insight could be obtained by intracellular recording (Table I): the cells appeared to be well polarized according to their resting membrane potentials (RMP). Local stimulation triggered synaptically driven single action potentials and some antidromic spikes. The cells did not fire spontaneously. The membrane time constant measured in a few cells from small depolarizing and hyperpolarizing pulses was found to be almost equal to the decay time constant of the EPSP. We therefore consider the time constant of the EPSP as an approximate indication for the value of the passive membrane time constant. The mean time constant of 9.4 msec is comparable to the value reported from in vivo experiments2L Normal evoked field responses, extracellular spikes, and intracellular recording could be obtained from a central layer of the thin slices. This layer was about 100 #m thick. Normal electrophysiological responses of these three types were less easy and less common to find from the top and bottom layers of the thin slices. In a few thin slices, the normal physiological responses were few or absent. In these slices there were few normal-appearing nerve cells and much swelling (see below). TABLE I
Characteristics o f neurons in the neostriatal slices recorded intracelhdarly Values are the mean value and standard deviation obtained from 31 neurons. For the resting membrane potential, minimum was 40 and maximum 67 inV.
( A t resting membrane potential 50 m V) Action potential
Amplitude Orthodromic Antidromic Threshold voltage
53 =k 10.4 mV 100~ 26 ~ 7.9 d: 2.9 mV
EPSP
Latency Amplitude Duration Time to peak Time constant
2.0 7.3 17.7 3.8 9.4
i ± ± :k ±
0.6 msec 2.8 mV 5.9 msec 1 msec 3.3 msec
344
Fig. I. Light micrograph taken from horizontal section of the middle layer of a 300/~m thin slice incubated for 2 h, demonstrating well-preserved neostriatal cells. FB, fiber bundle in cross-section; arrows, dark cells. Fig. 2. Light micrograph taken from horizontal section of the middle area of a 700/~m thick slice incubated for 2 h, demonstrating all swollen cells with large cytoplasmic vacuoles and shrunken nuclei. Note swollen neuropile.
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Fig. 3. Distribution of intact, swollen, and dark cells throughout the entire thickness of thin (A) and thick (B) neostriatal slices. Data was expressed in percentage of the three cell types after cell counts of every 25th section from horizontal serial section of 300/zm and 700/~m slices. --, intact cells; . . . . , swollen cells; . . . . , dark cells. Normal physiological responses were infrequently obtained from the thick slices at any depth of recording. When one normal unitary response was found, it was not uncommon to find another neuron close by. Morphologically, the description of a neuron in a slice bathed in an artificial medium as being 'well-preserved' or 'intact' has to be delineated by comparison to cells in neostriatal tissue obtained from perfused animals described in earlier studies 1. Intact neurons in the slice can be defined morphologically by the following characteristics: (a) lack of swelling or shrinkage of their overall sizes (the average diameter of the cells in our preparations was 15-20 #m) (Fig. 1), (b) homogeneous nucleoplasm with distinct nucleoli, (c) mitochondria with parallel cristae and dense mitochondrial matrix, (d) manifest cell organelles (endoplasmic reticulum, Golgi apparatus) in the cytoplasmic matrix, and (e) smooth and uninterrupted cell and nuclear membranes (Fig. 5).
Thin slices The group of 300 # m slices demonstrated well-preserved cells, which in crosssections of the slice were localized in the middle layer (Fig. 4). A fewer number of intact cells was seen in both outer layers where more swollen cells and darkened cells were found. Neuropil in the inner layer was not swollen compared to the surface layers (Fig. 4). Neuropil swelling in the outer layers appeared as vacuoles which were identified as glial cell processes and less frequently as dendritic processes.
346 i
Fig. 4. Low magnification light micrograph prepared from a cross-section of a 300/tin thin neostrialal slice incubated for 2 h, demonstrating a middle layer of intact cells and well-preservedneuropile sandwiched by outer layers consisting of dark cells, swollen cells and swollen neuropile. Vertical arrow, dark cells; horizontal arrow, swollen cells. In serial horizontal sections more detailed information was obtained concerning the distribution of intact and abnormal cells. Of the total number of cells in the middle layer 88 ~ were intact (Fig. 3). This middle layer measures 100 # m in the center of the slice (Fig. 4). In both surface layers most of the cells were swollen. Besides the swollen cells, there were a number of dark, shrunken cells in the surface layers and a few such dark cells in the middle layer (Figs. 3 and 4). Electron microscopic examination of the middle layer revealed that the majority of cells had well-preserved cytoplasmic organelles such as mitochondria in the perikaryon (Fig. 5a). The nucleoplasm was also well preserved, showing the presence of a nucleolus and evenly distributed chromatin (Fig. 5a). In the neuropil there were well preserved neuronal elements and there were numerous nerve terminals synapsing on dendrites and more frequently on dendritic spines (Fig. 5b). Post-synaptic thickenings were apparent. On the pre-synaptic side there were synaptic vesicles, some closely associated, some fused to the membrane. The synaptic vesicles were mainly spherical and measured an average of 50 nm in diameter (Fig. 5c). The middle layer also had some swollen axonal, dendritic and glial cell processes, and in such elements we noted swollen mitochondria and various vacuolization. Such morphological changes were more frequent in the surface layer (Fig. 4).
Fig. 5. A: an electron micrograph taken from a 300 #m thin slice incubated for 2 h, demonstrating a well-preserved neostriatal medium-sized cell and surrounding neuropile. N, nucleus; No, nucleolus; G, golgi; M, mitochondria; D, dendrite. B: an electron micrograph taken from the middle layer of a 300/~m slice incubated for 2 h, demonstrating well-preserved neuropile with many axo-spinous synapses and clear post-synaptic thickenings. C: a high magnification electron micrograph prepared from a 300 /tm thin neostriatal slice incubated for 2 h, demonstrating well-preserved synapses. Post-synaptie thickening is apparent and numerous synaptic vesicles are present in the nerve terminal.
348
Thick slices A massive swelling effect was seen in the 700/~m slices. Most of the cells were swollen and only small patches of intact cells were observed. This was especially obvious in the horizontal serial sections (Fig. 2). Most of the intact cells seen in the thick slices were found at a depth of 50-150/~m and very few were localized in the middle portion of the slice (Fig. 3). This was about the same depth at which intact cells were found in the 3130/zm slices. This zone of patches of intact cells was often found in only one side of the 7130/zm slice rather than both sides. The rest of the slice contained 95-98 ~ swollen cells and some darkened cells (Fig. 3). Electron micrographs of the 700/~m slice further depicted swollen cells which were approximately twice as large as normal cells and had large cytoplasmic vacuoles. These cytoplasmic vacuoles were sometimes membrane bound (Fig. 6). Mitochondria and Golgi apparatuses were swollen with loss of their internal matrix (Fig. 6). The nuclei were markedly reduced in size with irregular nuclear membranes, and in the nucleoplasm chromatin was more aggregated making several patches. These characteristics were also clearly seen in low power light micrographs (Fig. 2). Alteration of the incubation temperature from 36 °C to room temperature (25 °C) did not improve the morphological appearance of the thick slices (700/zm). With extracellular recording, the thick slices showed very little electrical activity, and this was true for those maintained at room temperature or at 36 °C. Field potentials could be monitored only in very restricted portions within the tissue, the maximal amplitudes of the population spikes approached only 0.5 mV contrasting with the 1 2 mV amplitudes in thin slices. The incubation effects on the thick and thin slices were studied at varying incubation times: 0, 10, 60, 120 and 300 min. Each slice was checked for field potential just prior to fixation, except 0 and 10 rain 300 # m and 700 /zm slices. The 0 rain group, which was fixed immediately after removal from the brain, demonstrated no swollen cells. Both thick and thin slices, however, showed dark cells, mostly localized in the outer layers. These dark cells did not alter in number with prolonged incubation time. Most of the dark cells were pycnotic with electron-dense cytoplasm (Fig. 7). A second type of dark cell had moderately darkened cytoplasm without a change in cell contour and a third type was extremely shrunken and fragmented and often engulfed by glial cells. After 10 rain of incubation, both slices had minor swelling of some cell bodies and neuropil only in surface areas. In the 60 min incubation group, the 300/zm slices began to show a slightly swollen neuropil with darkened cells and swollen cells in both outer layers. Under low power microscopy, a reasonably normal central layer could be seen sandwiched between the pathological layers. Thin slices incubated for 120 min depicted few changes as compared to the 60 min group, except that the outer layers showed slightly more swollen neuropils (Fig. 4). With prolonged incubation, (4-5 h) the thin slice groups show very little change from the 2 h incubation group with a surprising number of intact neurons in the inner layer. The 700/~m thick slice group incubated for 60 min or 120 min showed swelling throughout the section and in the 120 min incubation group the slices showed an overall increase in swollen cells and neuropil relative to the 60 rain incubation.
349
Fig. 6. An electron micrograph prepared from a 700 ~ m thick slice incubated for 2 h, demonstrating a swollen cell with large cytoplasmic vacuoles. V, cytoplasmic vacuoles. Fig. 7. An electron micrograph prepared from a 300/~m thin neostriatal slice incubated for 2 h, demonstrating a dark cell with shrunken nucleus, wrinkled nuclear and cell membranes and darkened cytoplasm.
350 Thin and thick slices were also compared for their acetylcholine (ACh) levels since it had been found that neostriatal slices suitable for recording electrical activity can synthesize ACh to achieve concentration up to 10-fold higher than those in found in vivo 3o. For the thick slice at 36 °C after 1 h incubation, the mean ACh level was 3.85 :!: 0.25 retool/rag protein (n = 7), and at 25 :C the ACh level was 2.16 ~ 0.23 nmol/mg protein (n == 4). In the thin slices the ACh level after 1 h incubation was 8.34 ± 1.63 nmol/mg protein at 36 °C and at 25 °C the level was 3.16 :t: 0.35 nmol/mg protein (n = 4). Thus, the ACh level in the thick slices was lower than that in thin slices at either of the two temperatures (36 ~'C and 25 °C). DISCUSSION The present results indicate that neurons of neostriatal tissue can be well preserved in vitro if slices are appropriately prepared and maintained so that electrical potentials can be evoked by local stimulation. Two types of neuronal deterioration, however, were found, i.e. 'swollen' neurons and 'dark, pycnotic' neurons. Some cues for the understanding of the factors possibly responsible were seen in the distribution pattern of well preserved and deteriorated neurons in the slices, and in the different time course of appearance of these changes. The discussion will focus on the following two aspects: (a) significance of the morphological preservation of neostriatal elements for the interpretation of physiological investigations and (b) the significance of the types of neuronal disintegration observed to the preparation and maintenance of neostriatal slices. A substantial number of cells examined by the electron microscope were found to be well preserved, using the definition of well preserved cells in terms of their ultrastructure given in the Results section. This supports the conclusion drawn from the electrophy~iological recordings. Because of the data obtained by intracellular recording 22 (Table I) it was inferred that the neurons were functionally well preserved. Further, in slices eliciting no or only poor electrical activity following the local stimulation, none or only a few cells with well preserved ultrastructure could be found. There also is correlation in the evocation of synaptic potentials by local stimulation 2~ and in the preservation of synapses in terms of their ultrastructure. The parallel between the ultrastructural and functional preservation of neuronal elements in slices is in accordance with findings of Yamamoto et al. ~2 that olfactory slices appropriately maintained to show electrical activity also preserve their morphological integrity, and that ultrastructural abnormalities are associated with suppression of electrical activity. Electrophysiological analysis revealed that local stimulation in the neostriatum slice activates synapses of intrinsic neurons 22 which use ACh as a transmitter to excite their target neurons 2t-23. So far the target neurons have not been identified. Although this question cannot be settled by the present study, morphological analysis offers some hints. Considering the intact cells only, cells of various sizes were observed, including the rare large cells z. Based on the observation of varying cell sizes, it appears unlikely that the intact cells in the slice belong to only one group of neostriatal
351 neurons. Most probably they are representatives of various groups of the neuronal subgroups which have been classified in the neostriatum 1,1°,11,1s,24. Medium-sized cells, as defined with ordinary stains and with light microscopy, consist of several types, including at least one efferent type 7. Since about 50 ~ of neostriatal neurons are efferent neurons TM, and since in the middle layers of the thin slices most neurons are intact morphologically, there is a good chance that some neurons preserved in the slice are efferent neurons. This view is also supported by the following observations: the number of intact cells found in the inner layer had not been decreased when compared to the number of neurons found in a comparable field of normal perfused tissue, and the deteriorated cells were not distributed evenly but were predominantly in the outer layers. This implies that efferent neurons can survive, at least for a while even though their axons have been severed. Thin (300 ~m) slices displayed three distinct layers. In the middle layer the intact neurons prevailed, whereas in the outer layers deteriorated cells outnumbered the intact cells (Fig. 3). Two types of deteriorated cells were distinguished, the 'swollen' cells and the 'dark, pycnotic' cells. A considerable number of dark cells was already found in slices fixed at time 0, i.e. in slices which were not incubated before fixation, and their probability of occurrence did not increase with incubation time. Further, in thick (700 #m) slices they were found in relatively the same number. These 'dark, pycnotic' cells have also been observed in tissue fixed by perfusion, especially when the tissue was badly handled 5 (Bak, personal observation). We would therefore tend to consider these changes due to mechanical stress. On the other hand, the relative number of swollen neurons increased with time of incubation. In the thick slices only a few intact cells were found close to the surface. The middle of the thick slices was composed mainly of swollen cells (Fig. 3). Therefore, we believe that swelling of neurons occurs when maintenance conditions for the slices are inappropriate, the oxygen and glucose supply probably being the most important factors. In olfactory cortex slices it was found that by reducing the temperature of the surrounding medium and thereby reducing the oxygen consumption it was possible to employ sufficiently thick slices to incorporate more complete neural circuitries and thereby to preserve inhibitory elements 15. This approach appeared not to be suitable for neostriatal slices since thick slices maintained at room temperature had neurons more swollen than neurons in thick slices maintained at 36 °C. It follows that slice thickness, incubation temperature and mechanical stress are important considerations in the study of rat neostriatal slices, and probably in the application of the slice technique to other brain tissues as well. ACKNOWLEDGEMENTS The authors are grateful to Drs. C. H. Markham and D. J. Jenden for critical comments and revision of the manuscript. This study has received support from the Scott Trust.
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