Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4706-4711, May 1996 Neurobiology

Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system (/CA3 region/presynaptic terminals/spines) DANNY BARANES*, JUAN CARLOS L6PEZ-GARCIA*, MARY CHEN*, CRAIG H. BAILEY*, AND ERIC R. KANDEL*t tHoward Hughes Medical Institute and *Center for Neurobiology and Behavior, College of Physicians and Surgeons of Columbia University, 722 West 168th Street, New York, NY 10032 Contributed by Eric R. Kandel, December 20, 1995

ABSTRACT of the hippocampal mossy fiber vitro granule-to-pyramidal connection exhibits NMDA- pathway exhibit several characteristic features, including a independent LTP similar to that evident in vivo. unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, MATERIALS AND METHODS a complex postsynaptic architecture, and sprouting in re- Cell Culture. Hippocampi of P1-P5 rat pups were split sponse to seizures. However, these connections have proven longitudinally along the midportion ofthe CA3 region (see Fig. difficult to study in hippocampal slices because of their 1A). Tissue was treated for 30 min at 37°C with 0.25% trypsin relative paucity (<0.4%) compared to commissural-collateral (type XI; Sigma), and then gently triturated and the dissociated synapses. To overcome this problem, we have developed a cells either plated at a concentration of 2 x 105/ml onto novel dissociated cell culture system in which we have en- poly-D-lysine (20 ,ug/ml; Sigma)- and laminin (10 ,tg/ml; riched mossy fiber synapses by increasing the ratio ofgranule- Collaborative Research)-coated glass coverslips or sedimented to-pyramidal cells. As in vivo, mossy fiber connections are through a Percoll gradient before plating as described (9). composed of large dynorphin A-positive varicosities contact- Cells were plated in Eagle's minimal essential medium ing complex spines (but without a restricted localization). The (MEM) containing 10% heat-inactivated fetal bovine serum elementary synaptic connections are , inhibited (HyClone), 2 mM glutamine, and 0.76% glucose. On the by dynorphin A, and exhibit N-methly-D-aspartate-indepen- following day, the medium was replaced with fresh SF1C dent long-term potentiation. Thus, the simplicity and exper- medium (10), including B-27 supplements (GIBCO). The cells imental accessibility of this enriched in vitro mossy fiber were grown for up to 12 weeks in a humid incubator containing pathway provides a new perspective for studying nonassocia- 5% C02/95% air at 37°C. tive plasticity in the mammalian central . Cell Type-Specific Labeling. Cells were intracellularly in- jected with 6% carboxyfluorescein by using sharp electrodes (70 MfQ resistance). Impaled were allowed to fill by Several features of the hippocampal mossy fiber (MF) pathway diffusion for -2-3 min. are interesting for studying (1). First, repeated The dentate gyrus area was labeled following its selective activation of MFs results in long-term potentiation (LTP) in the dissection. Cells were concentrated to 1 dentate gyrus/0.5 ml CA3 subfield (2) that does not require activation ofthe N-methyl- and incubated for 1 h with 40 ,uM DiI (Sigma) in plating D-aspartate (NMDA)-type glutamate receptor. Second, MFs are medium, washed, and plated at 500 cells/cm2. capable of extensive sprouting following (3) and con- To label CA3 cells, microspheres (0.2 ,tl; Lumafluor, New tribute to the susceptibility of CA3 neurons to epileptic seizures York) were injected into the CA3 subfield of PO-Pl pups, 0.5 (4, 5). Finally, the enpassant connections established by MF with mm posterior to lambda and 2.5-3 mm lateral to the sagittal dendritic spines of CA3 pyramidal cells have unique morpholog- suture at a depth of 2-3 mm as described (11). After 48 h, the ical characteristics. The presynaptic endings of the MF are injected was removed, fixed for 4 h with 4% among the largest in the brain, and the postsynaptic target of the paraformaldehyde, and incubated overnight at 4°C with 30% MFs often take the form of giant, multi-invaginating spines sucrose. Frozen sections were taken at 30 ,um intervals in the termed thorny excresences, making these connections readily coronal plane and examined with the fluorescence microscope. identifiable (1, 6, 7). The contralateral hippocampus was coronally sliced, and cul- Study of the MF pathway in hippocampal slices has been tures were prepared from those cases demonstrating retro- limited by the complex circuitry of the CA3 region and the grade transport to the CA3 subfield. extremely low incidence of MF synapses (1, 2). Here we Immunocytochemistry. Cells were immunolabeled as de- describe a dissociated cell culture system where mossy fiber scribed by Craig et al. (12). The mouse antibodies used in this plasticity can be characterized at the level of individual neu- study were monoclonal anti-MAP2 (5 ,ug/ml; Sigma) and rons and elementary synaptic connections. In culture, the MF monoclonal anti-synaptophysin (5 Ag/ml; Boehringer Mann- connections retain the major morphological and immunocy- heim). The rat antibodies were a-MAPs (Sigma), a-dynorphin tochemical characteristics ofthe MF pathway in vivo. Thus, this A (10 p.g/ml; Chemicon), and a-glutamate receptor Rl reduced preparation provides an experimentally accessible, in (GluRl; provided by R. L. Huganir, Johns Hopkins University vitro model of the MF pathway that allows the study of its School of Medicine). For dynorphin A and GluRl double cellular and molecular properties and plastic capabilities at the labeling, cells were first incubated for 48 h with a-GluR1, level of individual, identified synapses. In a companion study, labeled with secondary Cy3-conjuaged antibody, fixed with 4% Lopez-Garcia et al. (8) have further demonstrated that the in paraformaldehyde, and then labeled with a-dynorphin A

The publication costs of this article were defrayed in part by page charge Abbreviations: MF, mossy fiber; LTP, long-term potentiation; DG, payment. This article must therefore be hereby marked "advertisement" in dentate gyrus; NMDA, N-methyl-D-aspartate; mEPSC, miniature accordance with 18 U.S.C. §1734 solely to indicate this fact. excitatory postsynaptic current.

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...... A3 t - 35% , .. CA3 60% Downloaded by guest on September 28, 2021 4708 Neurobiology: Baranes et al. Proc. Natl. Acad. Sci. USA 93 (1996) Table 1. Morphological and electrophysiological characteristics of granule and pyramidal neurons in dentate gyrus-CA3 culture Pyramidal, Granule, [km ± SEM Am ± SEM Width of cell body 28.5 ± 5.7 11.4 ± 0.9 Length of cell body 29.2 ± 2.5 13.4 ± 1.3 No. of apical 1 1 No. of basal dendrites 2-6 0-1 Length, apical 194.0 ± 14.5 113 ± 8.0 Length, basal 107.6 ± 17.9 96.1 ± 8.9 Branching points Apical 3-5 0-3 Basal 0-3 0-2 Oblique distance from apical origin 37.3 ± 15.9 62.6 ± 13.7 Oblique distance from basal origin 38.5 ± 10.2 106 ± 22.7 Width of apical , origin 5.7 ± 1.3 3.7 ± 0.5 Width of basal dendrite, origin 3.9 + 0.1 1.8 ± 0.1 Resting potential, mV -49.77 ± 2.63 -61.69 ± 0.43 Input resistance, Mfl 491.7 ± 58.3 658.3 ± 50.31 Cells were photographed with an MRC-1000 laser confocal micro- scope after staining with a-MAP2. Measurements of the width were taken from phase contrast pictures of identified neurons. Shown are the mean ± SEM of 50 (granule) and 15 (pyramidal) cells. Apical dendrite for both cell types was defined as the thickest and longest. In the case of pyramidal cells, this always occurred at the apex of the perikaryon. Measurements of resting membrane potential and input resistance were done using the perforated patch technique (n = 13). followed by IgG-fluorescein isothiocyanate-conjugated label- ing. Cells were examined with an Axiovert-100 Zeiss fluores- cent microscope. Pictures were taken using a MC-80 Zeiss camera, a Hamamatsu (Middlesex, NJ) C-2400 SIT camera, or a MRC-1000 Laser confocal microscope (Bio-Rad). Electron Microscopy. Cells were allowed to grow for 18-28 days on poly-D-lysine and laminin-coated Aclar 33c coverslips. Cultures were fixed in place by slow perfusion with a trialde- hyde solution containing 1% paraformaldehyde, 1% acrolein, 2.5% glutaraldehyde, and 2.5% dimethylsulfoxide plus CaCl2 (0.05%) in 0.1 M cacodylate buffer (pH 7.4). After 1 h at room temperature, this fixative was replaced with 2% paraformal- dehyde and 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 16-20 h at 4°C. The cultures were treated with 2% OS04 in 0.1 M cacodylate buffer (pH 7.4) for 1 h at room temperature. After the cultures were embedded in Epon 812, serial thin sections (50-100 sections per block) were cut parallel to the substrate surface, stained with lead and uranyl acetate, and photographed with a Phillips model 301 electron microscope (Phillips Electronic Instruments, Mahwah NJ). Electrophysiology. Recording of spontaneous activity was done by whole-cell patch clamp in the presence of 0.5 ,uM FIG. 2. In vitro dentate gyrus neurons have MF-like axons. (a) tetrotodotoxin (8). Five minutes after gaining stable access Dynorphin A (green) is expressed in -like processes but not in into the cell, the occurrence of spontaneous miniature exci- dendrites (red). Arrows point to filopodia-like structures. (b) Vari- cosities of a dynorphin A-positive axon in putative contact with tatory postsynaptic currents (mEPSCs) was recorded for 1 min pyramidal cells on their apical dendrite (arrowheads), the basal using pCLAMPx 6 (Axon Instruments, Foster City, CA), and dendrite (arrow), and cell body (open arrow). (c) MF varicosities in the frequency and amplitude of the events were analyzed by close association with a nonpyramidal cell. Arrows indicate varicosities visual inspection of the traces. with a diameter of 4-4.5 ,um. (Bar = 20 ,um.)

FIG. 1. (On previous page) Hippocampal culture enriched for dentate gyrus granule versus CA3 pyramidal cells. (Al-A3) Hippocampi of P1-P5 rat pups were split through the longitudinal axis of the CA3 region close to the dentate gyrus region. (A4) In several experiments, enrichment was increased by separation through a Percoll gradient. MAP2 staining of 2-week-old CA3-CA1 culture (B]) and dentate gyrus-CA3 culture (B2). (B3) Higher magnification of the area boxed in B2. The arrow indicates a . A pyramidal (B4) and a granule (BS) from dentate gyrus-CA3 culture intracellularly filled with carboxyfluorescein. Arrow points to the apical dendrite. (B6) A of the dentate gyrus-CA3 culture identified by prelabeling the dentate gyrus with DiI. (C1) Microspheres taken up by cells positioned in the CA3 subfield of a hippocampus contralateral to the one injected in the CA3 region. (C2) MAP2 staining of dentate gyrus-CA3 culture prepared from the section shown in Cl. (C3) Only the pyramidal cell, indicated by an arrow in C2, contains microspheres (arrow). (Bars: B] and B2 = 90 ,tm; B3-B6, C2, C3 = 35 ,uLm; Cl = 500,m.) Downloaded by guest on September 28, 2021 Neurobiology: Baranes et aL Proc. Natl. Acad. Sci. USA 93 (1996) 4709

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0 10 20 30 a 10 20 30 Amplitude (pA) FIG. 3. Granule-to-pyramidal connections are formed later than pyramidal-pyramidal connections. Seven-day-old dentate gyrus-CA3 (A) and CA3-CA1 (B) cultures double-labeled with a-MAP2 (green) and a-synaptophysin (yellow). (Bar = 30 ,um.) (C) Correlation between density of presynaptic terminals (synaptophysin dots) and frequency of spontaneous activity. To quantify the level of presynaptic terminal formation, images of 20 fields containing between two and five cells from both cultures were digitized, counted for synaptophysin-positive spots ranging between 0.5-4.0 ,um in diameter, and then the number was divided by the number of neurons found in the field (MAP2 positive). The frequency of mEPSC was normalized to the number of neurons per dish (MAP2 positive) (n = 12 of each culture). RESULTS We found that during the second week in vitro, dynorphin A staining was present in the axons of dentate gyrus-CA3 cells Enrichment of Granule Cells Versus Pyramidal Cells in Cul- (Fig. 2). By contrast, examination of CA3-CA1 cultures re- ture. To enrich primary neuronal cultures for MF connections vealed no significant dynorphin A staining, suggesting that the and to eliminate as much as possible the collateral connections positive labeling in the dentate gyrus-CA3 culture is due to between the pyramidal cells, we increased the ratio between granule cell axons. In support of this view, dynorphin A was granule cells and CA3 pyramidal cells by coculturing the entire found to inhibit synaptic transmission of the MF in the dentate dentate gyrus with only a portion of the CA3 subfield (dentate gyrus-CA3 culture (8) as reported for these synapses in the gyrus-CA3 culture) (Fig. 1A). In dentate gyrus-CA3 cultures, intact hippocampus (18). only 6.3% (±2% SEM) of the total neurons displayed a pyrami- Similar to their in vivo counterparts, the MEF axons in culture dal-like shape, whereas 50.6% (±2.8% SEM) of the cells were displayed varicosities ofvarious shapes and sizes (1-4.5 ,Lm) (Fig. granule-like (Fig. 1B2; Table 1). The remainder of the cells were 2a) and filopodia-like structures (1). However, unlike the situa- classified as nonpyramidal, nongranule cells. Ten to 15% of the tion in vivo, MF contacts were not restricted to the proximal nonpyramidal, nongranule cells were decarboxyl- portion of the CA3 cell apical dendrite (Fig. 2b). Perhaps, as ase-positive (not shown) and presumably represent inhibitory suggested by Bossetal. (13), the correct localization ofmossyfiber . The average ratio of putative granule to pyramidal connections in vivo is extrinsically regulated and this control is cells under these conditions was 13:1 (measured by shape and absent from isolated cells in dissociated culture. dimension of cell body and dendritic ramification; see below). By One of the features that distinguishes the MF pathway from contrast, in CA3-CA1 cultures, pyramidal-like neurons occupy other hippocampal excitatory pathways is a late onset of -60% of the total neuronal population (Fig. l18; Table 1) with activity due to the late generation of granule cells (19). We granule-like cells making up <6%, probably reflecting contam- found that at 7 days in vitro, synaptophysin-positive presynaptic ination from the dentate gyrus. structures and mEPSC frequency (Fig. 3 B and C) were To verify that pyramidal cells in dentate gyrus-CA3 cultures approximately four times lower in the dentate gyrus-CA3 originate from the CA3 region, cells from the CA3 subfield compared to the CA3-CA1 culture (Fig. 3C). By contrast, no were retrogradely labeled with fluorescent microspheres differences were seen in the amplitude distribution of the through the commissural pathway and were found to exhibit spontaneous miniature synaptic potential between the two CA3 pyramidal-like structure when placed in culture (Fig. 1C). cultures, with both showing a peak at 4 pA in the amplitude The pyramidal cells in culture acquired the appearance of their histograms (Fig. 3C). At 14 days in vivo, the concentration of mature counterparts, having large triangular cell bodies, one synaptophysin-positive terminals was equal between the two major apical dendrite, and 2-6 basal dendrites (Fig. 1 B3 and cultures but the differences in the frequency of mEPSC B4; Table 1). As first shown by Boss et al. (13), dentate gyrus persisted (not shown). Thus, in culture as in vivo, the granule granule cells labeled with DiI (Fig. 1B6) appeared immature, to pyramidal cell connections are formed later than the exhibiting smaller, round, or oval cell bodies as in vivo (14), and pyramidal to pyramidal cell connections. 90% bore a basal dendrite (Fig. 1B2, B3, and B6; Table 1), Characterization ofthe Postsynaptic Site. Similar to their in similar to the immature granule cells described in rats (15), vivo counterparts, the spine repertoire of the dentate gyrus- monkeys, and humans (16). Ten percent of the granule cells CA3 culture included several morphologically identifiable lacked basal dendrites (Fig. 1B5). A second approach to verify types, including thin, stubby, and mushroom (Figs. 4 and 5). that granule cells survived in dentate gyrus-CA3 culture was Spines in close contact with MFs tended to be more complex the use of dynorphin A as a specific marker for MF axons (17). (Fig. 4A and B) but were not restricted to the proximal portion Downloaded by guest on September 28, 2021 4710 Neurobiology: Baranes et al. Proc. Natl. Acad. Sci. USA 93 (1996)

FIG. 4. Characterization of postsynaptic spines at mossy fiber connections in vitro. Light microscopy. (A and B) Cells were double-labeled with a-dynorphin A (green) and a-GluR1 (red) antibodies. Dynorphin A varicosity innervates the spine head near the GluRl hot spot (large arrow in A; arrowhead in B) resulting in a yellow color. Small arrows inA and large arrows in B indicate protrusions rising out of the spine head. Electron microscopy. (C) A simple, thin spine. (D) A more complex, mushroom-type spine. (E) Apparent spinule (arrow) projecting out of a perforated . (Bars: A and B = 0.5 ,m; C-E = 0.25 ,um.) of apical dendrites of pyramidal cells. They often consisted of Similarly, in culture, we observed a 53% higher concentration of a widened neck and an enlarged head composed of several spines on the distal versus proximal portions of pyramidal cells protrusions, resembling immature "thorny" spines (1). Elec- (Fig. 5 b and c;-Table 2), suggesting that CA3 neurons have an tron microscopy confirmed these different classes of spines intrinsic capability to enhance spine formation at the distal part (Fig. 4 C and D) and also revealed evidence of possible of their apical dendrite but are incapable of concentrating thorny structural remodeling in the form of "spinules" and perforated spines of the mossy fiber pathway on their proximal segment. synapses (Fig. 4E), both of which have been suggested to play a role in some types of synaptic plasticity (20-22). DISCUSSION The absence ofa restricted localization ofcomplex spines to the We have been able to develop the procedures necessary for proximal portion of pyramidal cell apical dendrites might be due reconstituting the MF pathway in dissociated cell culture. In this to an abnormal spine distribution in culture. We have found this culture the presynaptic granule cells and their connections with is not the case. In vivo, hippocampal pyramidal cells have the CA3 pyramidal cells are enriched. Moreover, both the pyramidal highest spine frequency on distal parts of the dendrite (23). and granule cells, identified by independent criteria, are readily Downloaded by guest on September 28, 2021 Neurobiology: Baranes et al. Proc. Natl. Acad. Sci. USA 93 (1996) 4711 Table 2. Spine distribution on granule and pyramidal cells Distance Ratio of spine from cell Spines/,um concentration body (,um) Pyramidal Granule Pyramidal Granule Apical Proximal 0.32 ± 0.07 0.59 ± 0.08 dendrite (0-60) Distal 0.47 ± 0.1 0.67 ± 0.21 (60-200) Distal/ 1.53 1.14 Proximal Basal (0-200) 0.45 ± 0.5 0.53 ± 0.19 dendrite Apical/ 0.88 1.19 Basal Spines were counted visually in GluRl stained cells (n = 20 of each cell type). Note that the concentration of spines on both the apical and basal dendrites of granule cells is higher than that of pyramidal cells. However, the ratio ofspine concentration between the distal versus the proximal parts along the apical dendrite is 3.9-fold higher on pyramidal than on granule cells (calculated as: [distal/proximal (pyramidal) - 1]/[distal/proximal (granule) - 1]). The concentration of spines on the basal dendrite of pyramidal cells is higher than that of the apical dendrite. For granule cells, this distribution is reversed. The ratio for apical/basal was calculated as (average between proximal and distal along the apical portion of the dendrite)/(spine concentration on the basal dendrite). large size ofthe MF terminals and their ability to sprout following seizure provides a novel and potentially useful in vitro model for examining the functional and structural components of learning- related plasticity at a mammalian nonassociative . We thank Drs. Mark Mayford, Robert D. Hawkins, Ottavio Arancio, and Steven A. Siegelbaum for critical comments on earlierversions ofthis manuscript and Harriet Ayers for typing the manuscript. We also thank Drs. Steven Rayport and Ottavio Arancio for technical advice in cell culturing and Mr. Len Zablow and the Presbyterian Hospital Photo Department for processing the pictures and images..This research was FIG. 5. Spine distribution on isolated pyramidal and granule cells in supported by the Howard Hughes Medical Institute (E.R.K.) and Na- culture. Spines and cell type were detected using a-GluR1 (yellow) and tional Institutes of Health Grants MH37134 and GM32099 to C.H.B. a-MAP2 (green) antibodies. (a) Granule cell. (b) Pyramidal cell. The large arrow in both pictures indicates the apical dendrite. Small arrows 1. Amaral, D. G. & Dent, J. A. (1981) J. Comp. Neurol. 195, 51-86. point to thin and stubby spines extending from the proximal area of the 2. Claiborne, B. J., Xiang, Z. & Brown, T. H. (1993) Hippocampus 3, dendrite. (c) The distal part of an apical dendrite (open arrow) of the 115-122. pyramidal cell has a higher frequency of spines (small arrows) compared 3. Tauck, D. L. & Nadler, J. V. (1985) J. Neurosci. 5, 1016-1022. to the area proximal to the oblique (B). (Bar = 15 ,um.) 4. Nitecka, L., Tremblay, E., Charton G., Bouillot, J. P., Berges, M. L. & Ben-Ari, Y. (1984) Neuroscience 13, 1073-1094. distinguishable upon a visual inspection alone. This provides a 5. Okazaki, M. M. & Nadler, J. V. (1988) Neurocience 26, 763-781. 6. Claiborne, B. J., Amaral, D. G. & Cowan, W. M. (1986) J. Comp. significant experimental advantage over classical cell cultures Neurol. 246, 435-458. prepared from the entire hippocampus, and designed to recon- 7. Chicurel, M. E. & Harris, K M. (1992)J. Comp. Neurol. 325,169-182. stitute the pathway, where one cannot identify 8. L6pez-Garcia, J. C., Arancio, O., Kandel, E. R. & Baranes, D. (1996) the presynaptic CA3 neuron from the postsynaptic CAl cell. Proc. Natl. Acad. Sci. USA 93, 4712-4717. not all 9. Hatten, M. E. (1985) J. Cell Biol. 100, 384-396. However, the morphological features characteristic of 10. Rayport, D., Sulzer, W.-X., Samasdikosol, S., Monaco, J., Batson, D. mature MF synapses in vivo (for example, multibranched, thorny & Rajendran, G. (1992) J. Neurosci. 12, 4264-4280. spines) were observed in this culture. Thus, the dentate gyrus- 11. Buchhalter, J. R., Fieles, A. & Dichter, M. A. (1990) Dev. Brain Res. CA3 culture appears to represent a young MF pathway, perhaps 56, 211-216. most similar to that found in rats during their first 2 weeks after 12. Craig, A. M., Blackstone, C. D., Huganir, R. L. & Banker, G. (1993) Neuron 10, 1055-1068. birth (1). Nevertheless it appears that synaptic connections in the 13. Boss, B. D., Gozes, I. & Cowan, W. M. (1987) Dev. Brain Res. 36, dentate gyrus-CA3 culture are large enough to allow their 199-218. structural dynamics to be characterized, even at the light micro- 14. Rihn, L. L. & Claiborne, B. J. (1990) Dev. Brain Res. 54, 115-124. scopic level. 15. Seress, L. & J. Pokorny, J. (1981) J. Anat. 133, 181-195. The relative simplicity and experimental accessibility of the 16. Seress, L. & Mrzljak, L. (1987) Brain Res. 405, 169-174. 17. Chavkin, C., Chavkin, C., Shoemaker, W. J., McGinty, J. F., Bayon, A. dentate gyrus-CA3 enriched culture now allows study, on an & Bloom, F. E. (1985) J. Neurosci. 5, 808-816. elementary level, of the development and plastic properties of 18. Weisskopf, M. G., Zalutsky, R. A. & Nicoll, R. A. (1993) Nature both the MF connections between dentate granule cells and (London) 362, 423-427. hippocampal pyramidal cells from the CA3 region as well as the 19. Bayer, S. A. (1980) J. Comp. Neurol. 190, 87-114. axon collateral connections between the pyramidal cells from the 20. Tarrant, S. B. & Routtenberg, A. (1977) Tissue Cell 9, 461-473. 21. Schuster, T., Krug, M. & Wenzel, J. (1990) Brain Res. 523, 171-174. CA3 region (associational-commissural). Indeed, the ability to 22. Geinisman, Y., Morrell, F. & deToledo-Morrell, L. (1991) Brain Res. produce MF LTP under controlled conditions while simulta- 566, 77-88. neously monitoring identified connections combined with the 23. Homer, C. H. (1993) Prog. Neurobiol. 41, 281-321. Downloaded by guest on September 28, 2021