In this report, we showed a method for preparation of hippocampal slice culture and consecutive analysis of Aβ. The cultured hippocampal slice preserved tissue architecture, neural circuits, and synaptic dynamics 6,27,28. Here, we showed that the existence of microglia, astrocyte, and neuron in a cultured slice for several weeks or potentially several months (Fig. 2). Our chronological immunoblotting analysis indicated that glial cells have proliferated for 1 to 2 weeks, and then be maintained for several weeks (Fig. 2G). Besides, galectin-3, which required for microglia activation and proliferation in injured brain 26, was elevated for1 week after preparation and then declined (Fig. 2I). These data may indicate that microglia were activated to eliminate dead or apoptotic cells. The expression levels of synapse marker proteins, synaptophysin and PSD95 decreased from 1 to 4 days, and increased after 7 days 9. Quantitative analyses suggested that expression levels of APP were not changed significantly, and expression levels of BACE1 were stable after 7 DIV (Fig. 3). Since BACE1 is mainly expressed in neuron, the expression level of BACE1 decreases due to neuronal cell death in the first week of culture (Fig. 2F). Immunoblotting analysis indicated that the molecular weight of BACE1 changed slightly after 4 DIV, which may represent the effect of the BACE1 maturation process. Newly synthesized BACE1 is cleaved at its prodomain before transport to the trans-Golgi network 32. These results indicated that slice culture may be suitable for continuous ex vivo analysis of APP cleavage. These results provide that slices cultured after 2 or 3 weeks are suitable for analysis of neural functions and APP processing.
Aβ is formed after sequential cleavage of the APP by β- and γ-secretase, accumulates in the central nervous system and subsequently initiates the neural dysfunction. An excessive accumulation of Aβ results in aggregation and amyloid deposition in the brains of AD patients 33. The γ secretase cleaves within the transmembrane region of APP and can generate a number of isoforms of 30–51 amino acid residues in length 34. The Aβ40 and Aβ42 are the most common isoform of Aβ in brain and the main component of the amyloid plaques found in the brains of AD. The Aβ40 form is the more common of the two, but Aβ42 is the more fibrillogenic and is thus associated with disease states 35.
Since the pathological changes of AD are progressing slowly, a model for continuously analyzing central nervous function for a long period is required. In this report, we evaluated the method to investigate Aβ formation continuously using cultured hippocampal slices. To estimate Aβ production, we measured Aβ40 and Aβ42 secreted from hippocampal slices using a two-site ELISA assay. In culture medium, four cultured hippocampal slices secreted 200–400 pg Aβ40 and 23–50 pg for 24 h 9. These data indicated that 2.0–4.0 pg Aβ40 and 0.2–0.5 pg Aβ42 were secreted by a cultured hippocampal slice in 1 h. In accordance with previous reports 13, the ratio of Aβ42 to Aβ40 was between 10 to 17 % (Fig. 4). As a result of examining the differences between species, there was no difference between the amount of Aβ40 and sAPP secretion in mouse and rat hippocampal slices (Fig. 5).
Here we tried to optimize the incubation period and the amount of slices for analysis of Aβ. We placed 1 to 6 hippocampal slices on a membrane cup for one week culture, and incubate these slices for 24h with fresh SCM for Aβ assay. The concentration of Aβ linearly increases corresponding to the number of slices (Fig. 4). For time-lapse analysis of Aβ secretion, we cultured four hippocampal slices and incubate for 1 to 90 h with fresh SCM after washing three times with 1 mL fresh SCM. Our results indicate that single hippocampal slice secreted 2.68 ± 0.82 pg for 1h (Fig. 4B, C). The dispersion of Aβ assay indicates that the carryover medium significantly impacts the measurement results with short term incubation. When many slices are placed on a membrane cup, it is easy to analysis of Aβ production, but proper placement of slices is difficult. We suggest that it is appropriate to incubate two to six hippocampal slices in a membrane cup.
The α-secretase family alternatively cleaves the APP within the Aβ sequence. Thus, α-cleavage precludes Aβ formation and is considered to be part of the non-amyloidogenic pathway in APP processing 36. In cultured hippocampal slice, alternative cleavage of APP is conserved (Fig. 3), and thus application of the α-secretase inhibitor increased secretion of Aβ 9.
Synaptic plasticity is activity dependent changes in synaptic function that is thought to play a crucial role in learning and memory 37. Two forms of long-term plasticity, long-term depression (LTD) and long-term potentiation (LTP) are involved in long term memory and induce in acute brain slice. In cultured hippocampal slice, the synaptic plasticity was occurred 28, and synapse formation or elimination is induced by repetitive induction of LTP or LTD 7,8. Because synaptic plasticity is disturbed in neuropsychiatric disorder model including AD 38,39, rodent hippocampal slice culture is a good in vitro experimental model for pathological changes in the central nervous system. Here we showed no difference in Aβ secretion between mouse and rat hippocampal slice culture (Fig. 5).
Because of individual differences and difficulty of manipulation in animal models, comparative analyses before and after administration in same preparation are not easy. To evaluate drug efficacy, however, this comparative analysis between before and after administration in same cultured preparation is meaningful. Because primary dissociated neural cultures are vulnerable to drug washout during media changes, comparative analysis using the same preparation is extremely difficult in actuality. Filter membrane cups allow easy handling of preparations; therefore, we were able to collect and change culture media without damage and perform comparative analyses using the same preparations (Fig. 4). Because the membrane cup is designed for disposable use, slice culture is a costly method. Using self-made devices and filter membranes can reduce costs 40.
Slice culture are usually derived from early postnatal or embryonic rodents. Because essential cytoarchitecture are already established but neural circuits are still immature, early postnatal periods (day 0 to 10) are ideally suited for culturing. Some attempts have been made to culture adult tissue of model animal for elucidating age-related neurodegenerative diseases 41.
Slice culture have been used for wide research field ranging from physiology, pharmacology, endocrinology, biochemistry, and development to pathology. We anticipate that hippocampal slice culture will be used to reveal molecular and cellular basis of neuropsychiatric disorder as ex vivo model.
We provide a method of consecutive analysis protein secretion from brain slices to study neurodegenerative disease. Hippocampal slice culture is an experimental system that effectively keeps the in vivo neuronal network and seems to be a convenient method to study the cellular and molecular mechanism underlying the neuropsychiatric disorders including AD, and to evaluate therapeutic approach for such diseases.