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1 Laboratory of Molecular Biology and 2 Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20814; and 3 Department of Neurobiology, The Weizmann Institute, Rehovot, 76100, Israel
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Collin, Carlos, Katsuyuki Miyaguchi, and Menahem Segal.Dendritic spine density and LTP induction in cultured hippocampal slices. J. Neurophysiol. 77: 1614-1623, 1997. Transverse hippocampal slices were cut from 8- to 9-day-old rats and maintained in an interface chamber for periods of 1-4 wk, in tissue culture conditions. Neurons in the slice preserved their spatial organization and connectivity. Dendritic spine density in CA1 neurons was very low at 1 wk in culture, and long, filopodia-like structures were abundant. Spine density increased in these neurons nearly threefold during the course of 3 wk in vitro, to approach values of those of the normal, in vivo hippocampus. The magnitude of long-term potentiation (LTP) of reactivity of CA1 to stimulation of CA3 neurons also increased during weeks in culture in parallel with the change in spine density. Chronic exposure of slices to drugs that interact with synaptic activity caused changes in their dendritic spine density. Blockade of the N-methyl-D-aspartate (NMDA) receptors with the receptor antagonist 2-aminophosphonovalerate (D-APV) or blockade of action potential discharges with tetrodotoxin (TTX) prevented dendritic spine development in immature cultures. Enhancing synaptic activity by blockade of GABAergic inhibition with picrotoxin did not affect spine density to a significant degree. D-APV-treated slices expressed larger LTP than controls. TTX-treated slices expressed smaller LTP than controls. Picrotoxin treated slices did not express LTP. It is proposed that LTP and dendritic spine density are correlated strongly during development, whereas they are not correlated in the more mature slice/culture of the hippocampus where spine density can be modulated by chronic exposure to blockers of synaptic activity, which will not affect LTP in a similar manner.
More than a century since their discovery, dendritic spines continue to be an enigma. Their strategic location as the postsynaptic site of most excitatory synapses in the brain, as well as their unique morphology, make the dendritic spine a probable locus for long-term changes associated with learning and memory in the brain (Bliss and Collingridge 1993 Hippocampal slices, 300-µm thick, were prepared from 8- to 9-day-old Sprague-Dawly rats as described before (Stoppini et al. 1991 Drug application
The following drugs were applied in the growth medium for periods ranging from 2 to 21 days: picrotoxin (RBI), 50 µM; 2-aminophosphonovalerate (D-APV), Sigma, 50 µM; tetrodotoxin (TTX, Sigma), 1 µM; 6,7-dinitroquinoxaline-2,3-dione (DNQX, RBI), 10 µM. Drugs were refreshed every 2-3 days with the change of medium. Most of the experiments were conducted with long-term exposure to the drugs (14 days), starting 2-3 days after plating of the cultures. No drugs were added to the recording medium.
Confocal microscope imaging
After intracellular recording, which verified that the cells display, normal activity and were not epileptic, staining of 2-5 cells in area CA1 of the slice with LY was accomplished by injecting negative current pulses through the recording micropipette (200 ms pulses of
Electrophysiology
Extracellular recordings were made from strata pyramidale and radiatum of the CA1 region of the slices. Stable responses to stimulation of stratum radiatum or the adjacent CA3 area was recorded, an input/output curve was constructed, and the intensity of stimulation that evoked two maximal responses were chosen for later experiments. Stimulation was applied at a rate of 0.1 Hz, and the responses to all stimulations were stored for later analysis.
Electron microscopy
Slices of different ages or drug treatments were fixed in a solution of 2.5% glutaraldehyde in 0.1 M phosphate buffer (PBS) for 1 h. After a rinse in PBS, they were postfixed in 1% osmium in PBS for 1 h. The slices were then dehydrated, embedded in plastic, thin sectioned and contrasted. Alternatively, slices were frozen rapidly on a copper block (Life Cell) and freeze substituted as described elsewhere (Bridgman and Reese 1984 Development of dendritic spines in slice culture
In the first series of experiments, the density of dendritic spines in neurons grown in slice cultures for periods of 1-4 wk were studied. There was a clear distinction between genuine dendritic spines, consisting of a thin and short (1-2 µm) neck and a distinct head, and filopodia, being very thin and long without a distinct head. The two types of protrusions were seen side by side and were counted separately in some experiments. Significant regional and age variability also was observed. Although in some branches, thin filopodia were predominant (Fig. 1B), at 1 wk in culture, already two-thirds of protrusions were spines (Fig. 2). Because of the relative abundance of filopodia and the smaller diameter of dendritic branches at this age, proper identification of spines was only accurate when confocal sections were analyzed individually. The filopodia were not counted in later drug experiments (see below). A marked age-related increase in spine density during the 1 mo of growth in culture was seen and a concomitant decrease in the relative abundance of filopodia. The majority of spines in the 4-wk-old slices were shorter, had a distinctly larger spine head, and were about three times more numerous per length of dendrite compared with those in the younger cultures (Fig. 2). The mean spine density of 0.399 ± 0.031 spines (mean ± SE) per 1 µm length of dendrite (out of a total of 0.621 ± 0.025 protrusions (spines + filopodia) per micrometer of dendrite) in 1-wk-old cultures, increased to 1.159 ± 0.063 spines per 1 µm dendrite out of a total of 1.176 ± 0.061 (spines + filopodia) in 4-wk-old cultures (ANOVA for comparison of spine density across four age groups, F = 84.5, P < 0.01). Thus although spine density increased by about threefold across 3 wk in culture, the density of filopodia decreased from 0.222 to 0.017 per 1 µm in this time period. The increase in spine density was associated with an increase in dendritic arborization and cell volume, but these parameters were not studied systematically. Suffice to say that the increase in both spine density and dendritic length, contributed to an actual larger increase in total number of spines per neuron in the slice. We analyzed also changes in shape and size of the spine head during development, but the variability within cells and cultures was too large in this sample. An electron microscopy (EM) analysis indicated that young filopodia like structures often contained synaptic junctions and smooth endoplasmic reticulum (SER), (Fig. 3, A and B), indicating that these filopodia eventually might become genuine spines. Mature spines with large heads and necks, as shown in Fig. 3C, became dominant ~2 wk in culture. Concave-shaped spine synapses (Miyagushi 1994
Development of electrophysiological properties of CA1 neurons in slice cultures
Input/output relationships of excitatory postsynaptic potentials (EPSPs) were recorded in stratum radiatum (Fig. 4A, top) and stratum pyramidale (SP; Fig. 4A, bottom) at 1 (n = 10), 2 (n = 6), 3 (n = 10), and 4 (n = 8) wk in culture. Bipolar electrodes were placed in CA3-CA4 pyramidal layer and 100-µs pulses of increasing intensity were applied. Maximum EPSP amplitudes recorded in SP were 3.63 ± 0.93, 4.23 ± 1.12, 5.81 ± 1.59, and 5.39 ± 1.3 mV at 1, 2, 3, and 4 wk, respectively.
Activity dependent change in dendritic spine density
Slices were exposed for 3-21 days to drugs known to affect spike and synaptic activity. These include blockade of action potential discharges with TTX, selective blockade of the N-methyl-D-aspartate (NMDA) receptor with D-APV, blockade of both types of ionotropic glutamate receptors with D-APV/DNQX, and activation of the hippocampal circuit with the
Activity-dependent changes in ability to express LTP
D-APV treatment for 2 wk (n = 15 slices) caused both a larger STP and an apparently larger LTP compared with controls (Fig. 6A). Exposure of the slices to D-APV/DNQX caused a large enhancement of spontaneous synaptic activity when slice cultures were transferred to the recording chamber and the drugs were washed away. In these cases, tetanic stimulation triggered massive epileptiform discharges. Picrotoxin-treated slices (n = 15) expressed a marked reduction in the magnitude of STP and a complete elimination of LTP. TTX treatment (n = 12) did not affect STP but sustained potentiated levels were smaller than controls. STP was108.7 ± 5.81% more than baseline in control slices vs.146 ± 6.03% in D-APV-treated slices (t = 4.09, P < 0.001),52 ± 5.52% in picrotoxin (t = 6.89, P < 0.0001), and96.8 ± 6.72% (t = 1.31, NS) in TTX-treated slices. EPSP magnitudes at 30 min were 51.4 ± 0.9.7% more than baseline in control and 68.7 ± 0.8.0% in D-APV-treated slices. Smaller LTP was obtained in TTX-treated slices: 28.3 ± 10.1%; although insignificant potentiation in the picrotoxin-treated slices: 6.5 ± 9.2% (ANOVA for LTP differences between control and the 3 drug treatments was F = 17.82, P < 0.0001).
The present results confirm and extend earlier observations that demonstrated the potential use of slices of developing hippocampus maintained in culture for the study of long-term regulation of spine morphology and for the study of physiological functions of the hippocampus (Debanne et al. 1995 Drug effects on spine density and LTP
Drugs that block or enhance ability to express LTP produce different effects on spine density. The NMDA antagonist D-APV enhanced LTP magnitude, either because it prevents degeneration of neurons (Pozzo-Miller et al. 1994) or because it prevented saturation of LTP-generating mechanism by ongoing activity in the slice. Hyperexcitability and epileptic activity observed in slices exposed to a combination of D-APV and DNQX could be explained by the washout of excitatory antagonists when cultures were transferred to the normal recording medium and also may be similar to epileptiform activity induced by the removal of the glutamate antagonist kynurenic acid and elevated magnesium shown previously in neuronal microcultures (Furshpan and Potter 1989 Roles of dendritic spines in neuronal integration
Numerous models and theories ascribed roles for dendritic spines in long-term plasticity and neuronal integration in mature animals (Bliss and Collingridge 1993
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). Their minute size, at the limit of optical resolution, renders them extremely difficult to study in a systematic manner and causes a great deal of controversy with respect to the exact nature of the changes the spines undergo in plasticity-related processes (Chang and Greenough 1984; Desmond and Levy 1988; Fifkova and Van Harreveld 1977; Lee at al. 1980; Moser et al. 1994). The recent use of dissociated neuronal cultures allow the exploration of the rules governing morphological plasticity in dendritic spines (Papa et al. 1995). Dissociated cultures, however, suffer from the lack of the typical organization of the hippocampus in vivo; neurons in the dissociated culture do not express the typical apical/basal orientation, with a long apical dendrite and a few shorter, basal dendrites, and they lack a clear lamination of inputs. Also, the density of spines in dissociated cultures is lower than in vivo. The hippocampal slice, grown for weeks in tissue culture condition (slice culture) offers both the advantages of cultured neurons, i.e., the ability to control the growth conditions of the cells, and easy access to the cell, but it also maintains the organization typical of the in vivo hippocampus (Li et al. 1994; D. Muller et al. 1993; Pozzo Miller et al. 1993; Stoppini et al. 1991 1993). We have used the hippocampal slice culture to explore some long-term drug effects on dendritic spine formation and on physiological functions assumed to be associated with the spine, i.e., long-term potentiation (LTP) of reactivity to afferent stimulation.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Briefly, slices were placed on a transparent porous filter (Millicell CM, Millipore), in a 37°C, 5% CO2 humid incubator, and fed twice a week with a medium containing 50% minimal essential medium, 25% Earle's balanced salt solution (EBSS), and 25% heat-inactivated horse serum supplemented with 7 mg/ml glucose, penicillin, and streptomycin (100 U/ml each). Throughout their growth period, the slices were kept at an interface between the growth medium and the humid atmosphere. Slices were taken to the recording chamber where they were superfused with the recording medium in a submerged slice configuration. The recording medium contained (in mM) 125 NaCl, 3 KCl, 2.4 CaCl2, 10 glucose, 1.25 NaH2PO4, and 26 NaHCO3, pH 7.4, and was saturated with a 95%O2-5% CO2 gas mixture. Extracellular recording were made with a wide tip (2-4 µm) micropipette filled with the recording medium. Intracellular recording was made with sharp (60-100 M
) micropipettes backfilled to the tip with 3% Lucifer yellow (LY) Li salt. The rest of the electrode then was filled with 1 M potassium acetate (KAc). Different slices of the same batch were used for the electrophysiological and morphological experiments so as to minimize the delay between the drug treatment and the fixation of the slices assigned to the morphological study. Signals were amplified with an Axoclamp 2A amplifier, digitized, and stored in an IBM-compatible computer using standard Axon Instruments software.
0.5 nA at 1 Hz for 10 min) into the cell. After injection, viability of the cell was evaluated again, and only healthy cells were used for the morphological study. The slice was fixed in 4% paraformaldehyde in phosphate-buffered saline and mounted on a glass slide. Slices were imaged in an inverted confocal laser scanning microscope (Zeiss) equipped with an argon-ion laser, using an oil immersion, 100 × 1.3 NA neofluar objective. Low power images of the fluorescent cell somata were taken (Fig. 1A), followed by high-power, zoomed serial optical sections taken at 1-µm intervals through the imaged dendrites and stored for later analysis. In all cases, secondary dendrites branching off the main apical dendrite or off the primary basal dendrites were imaged. For good resolution, only dendritic branches close to the surface were selected for analysis. The dendrites were reconstructed in three dimension using Metamorph software (Universal Imaging; Fig. 1B), and the spines on given lengths of dendrites were counted by an independent observer, who was unaware of the experimental treatment of the tissue. At least two cells in each of two experiments were used for the analysis of each experimental group. For each cell, 8-10 fields were reconstructed, and the number of spines were counted on each of several clearly focused dendritic segments seen in these fields. Initially, representative dendritic segments from both the apical and basal dendrites were sampled and analyzed. As there were no significant differences between apical and basal dendrites, the results of the two groups were pooled. Data were analyzed with analysis of variance (ANOVA) or t-tests when applicable. No correction for hidden spines was made, but while counting the spines in the reconstructed images, position and verification of spines were aided by the images in the single plane, to assure that all seen spines were counted. Also, no correction for shrinkage of the tissue was made. In fact, because no clearing procedures were applied, shrinkage of the tissue was minimal.
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FIG. 1.
Dendritic spines in slice cultured hippocampal neurons stained with Lucifer yellow. A: low power image (×20) of a 3-dimensional (3-D) reconstructed CA1 pyramidal neuron in a 4-wk culture. B: high power (×100) image of a 3-D reconstructed apical dendrite at 1 wk in culture showing abundant long and thin, filopodia-like projections. C: within 4 wk in culture, dendrites are populated by mature spines only. D: 3 days after addition of 50 µM picrotoxin to a mature (3-wk-old) culture, spine density and morphology were not significantly changed. E: maturation of dendritic spines was retarded in cultures raised for 2 wk in presence of 1 µM tetrodotoxin (TTX). F: 2-aminophosphonovalerate (D-APV) treatment causeda reduction in spine density but no major alteration in spine morphology. Scale bar in A = 75 µm, B-F = 5 µm.
; Pozzo Miller and Landis 1993).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
) in which cytoplasmic extensions from the postsynaptic density region embraced the presynaptic terminals also became prominent after 3 wk in culture (Fig. 3C).
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FIG. 2.
Changes in dendritic spine density across weeks in culture. Spines were counted in 40-50 segments of 3-D reconstructed images for each age group. One-week cultures had a low density of spines but a large proportion of filopodia (the difference between spine counts and total counts). Spine density increased nearly threefold between 1 and 3 wk in culture and remained unchanged at 4 wk. Filopodia were abundant in young cultures and nearly nonexistent in older ones.
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FIG. 3.
Age- and drug-induced changes in spines as seen at ultrastructural level. A: CA1 region of chemically fixed slice culture after 1 wk in vitro. Dendrites have filopodia-like protrusions ( 
). Bar, 500 nm. B: higher magnification of a filopodia seen in A, having a synaptic junction. C: rapidly frozen and freeze-substituted CA1 area of a slice culture after 4 wk in vitro, showing a dendrite with a mature spine, which typically had a concave-shaped synapse, in which cytoplasmic extensions from the postsynaptic density region embraced the presynaptic terminals. D: CA1 region of a chemically fixed slice culture, 4 wk in vitro, to which TTX was added between 1 and 4 wk. Dendrites often have immature-like filopodia with synaptic junctions. B-D: synapses (long arrows), smooth endoplasmic reticulum (SER; arrowheads), microtubules (short arrows), bar = 200 nm.
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FIG. 4.
Development of plastic properties of hippocampal neurons in slice cultures. A: population excitatory postsynaptic potential (EPSP)'s amplitude measured in stratum radiatum (top) and pyramidale (bottom) of 1-wk (left)- and 4-wk-old (right) cultures. Responses were elicited after exploration of stimulation sites in CA3 and recording sites in CA1. Superimposed traces were elicited by increasing intensity of CA3 stimulation. B: development of ability to produce long-term potentiation (LTP). EPSPs were recorded in stratum pyramidale every 60 s and, after 10 min of baseline recordings, a single, 100-Hz, 1-s tetanus was applied. EPSPs were recorded and plotted for 30 additional minutes. Data points are averages ± SE of 1-wk- (n = 10), 2-wk- (n = 8), 3-wk- (n = 8), and 4-wk- (n = 10) old cultures.
) especially after 4 wk in vitro. In nearly all slices tested, the tetanic stimulation (100 Hz for 1 s) could evoke STP of the EPSP amplitude, lasting 
5 min after application of the tetanus. The short-term potentiation had about the same magnitude in all age groups. In only 2 of the 10 young slices could the stimulation evoke a robust (
20% increase more than baseline, lasting for 30 min) LTP. This increased to 50, 60, and 75% of the slices in 2, 3, and 4 wk in culture, respectively. The mean change in EPSP size 30 min after tetanic stimulation (including slices with failures of LTP, but not including slices that did not express short-term potentiation) amounted to 20.3 ± 8.7% at 1 wk, 35.1 ± 8.8% at 2 wk, 51.4 ± 4.1% at 3 wk, and 68.9 ± 5.3% at 4 wk in culture (Fig. 4B). This difference across age of culture was highly significant when tested by ANOVA (F = 196.57 P < 0.0001).
-aminobutyric acid (GABA) antagonist picrotoxin. Chronic exposure to D-APV for 2 wk (Figs. 1F and 5, left), or D-APV/DNQX for 3 wk reduced spine density by ~25-30%. (For the APV treatment, 0.55 ± 0.018 compared with control, 0.71 ± 0.031 spines per 1 µm, and for the APV/DNQX treatment, 0.817 ± 0.019 in the drug-treated slices, compared with 1.176 ± 0.061 spines per 1 µm in the control group, F = 78.2, P < 0.01.) There was no apparent change in the shape of spines after exposure to D-APV or D-APV/DNQX. TTX, treated for 2 wk (Fig. 1E), prevented development of the spines into a mature form, as indicated by the presence of long, filopodia-like structures. The spine density was reduced by about the same magnitude, 30%, as seen with D-APV (0.65 ± 0.057 of TTX-treated slices compared with 0.915 ± 0.041 of age-matched controls, F = 11.7, P < 0.01, Fig. 5, right).
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FIG. 5.
Drug effects on dendritic spine density in slice cultures. A: exposure of cultured slices to D-APV for 2 wk caused a significant reduction in spine density by ~30% compared with age-matched controls. B: chronic treatment with TTX prevented age-dependent increase in spine density. Drugs were added to culture medium at the third day in culture, and maintained for 3 wk. Bars are means ± SE of 2-4 cases.
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FIG. 6.
Drug effects on ability to produce LTP in slice cultures. A: sample traces recorded in stratum pyramidale in control, top, picrotoxin (PTX)-treated, middle, and TTX-treated slices, bottom, in baseline condition, left, at peak of response after tetanic stimulation at time 0, middle, and 30 min after tetanic stimulation, right. B: percent change in EPSP amplitudes after a single 100-Hz tetanus of 1-s duration was evaluated in 3-wk-old cultures exposed to drugs. D-APV-treated cultures (n = 15) showed larger short-term potentiation (STP) and LTP compared with controls (n = 15). TTX treatment (n = 12) did not affect STP and only caused a small reduction of LTP compared with controls. PTX treatment (n = 15) caused not only a marked reduction of STP but also completely blocked the ability to induce LTP. B: EPSP amplitudes were normalized between controls and STP levels to separate STP from LTP changes. This analysis shows that D-APV did not enhance LTP relative to STP. Data points are means ± SE.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Emptage et al. 1995; D. Muller et al. 1993; Pozzo Miller et al. 1993; Stoppini et al. 1993). Thus we found similar developmental changes in physiological properties of the slices, measured in both the percentage of slices expressing LTP and the magnitude of the sustained change. In addition, the slice/culture allows a detailed analysis of factors that regulate the formation of dendritic spines. As hasbeen shown before, the slice develops in vitro in a surprisingly ordered manner and maintains the morphology of the cells as well as the connectivity among regions of the slice. In the absence of extrinsic afferents, e.g., those originating in the contralateral hippocampus and the entorhinal cortex, the local afferents to CA1, coming from CA3 area, proliferate and extensively innervate the basal dendritic regions of cells in CA1 (Sacaguchi et al. 1994). Thus the cells in CA1 area are innervated sufficiently to allow extracellular recording of population EPSPs generated by stimulation of CA3 neurons. Incidentally, the fact that the basal dendritic area (stratum oriens) is innervated heavily by fibers of CA3 origin may underlie the lack of difference seen here between apical and basal dendritic spines. In the absence of afferentinnervation of basal dendrites, they are expected to shrink and reduce density of spines.
and Dailey and Smith (1996) but not with studies in dissociated hippocampal cultures, where filopodia were not associated with synapsin-stained particles (Papa et al. 1995).
; Segal and Furshpan 1990). The enhanced activation resulting from removal of the glutamate antagonists may indicate that the chronic blockade causes an upregulation of the glutamate receptor. This needs to be further examined using more direct methods of estimating glutamate receptor functions.
), or by the possible saturation of the LTP generating mechanism by the blockade of inhibitory activity, which will enhance excitability of the slice. It is not likely that picrotoxin affected viability of the slices, because the labeled cells in the picrotoxin-treated slices appeared normal and had about the same spine density as the control slices. These results contradict those of M. Muller et al. (1993), who found that picrotoxin causes elimination of spines in thin hippocampal slice cultures, and those of Papa and Segal (1996), who found that picrotoxin causes an increase in spine density and shortening of spine length in dissociated cultured hippocampal neurons. In the intact brain, Bundman et al. (1994) found that seizure activity produces new spines in the dentate gyrus. In mouse somatosensory cortex organotypic cultures, picrotoxin also was found to increase spine density in pyramidal neurons (Annis et al. 1994). These discrepancies may be due to the type of preparation employed in the different studies. However, it is also likely that cells in our slice culture are more intrinsically active, and the added blockade of inhibition does not make a morphological difference. In support of this are the results of the TTX experiment. TTX blocks action potential-related synaptic activity and could be expected to enhance the ability to express LTP for the same reasons as those of D-APV. In fact, TTX caused a different effect on both spine morphology and ability to express LTP than those of D-APV/DNQX. TTX prevented or markedly retarded morphological development of spines, characterized by abundant filopodia-like structures, akin to those of the immature cells in the slice culture (Annis et al. 1994; Cooper and Smith 1992). This structural difference may explain the difference in ability to produce LTP between the TTX- and D-APV-treated slices. At any rate, after development is completed in our mature cultures, there is no simple correlation between spine density and ability to generate LTP.
; Gold and Bear 1994; Koch and Zador 1993; Wickens 1988). The fact that neurons respond to ambient afferent input by changes in spine density as well as spine morphology indicates that spines are under continuous afferent control. Hormonal factors, such as the estrus cycle in female rats, also have been shown to cause a 30% variation in spine density. Because of the pivotal role that elevation of intracellular calcium concentration plays in synaptic plasticity, dendritic spines can provide the anatomic substrate for temporal and spatial segregation of these signals. In fact, it has been shown recently that the spine is a unique calcium compartment, capable of accumulating high concentrations of calcium, relative to its parent dendrite (Guthrie et al. 1991; Muller and Connor 1991; Petrozzino et al. 1995; Segal 1995). Our results show a strong correspondence of the density of mature looking spines to the magnitude of LTP as it develops between 1 and 3 wk in culture. However, large changes in LTP magnitude also were observed in the absence of any significant change in spine density or morphology after picrotoxin treatment.
obtained LTP for EPSCs evoked by impulses in single CA3 neurons, where the number of spines involved may be very small. In fact, LTP-like plasticity in nonspiny synapses has been reported recently in immature cells (Durand et al. 1996). However, the ability to produce LTP can be strongly correlated with development and maintenance of neuronal architecture. Thus the slice culture offers an excellent controlled condition for a systematic analysis of factors regulating development and adult plasticity in a central network.
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ACKNOWLEDGEMENTS |
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We thank B. Zoltick and H. Shah for excellent technical support, Dr. C. Smith for help with the NINDS imaging facility, and Drs. R. McKay, T. S. Reese, L. C. Pozzo Miller, and J. Connor for critical reading of the manuscript.
This work was supported in part by a grant from the United States-Israel Binational Science Foundation to M. Segal.
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FOOTNOTES |
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Address for reprint requests: C. Collin, LMB, Building 36, Room 3D02, NIH, Bethesda, MD 20814.
Received 18 July 1996; accepted in final form 9 December 1996.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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