Organotypic brain cultures, including slice cultures, are used to study the central nervous system owing to their advantages over both in vivo and in vitro research platforms (Cho et al., 2007; Uesaka et al., 2018). Sophisticated cultured brain slices retain tissue structure, neural circuit, and extracellular environment and replicate the native status of the in vivo context. Hippocampal slice cultures are widely used to investigate the effects of pharmacological, biochemical, physiological, and genetic manipulation on neuronal and glial cells (Bergold et al., 1993; Ikegaya, 1999). Three typical methods are used to maintain neural tissue in vitro: roller-tube cultures, matrix-embedded cultures, and membrane interfaces (Gähwiler et al., 1997). The membrane interface brain slice culture was improved for practical use by Stoppini et al. (1991). Since a sterilized filter membrane cup is designed to easily change the medium, this apparatus as an interface is helpful for the long-term maintenance of the explant tissue. Cultured hippocampal slices on the filter membrane cup were amenable to complete replacements of the culture medium. These advantages allowed us to keep the brain slices in culture for several months. Previously, we reported on neural circuits, synaptic plasticity, neurodevelopment, and neurodegenerative diseases using cultured hippocampal slices (Tominaga-Yoshino et al., 2002; Shinoda et al., 2005; Kamikubo et al., 2017).

Alzheimer’s disease (AD) is a chronic neurodegenerative brain disorder that usually starts slowly and worsens over a long period and is the most common cause of dementia in the elderly. Numerous genetic and biochemical studies have supported the hypothesis that excessive accumulation of amyloid β peptide (Aβ) results in aggregation and amyloid deposition in the brains of patients with AD (amyloid hypothesis) (Hardy and Selkoe, 2002). Aβ is derived from the amyloid precursor protein (APP), an integral type I membrane protein expressed in many tissues and concentrated in synapses. APP is commonly cleaved by membrane proteases in the secretase family: α-, β-, and γ-secretase. Aβ is generated by sequential cleavage by β-secretase (BACE1) (Vassar et al., 1999) and γ-secretase, whereas alternative cleavage by α-secretase prevents Aβ production (De Strooper and Annaert, 2000). Since neuronal activity is known to be an important regulator of APP β cleavage (Kamenetz et al., 2003; Sakurai et al., 2008), the regulation of cleavage, accumulation, and elimination of Aβ have been explored as a target for the therapy and prevention of AD.

Aβ is the main component of highly insoluble senile plaques in AD brain, but it is also known to form soluble and highly toxic aggregate, oligomers. Although amyloid plaques have been the first focus of study in the amyloid hypothesis, it has recently become clear that many toxic factors, including Aβ oligomers (Cline et al., 2018), are present. Aβ and other peptide oligomers have been reported to cause cytotoxicity and neurodegeneration (Caruso et al., 2019). On the other hand, APP cleavage products other than Aβ have been reported to be neurotoxic or neuroprotective (Chasseigneaux and Allinquant, 2012; Willem et al., 2015; Kaneshiro et al., 2022). Furthermore, previous studies showed that Aβ itself induces the secretion of neuroprotective factors (Zimbone et al., 2018). To elucidate the early pathogenesis of AD, it is necessary to clarify the function and behavior of Aβ monomers, oligomers, and other APP cleavage products.

A large amount of data from cell line model-based assays has revealed the molecular mechanism of Aβ secretion to understand AD (Hardy and Selkoe, 2002). Although cell line models are powerful tools for molecular biological studies, discordance has been observed between cell lines and animal models. Since neuronal and glial cells are morphologically and functionally specialized, dissociated primary cultures of neuronal tissues have the advantage of revealing the pathological processes and molecular mechanisms of neuropsychiatric disorders. Although experiments using cultured neurons provide important information, they exhibit loss of the tissue architecture of the brain. Since neural circuits, glial networks, and extracellular environments are essential in neurodevelopment, and neurodegenerative diseases, including AD, analysis using cultured neurons alone is insufficient (Vassar et al., 1999; Ichikawa-Tomikawa et al., 2012; Irie et al., 2012; Sethi and Zaia, 2017). However, several disadvantages are associated with animal model experiments, including the requirement of a long duration, substantial cost, and laborious monitoring of multiple parameters following manipulations. Furthermore, genetically engineered animals may be affected by compensatory and developmental changes (Kreiner, 2015). For this reason, hippocampal slice culture may be a potential technique for linking animal models and neural cells.

In the current study, we describe the methods of organotypic hippocampal slice culture and consecutive analysis of Aβ and related products, which play a central role in the pathogenesis of AD. The hippocampus is thought to be one of the first brain regions to suffer damage in AD (Hyman et al., 1984). With the method reported here, we could produce nearly 100 slices in a single cultivation and maintain hippocampal slices for more than 1 month resulting in an increased analysis efficiency. Continuous collection and analysis of Aβ is a powerful approach for elucidating the cellular and molecular mechanisms underlying AD. Long-term and consecutive analyses are also required to develop and evaluate therapeutic agents and treatment approaches for AD.

Herein, we present a method for preparing hippocampal slice cultures and consecutive analyses of APP processing. In this study, we sliced the hippocampus to a thickness of 400 μm. In many studies using brain organotypic cultures, the brain was sliced at 250–400 μm thick (Stoppini et al., 1991; Tominaga-Yoshino et al., 2002; Uesaka et al., 2012; Humpel, 2015). When thicker slices, such as 600 or 700 μm, are used, refluxing of the culture medium is required (Rambani et al., 2009), and fewer slices can be made from the hippocampus. Stable results were obtained by culturing four to five 400 μm thick slices in one cup and analyzing them together (Figure 4). Cultured hippocampal slices preserve the tissue architecture, neural circuits, and synaptic dynamics (Stoppini et al., 1991; Buchs et al., 1993; Muller et al., 1993). Because of individual differences and the difficulty of manipulation in animal models, comparative analyses before and after administration of the same preparation are not accessible. However, to evaluate drug efficacy, this comparative analysis before and after administration of the same culture preparation is meaningful. As primary dissociated neural cultures are vulnerable to drug washout during medium changes, a comparative study using the same preparation is extremely difficult. Filter membrane cups allow easy handling of preparations; therefore, we were able to collect and change the culture media without da mage and perform comparative analyses using the same preparations (Figure 4). Because the membrane cup is designed for disposable use, slice culture is costly. The use of self-made devices and filter membranes can reduce costs (Koyama et al., 2007).

Here, we showed the existence of microglia, astrocytes, and neurons in slices cultured for 2 months (Figure 2). Our chronological immunoblotting analysis indicated that glial cells proliferated for 1–2 weeks and were then maintained for several weeks (Figure 2G). In addition, the level of galectin-3, which is required for microglial activation and proliferation in the injured brain (Lalancette-Hébert et al., 2012), was elevated for a week after preparation and then declined (Figure 2I). The expression patterns of APP and BACE1 in cultured hippocampal slices accumulated at the stratum radiatum, similar to the in vivo distribution (Supplementary Figure S1). The expression levels of the synapse marker proteins synaptophysin and PSD95 decreased from 1 to 4 days and increased after 7 days (Kamikubo et al., 2017). Since synapses and neural circuits have formed and matured after 14DIV(Muller et al., 1993; Tominaga-Yoshino et al., 2002), quantitative analysis indicates that APP and BACE1 expression levels are stable (Tukey’s Multiple Comparison Test, p > 0.05, versus 14DIV, Figures 3A–C). Similar to previous studies (Kamikubo et al., 2017), immunoblotting analysis indicated that the electrophoretic mobility of BACE1 changed slightly after 4 DIV, which may represent the effect of the BACE1 maturation process. The newly synthesized BACE1 is cleaved at its prodomain before being transported to the trans-Golgi network (Benjannet et al., 2001). Further analysis of BACE1 intracellular localization is required; however, Aβ production is determined not only by BACE1 expression and enzyme activity but also by α-secretase and γ-secretase activity. Our results indicate that slices cultured for 2 or 3 weeks are suitable for analyzing neural functions and APP processing. We evaluated the differences between the slices and dissociated cultures; DAPT treatment accumulated APP-CTF to the same extent in both dissociated and slice cultures (Figure 3D). The α-secretase family of proteins alternatively cleaves APP within the Aβ sequence. Thus, α-cleavage precludes Aβ formation and is considered part of the non-amyloidogenic pathway in APP processing (Lammich et al., 1999). In cultured hippocampal slices, alternative cleavage of APP is conserved (Figures 3F–H); thus, the application of the α-secretase inhibitor increases the secretion of Aβ (Kamikubo et al., 2017). On the other hand, the ratio of mature APP-FL (mAPP) to immature APP-FL (imAPP) was not altered by secretase inhibitor treatment (Figure 3H). This result indicates that secretase activity does not affect APP maturation. Secretase inhibition may result in accumulation of only mAPP or both mAPP and imAPP. Further investigation is needed to explain why secretase inhibition increases both mature and immature forms of APP. Since secretases are required for neuronal growth and maturation (Chow et al., 2010), inhibition of both secretases could have adversely affected neurons and reduced GAPDH expression (Figure 3E). Western blotting is at best a semi-quantitative method and dependent upon the proper choice of loading controls. Long-term incubation may alter the expression levels of β-actin and GAPDH, which are generally considered loading controls. Therefore, further study is needed to compare different culture periods. In this report, immunostaining of slice cultures was performed to confirm the survival of neurons and glial cells and their distribution over a 2-month period.

Aβ is formed after the sequential cleavage of APP by β-and γ-secretase, which accumulates in the central nervous system and subsequently initiates neural dysfunction. Excessive accumulation of Aβ results in aggregation and amyloid deposition in the brains of AD patients (Blennow et al., 2006). γ-Secretase cleaves within the transmembrane region of APP and can generate several isoforms of 30–51 amino acid residues in length (Olsson et al., 2014). Aβ₄₀ and Aβ₄₂ are the most common isoforms of Aβ in the brain and are the main components of amyloid plaques found in AD brains. Since the pathological changes of AD progress slowly, a model for continuously analyzing central nervous function for an extended period is required. This study evaluated a method to continuously investigate Aβ formation using cultured hippocampal slices. To estimate Aβ production, we measured Aβ₄₀ and Aβ₄₂ secretion from hippocampal slices using a two-site ELISA assay. In the culture medium, four hippocampal slices secreted 200–400 pg Aβ₄₀ and 23–50 pg Aβ₄₂ for 24 h (Kamikubo et al., 2017). 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. According to previous reports (Kamenetz et al., 2003), the ratio of Aβ₄₂ to Aβ₄₀ is between 10 and 17% (Figure 4). Neurodegenerative diseases are well characterized in primary cultures of rats or mice. However, differences between species are rarely analyzed. We, therefore, evaluated APP processing in hippocampal slice cultures from these rodents. Examination of the differences between species revealed no difference between the amount of Aβ₄₀ and sAPP secretion in mouse and rat hippocampal slices (Figure 5). Cultured rodent hippocampal slices are useful for analyzing APP processing. The Aβ₄₀ form is the most common of the two; however, human Aβ₄₂ is more fibrillogenic and is thus associated with disease states (Yin et al., 2007). Since rodent Aβ is poorly aggregated (Jankowsky et al., 2007), cultured hippocampal slices are not a good platform for analyzing Aβ aggregation and accumulation. The addition of chemically synthesized or recombinant human Aβ should be used to analyze the regulation of endogenous Aβ production by using antibodies specific to mouse/rat Aβ.

Furthermore, we attempted to optimize the incubation period and number of slices for the analysis of Aβ. We placed 1 to 6 hippocampal slices on a membrane cup for 1 week of culture and incubated these slices for 24 h with fresh SCM for the Aβ assay. Aβ concentration increased linearly with the number of cultured slices (Figure 4). For time-lapse analysis of Aβ secretion, we cultured four hippocampal slices and incubated them for 1–90 h with fresh SCM after washing three times with 1 ml fresh SCM. Our results indicate that a single hippocampal slice secreted 2.68 ± 0.82 pg for 1 h (Figures 4B,C). The dispersion of the Aβ assay indicated that the carryover medium significantly affected the measurement results with short-term incubation. When many slices are placed on a membrane cup, it is possible to analyze Aβ production, but proper placement of slices is difficult. We suggest that it is appropriate to incubate 2 to 6 hippocampal slices in a membrane cup.

Slice cultures are usually derived from early postnatal or embryonic rodents. Because an essential cytoarchitecture has already been established, neural circuits are still immature, and early postnatal periods (days 0 to 10) are ideally suited for culturing. Some attempts have been made to culture adult tissues of animal models to elucidate age-related neurodegenerative diseases (Mewes et al., 2012). Slice cultures have been used in various research fields, including physiology, pharmacology, endocrinology, biochemistry, and development of pathology (Ikegaya, 1999; Humpel, 2015; Hiragi et al., 2017; Richter et al., 2020). Since rodent Aβ has low aggregation properties, it is possible to analyze Aβ aggregation and toxicity by exogenous administration of human Aβ, which has high aggregation properties. Other groups have been able to reproduce pathological protein aggregates deposition by injection seed of Aβ aggregates, α-synuclein aggregates, or extracts from the brains of patients with neurodegenerative diseases (Wu et al., 2020). We cultured hippocampal slices for several months using the methods described in previous reports (Kamikubo et al., 2006, 2017). Using slice cultures of hippocampi derived from early-onset models of neurodegenerative diseases (Saito et al., 2014; Novotny et al., 2016; Scheiblich et al., 2021), we can reveal the early processes of these diseases. The use of brain slice cultures of wild-type rodents to construct a model of neurodegenerative diseases may avoid the compensatory mechanism of genetic modification. Furthermore, by using cultured brain slices, we can analyze embryonically lethal genetic modifications. Specific cells can be removed from cultured hippocampal slices with drug administration (Vinet et al., 2012; Richter et al., 2020). Co-culturing slices from the brains of mice with distinct genetic modifications enables analyses that are not feasible in vivo (Uesaka et al., 2012, 2018). Brain organ culture only applies to analyzing a limited number of brain regions, making diagnosing neurodegenerative diseases in a wide range of brain regions challenging. We anticipate that hippocampal slice culture can be used as an ex vivo model to reveal the molecular and cellular basis of neuropsychiatric disorders.

This study provides a method for consecutive analysis of protein secretion from brain slices to study neurodegenerative diseases. Hippocampal slice culture is an experimental system that effectively maintains the in vivo neuronal network and is a convenient method to study the cellular and molecular mechanisms underlying neuropsychiatric disorders, including AD, and to evaluate therapeutic approaches for such diseases.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Animal Experimentation Committee of Juntendo University.

YK conceived and designed the study and wrote and reviewed the manuscript. YK, YZ, HJ, KN, YH, and NT contributed to investigation and data analysis. YK and TS supervised the study. All the authors approved the final version of the manuscript.

This work was supported by JSPS KAKENHI Grant Numbers JP20K07765 and JP17K09040 (to YK) and grants-in-aid from the TERUMO LIFE SCIENCE FOUNDATION (to YK).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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