The transverse hippocampal slice,: a well-defined ... - Science Direct

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The transverse hippocampal slice,: a well-defined cortical structure maintained in vitro It has proved possible to evoke both population and unit potentials by electrical stimulation of slices prepared from the piriform cortex 17,1s,2z or the fascia dentata6,20, 21 and maintained in vitro. Recent evidence suggests that the hippocampus and the fascia dentata are organized in a series of parallel lamellae oriented transversely to the longitudinal axis of the hippocampusl,Z,5,10. These investigations suggested to us the possibility of isolating and studying such lamellae in vitro. The present paper is a description of this preparation, which has the advantages that the layers of the hippocampus and the fascia dentata are well preserved, and certain main afferent and intrinsic pathways can be easily identified (Fig. 1A). Thirty-nine guinea-pigs and 2 rabbits were used. The animals were stunned by a blow on the neck and killed by a second blow on the chest. The brain was removed and slices of 300-500 #m thickness were prepared from the hippocampus and the fascia dentata using a razor blade and a cutting guide 13. The slices were cut nearly perpendicularly to the longitudinal axis of the hippocampus, and efforts were made to keep the plane of section parallel to that of the hippocampal lamellal, 3. Less than 5 rain after the animal was killed the slices were placed in a constant-flow incubation chambed s on a silk mesh in artificial cerebrospinal fluid saturated with 95 ~ 02 and 5 ~ CO2. The same gas mixture was moistened and passed over the upper surface of the preparation. The composition of the fluid was (mM): NaC1, 134; KC1, 5; KHzPO4, 1.24; MgSO4, 1.3; CaC12, 0.75; NaHCOz, 16; and glucose, 10. Stimuli were delivered through a pair of lacquer-insulated tungsten electrodes with tip separation of less than 0.3 ram. The responses were recorded with conventional glass micropipettes and referred to an indifferent silver wire electrode which was immersed in the bathing medium and connected to ground. From each animal 4-5 slices were prepared. In all experiments population potentials were evoked in at least one slice. In general, activity could be evoked for at least 8-12 h. Fig. 1A shows a photograph of an unstained hippocampal slice. In the schematic drawing in Fig. 1B, the main pathways under study are heavily outlined. According to Ramdn y CajaP 5,16 and Lorente de N612, the hippocampal formation contains a fourmembered neuronal chain consisting of (1) entorhinal cells, (2) dentate granule cells, (3) CA 3 and (4) CA 1 pyramidal cells. Their respective axons form the following sequentially activated pathways: the perforant path (pp), the mossy fibres (mf), the Schaffer collaterals (Sch) and the alveus (alv). In the slices, such as that shown in Fig. 1A, the following structures can be identified by simple inspection: (1) the granule (gc) and the pyramidal (pyr) cell body layers; (2) the myelinated fibres of the alveus (alv); the perforant path (pp) and the Schaffer collaterals (Sch), appearing as white bundles of different density; (3) the hippocampal fissure (fiss); and (4) the fimbria (tim), here being torn off on the left side. Using the structures listed above as landmarks, the pathways of the four-membered neuronal chain could be stimulated selectively and the evoked activity recorded Brain Research, 35 (1971) 589-593

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Fig. 1. A, Photograph of a surface illuminated unstained guinea-pig transverse hippocampal slice. The border line between the hippocampal CA 3 and CA 1 areas is marked by an arrow. This borderline was sometimes more easily seen when a few drops of methyl blue, dissolved in artificial cerebrospinal fluid, were applied to the surface of the preparation. The procedure did not alter the evoked potentials, but sometimes facilitated the identification of the cell body layers. The outline of some individual cells on the surface of the slice could then be seen with high magnification. B, Schematic drawing of the slice, with the main pathways heavily outlined. Abbreviations: alv, alveus; tim, fimbria; fiss, hippocampal fissure; gc, granule cell body layer; mf, mossy fibres; pp, perforant path; pyr, pyramidal cell body layer; Sch, Schaffer collaterals.

either f r o m the cell body layers of the g r a n u l e or the p y r a m i d a l ceils, or from different levels along their dendrites. I n this way it was possible (l) to excite granule cells, a n d C A 3 a n d CA 1 p y r a m idal cells m o n o s y n a p t i c a l l y by stimulating the p e r f o r a n t path, the mossy fibres a n d the Schaffer collaterals respectively, (2) to activate the same cells a n t i d r o m i c a l l y by Bra#l Research, 35 (1971) 589-593

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stimulating the mossy fibres, the Schaffer collaterals (or the fimbria) and the alveus respectively, (3) to excite CA 3 and CA 1 pyramidal cells disynaptically by stimulation of the perforant path and the mossy fibres, and finally (4) to excite CA 1 cells trisynaptically by perforant path stimulation. In order to obtain di- and trisynaptic activation along the neuronal chain, it was necessary to increase the stimulus frequency from 1/sec to 10/sec. Trisynaptic activation was obtained in only one experiment. The findings support those obtained in vivo 1,3, which suggested that all 4 members of the described chain may be found within a correctly cut transverse hippocampal slice. Potentials similar to those evoked in the intact anaesthetized preparation 1-3,1°,11 were recorded from granule as well as from pyramidal cells. Examples of our observations are shown in Fig. 2. The evoked potentials (Fig. 2B-D, F) were recorded in the fascia dentata from the cut surface of the slice (Fig. 2A, Rec. syn. and Rec. c.b.). Following low voltage perforant path stimulation (Fig. 2A, Stim. pp), a short-latency monophasic negative wave appeared in the middle third of the dendritic layer of the granule cells (Fig. 2B, Syn.), where the perforant path synapses are localized 1°,14. At the same time a positive wave was recorded in the cell body layer (Fig. 2B, C.b.), thus demonstrating the same sink-source distribution as shown in vivo TM. With increasing stimulus strength a population spike appeared; this was negative in the cell body layer and positive in the synaptic layer (Fig. 2C), again as occurs in vivo10. The evoked potentials were rapidly attenuated, both when the recording electrode was moved away from the granule cells, and when the stimulating electrode was moved away from the white bundle interpreted as the perforant path, indicating that the perforant path-granule cell system was responsible for the activity. A

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Fig. 2. A, Diagram giving electrode positions for orthodromic (Stim. pp) and antidromic (Stim. mf) activation of granule cells. Potentials recorded in the middle third of the dendritic layer of the granule cells, where the perforant path synapses are located (Rec. syn.), are presented in the upper traces in B - D (Syn.). Corresponding activity evoked in the cell body layer (Rec. c.b.) is shown in the lower traces in B - D (C.b.). All evoked potentials were recorded from the surface of the slice. B and C were obtained by orthodromic, D by antidromic activation. E, Spontaneous unit activity of CA 3 cells. F, Repetitive firing of granule cells following perforant path stimulation.

Brain Research, 35 (1971) 589 593

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Antidromic invasion of granule cells (Fig. 2D) was produced by localized stimulation of the mossy fibres in the CA 3 area (Fig. 2A, Stim. mf) and appeared as a negative spike without any prepotential in the cell body layer, with a positive spike recorded in the dendritic region, indicating that the dendrites acted as passive sources. Single units could also be recorded, as exemplified by the spontaneously active CA 3 units in Fig. 2E. In experiments where the units were stimulus-locked, they coincided with population spikes, supporting the view that the population spike is due to simple summation of unitary action potentialsL The presence of postactivation facilitation11, inhibitory mechanisms8, 9, fre quency potentiation4, 7 and post-tetanic potentiation 7 suggests that fundamental properties of both granule and pyramidal cells are preserved in the slice. This notion is supported by intracellular records from CA 3 cells, obtained by Yamamoto 19 in experiments on the same type of preparation, performed in parallel with the present investigation. The electrophysiological properties of the slice were, however, found to differ in some respects from those of the intact hippocampus. Exceptionally large, negative population spikes (up to 25 mV) were occasionally recorded at temperatures of 3739°C, and the ceils often showed repetitive firing, a series of 10-15 population spikes not being unusual (Fig. 217). Potentials most comparable to those found in the intact hippocampus were recorded below 37°C or after low voltage stimulation. The factors underlying these discrepancies remain to be elucidated, but several possibilities might be suggested. Preliminary experiments indicate that the excitability of the preparation in vitro is highly dependent on the ionic composition of the bathing medium (GardnerMedwin and Lomo, personal communication). The orientation at which the slice is cut may affect the response characteristics. Since, in this study, the slices were cut transversely to the long axis of the hippocampus, it is possible that the influence of the longitudinally oriented pathways (e.g., the CA 3 association pathway12, and the basket cell system9 (Andersen et aL, unpublished) was disrupted, resulting in the deviating observations. In summary, this investigation shows (1) that the four-membered pathway from the perforant path to the alveus can be found intact and activated selectively in transverse hippocampal slices, (2) that the synapses involved in the described neuronal loop have a series of properties similar to those of the intact anaesthetized hippocampus and fascia dentata, but also (3) that the preparation displays properties that have not been demonstrated in the intact hippocampal formation. We wish to thank Dr. Per Andersen for help and criticism. We are also grateful to Dr. T. V. P. Bliss for discussions, and to Dr. C. D. Richards for demonstrating his constant-flow incubation technique. This work was supported in part by the Norwegian Research Council for Science and the Humanities, and by the British Medical Research Council. Institute of Neurophysiology, University of Oslo, Oslo 1 (Norway) Brain Research, 35 (1971) 589-593

KNUT KR. SKREDE ROLF H. WESTGAARD


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1 ANDERSEN,P., BLISS, T. V. P., LOMO,T., OLSEN,L. I., AND SKREDE,K. K., Lamellar organization of hippocampal excitatory pathways, Acta physiol, scand., 76 (1969) 4A-5A. 2 ANDERSEN,P., BLISS, T. V. P., AND SKREDE, K. K., Unit analysis of hippocampal population spikes, Exp. Brain Res., 13 (1971) 208-221. 3 ANDERSEN,P., BLISS, T. V. P., AND SKREDE,K. K., Lamellar organization of hippocampal excitatory pathways, Exp. Brahl Res., 13 (1971) 222-238. 4 ANDERSEN,P., AND LOMO, T., Mode of activation of hippocampal pyramidal cells by excitatory synapses on dendrites, Exp. Brain Res., 2 (1966) 247-260. 5 BLACKSTAD,T. W., BRINK, K., HEM, J., AND JEUNE, B., Distribution of hippocarnpal mossy fibers in the rat. An experimental study with silver impregnation methods, J. eomp. Neurol., 138 (1970) 433-450. 6 BLISS, T. V. P., AND RtCHARDS, C. D., Some experiments with in vitro hippocampal slices, J. Physiol. (Lond.), 214 (1971) 7-9P. 7 GLOOR, P., VERA, C. L., AND SVERTI,L., Electrophysiological studies of hippocampal neurons. III. Responses of hippocampal neurons to repetitive perforant path volleys, Electroeneeph. clin. Neurophysiol., 17 (1964) 353 370. 8 KANDEL, E. R., SPENCER, W. A., AND BRINLEY, F. J., JR., Electrophysiology of hippocampal neurons. I. Sequential invasion and synaptic organization, J. Neurophysiol., 24 (1961) 225-242. 9 LOMo, T., Nature and distribution of inhibition in a simple cortex (dentate area), Aeta physiol. scand., 74 (1968) 8A-9A, 10 Lotto, T., Patterns of aclivation in a monosynaptic cortical pathway: the perforant path input to the dentate area of the hippocampal formation, Exp. Brain Res., 12 (1971) 18-45. 11 LOMo, T., Potentiation of monosynaptic EPSPs in the perforant path-dentate granule cell synapse, Exp. Brain Res., 12 (1971) 46-63. 12 LORENTEDE N6, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the Ammonic system, J. Psychol. Neurol. (Lpz.), 46 (1934) 113-177. 13 MClLWAIN, H., AND RODNIGHT, R., Practical Neurochemistry, Churchill, London, 1962, pp. 109-120. 14 NAESTAD,P. H. J., An electron microscope study on the termination of the perforant path fibres in the hippocampus and the fascia dentata, Z. Zellforsch., 76 (1967) 532-542. 15 RAMdN Y CAJAL, S., Ober die feinere Struktur des Ammonshornes, Z. wiss. Zool., 56 (1893) 615-663. 16 RAMdN Y CAJAL, S., Histologie du SystOme Nerveux de l'Hotmne et des Vertdbrds, Vol. 2, Maloine, Paris, 1911, 993 pp. 17 RXCHARDS,C. D., AND SERCO~aE, R., Electrical activity observed in guinea-pig olfactory cortex maintained in vitro, J. Physiol. (Lond.), 197 (1968) 667 683. 18 RICHARDS,C. D., AND SERCOMBE,R., Calcium, magnesium and the electrical activity of guineapig olfactory cortex in vitro, J. Physiol. (Lond.), 211 (1970) 571-584. 19 YAMAMOTO,C., Synaptic transmission between mossy fiber and hippocampal neurons studied in vitro in thin brain sections, Proc. Japan Acad., 46 (1970) 1041-1045. 20 YAMAMOTO,C., AND KAWAI, N., Presynaptic action of acetylcholine in thin sections from the guinea pig dentate gyrus in vitro, Exp. Neurol., 19 (1967) 176-187. 21 YAMAMOTO,C., AND KAWAI, N., Generation of the seizure discharge in thin sections from the guinea pig brain in chloride-free medium in vitro, .lap. J. Physiol., 18 (1968) 620-631. 22 YAMAMOTO,C., AND MCILWAIN, H., Electrical activities in thin sections from the mammalian brain maintained in chemically-defined media in vitro, J. Neurochem., 13 (1966) 1333-1343. (Accepted October 4th, 1971)

Brain Research, 35 (1971) 589-593