Development of rat CA1 neurones in acute versus organotypic slices: role of experience in synaptic morphology and activity

Abstract

Despite their wide use, the physiological relevance of organotypic slices remains controversial. Such cultures are prepared at 5 days postnatal. Although some local circuitry remains intact, they develop subsequently in isolation from the animal and hence without plasticity due to experience. Development of synaptic connectivity and morphology might be expected to proceed differently under these conditions than in a behaving animal. To address these questions, patch-clamp techniques and confocal microscopy were used in the CA1 region of the rat hippocampus to compare acute slices from the third postnatal week with various stages of organotypic slices. Acute slices prepared at postnatal days (P) 14, 17 and 21 were found to be developmentally equivalent to organotypic slices cultured for 1, 2 and 3 weeks, respectively, in terms of development of synaptic transmission and dendritic morphology. The frequency of inhibitory and excitatory miniature synaptic currents increased in parallel. Development of dendritic length and primary branching as well as spine density and proportions of different spine types were also similar in both preparations,at these corresponding stages. The most notable difference between organotypic and acute slices was a four- to five-fold increase in the absolute frequency of glutamatergic (but not GABAergic)miniature postsynaptic currents in organotypic slices. This was probably related to an increase in complexity of higher order dendritic branching in organotypic slices, as measured by fractal analysis, resulting in an increased total synapse number. Both increased excitatory miniature synaptic current frequency and dendritic complexity were already established during the first week in culture. The level of complexity then stayed constant in both preparations over subsequent stages, with synaptic frequency increasing in parallel. Thus, although connectivity was greater in organotypic slices, once this was established, development continued in both preparations at are markably similar rate. We conclude that, for the parameters studied, changes seem to be preprogrammed by 5 days and their subsequent development is largely independent of environment.

Figure 1. Relative frequency of action potential-dependent and miniature currents in acute and organotypic slices during development

A, current traces showing the total synaptic activity at DIV21 in control solution (upper trace), miniature currents in TTX (1 μM, middle trace) and mEPSCs remaining when bicuculline (10 μM) was added to the bath solution (lower trace). B, frequencies of synaptic activity for organotypic and acute slices. Frequencies of both spontaneous and miniature currents increased during development (P < 0.05, n > 4 in each group). Hatched columns represent mIPSC frequency after subtraction of the bicuculline-insensitive mEPSCs (black columns). The composite columns (hatched + black) and error bars represent the mean ±s.e.m. recorded frequencies of total miniature currents. C, mEPSC frequencies on a larger scale. The mEPSCs become more frequent with development, with consistantly higher values for organotypic slices (P < 0.0005).

Figure 2. Amplitudes and kinetics of miniature currents in organotypic (blue) and acute slices (red) during development

A and E, cumulative relative amplitude distributions of mEPSCs and mIPSCs, respectively; bin width 1 pA. Numbers in parenthese refer to the number of slices. B and F, mean of the median amplitudes of mEPSCs and mIPSCs, respectively. C and G, mean rise times of mEPSCs and mIPSCs, respectively. D and H, mean decay time constants of mEPSCs and mIPSCs, respectively. *Significant differences at P ≤ 0.05 by two-way ANOVA. Asterisks below the x-axis indicate a significant difference between acute and organotypic slices; asterisks above the x-axis indicate significant changes during development. •, data from acute slices; □, data from organotypic slices.

Figure 3. Morphology of CA1 neurones in acute and organotypic slices

Note the overall greater complexity of neurones in organotypic slices throughout development. The scale bar in each panel represents 50 μm.

Figure 6. The spine density and shape change similarly during in vivo and in vitro development

A, a skeletonized neurone as used for fractal analysis. The different compartments of the dendritic tree are indicated. The colour key refers to A and B. B, spine density during development. Open symbols with dotted lines represent densities in organotypic slices and filled symbols with continuous lines are data in acute slices. C, a confocal micrograph from a DIV7 slice showing the five different spine shapes analysed. The colours for each spine type are the same as in D. D, plot of spine shape distribution in organotypic and acute slices. Each bar represents the mean percentage ±s.e.m. of spine type. The pattern of spine shape changes was very similar during development in vivo as seen in acute slices and in vitro. * Significant differences, P < 0.05.

Figure 4. Fractal dimension of CA1 neurones in organotypic (□) compared to acute slices (•)

No difference was found during development; organotypic slices featured a higher fractal dimension at all stages. * Significant difference between acute and organotypic slices, P < 0.0001.

Figure 5. Development of spines in CA1 neurones in organotypic and acute slices

Note the prevalence of filopodia at early stages and the increased density of spines at later stages. A variety of spine shapes can be seen throughout development. A, examples of resolution of primary dendrites in stratum radiatum. B, examples of spines in the branches of dendrites in statum radiatum or stratum lucidum, between which there are no significant differences. Scale bars 5 μm.