Biolistic transfection and expression analysis of acute cortical slices

Highlights

• Transfected genes can be assessed in acute slices without a need for long-term vitro culture.

• Fluorescent marker proteins are expressed in acute slices within 4 h.

• GECI-transfected acute slices can provide physiological readouts.

• Transfected acute slices display normal spontaneous neuronal activity.

Abstract

Background

Biolistic gene gun transfection has been used to transfect organotypic cultures (OTCs) or dissociated cultures in vitro. Here, we modified this technique to allow successful transfection of acute brain slices, followed by measurement of neuronal activity within a few hours.

New method

We established biolistic transfection of murine acute cortical slices to measure calcium signals. Acute slices are mounted on plasma/thrombin coagulate and transfected with a calcium sensor. Imaging can be performed within 4 h post transfection without affecting cell viability.

Results

Four hours after GCaMP6s transfection, acute slices display remarkable fluorescent protein expression level allowing to study spontaneous activity and receptor pharmacology. While optimal gas pressure (150 psi) and gold particle size used (1 μm) confirm previously published protocols, the amount of 5 μg DNA was found to be optimal for particle coating.

Comparison with existing methods

The major advantage of this technique is the rapid disposition of acute slices for calcium imaging. No transgenic GECI expressing animals or OTC for long periods are required. In acute slices, network interaction and connectivity are preserved. The method allows to obtain physiological readouts within 4 h, before functional tissue modifications might come into effect. Limitations of this technique are random transfection, low expression efficiency when using specific promotors, and preclusion or genetic manipulations that require a prolonged time before physiological changes become measurable, such as expression of recombinant proteins that require transport to distant subcellular localizations.

Conclusion

The method is optimal for short-time investigation of calcium signals in acute slices.

Introduction

Genetically modified mice (GMM) models are being used since three decades to understand brain functions and diseases. Well-designed GMM have successfully led to the identification of gene functions, the understanding of brain diseases, and the development of effective treatments. It is very frequent that researchers need to perform an experiment in a GMM that cannot be pursued in an animal model that has only a single genetic modification. Therefore, it is often required to introduce another genetic modification to properly address an experimental question. For instance, when considering to perform calcium imaging to record neuronal activity in a GMM model, it is indispensable to use a genetically encoded indicator (e.g. by means of fluorescence markers) in order to obtain a physiological readout. This can be achieved by either introducing a second genetic modification into the GMM model of interest or by transducing the GMM in order to label the targeted cells with a GECI (e.g. by means of viral transduction in a living animal). However, the creation of a second modification in the GMM is time consuming and requires permission of the procedures. On the other hand, in vivo methods for acute modulation of gene expression (for a review see (Cwetsch et al., 2018)) in living animals are fast and reliable, but they have several drawbacks. For instance, viral transduction can trigger immunogenicity, cytotoxicity and requires complicated surgical procedure.

As an alternative to the in vivo approach, researchers had employed acute slices and organotypic cultures (OTCs) of mammalian CNS to study neuronal activity. Acute slices and OTCs have their own advantages and disadvantages; for a review see (Lossi et al., 2009). In the OTC system, which has been first developed by Gähwiler (Gähwiler, 1981) and later modified (Gähwiler et al., 1997; Stoppini et al., 1991), there are many pharmacological and genetic manipulations, which have been demonstrated to be successfully reproducible (Cho et al., 2004; Ray et al., 2000). Moreover, there are some properties of these OTCs that substantially differ from the characteristics acquired during physiological brain maturation in vivo (Fenili and de, 2003). The main disadvantage of using the OTCs system is that not all brain areas allow culturing. Another disadvantage is that OTCs preparation is restricted around early development. Preparing OTC from older animals is feasible, however, the neuronal survival rate is low (Bruce et al., 1995). On the other hand, acute slices can be obtained from young and older animals and from many brain areas. The short living acute slices have the advantage over the OTCs that the neuronal network connection is maintained and structural modifications are low in comparison to OTCs cultured for days or weeks. However, the thick acute slices (∼ 350−400 μm) can be difficult to image if the transfected cells are located deep in the tissue. Moreover, some studies have shown that the acute slice preparation procedure can impair spinogenesis (Kirov et al., 2004) and thereby synaptic plasticity can be affected (Bourne et al., 2007).

Transfection is an effective analytical tool allowing to study gene and protein function in cells. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means to record neural activity in the CNS. To study the physiology of neural cells, acute brain slices explanted from mouse brain can be perfused with artificial cerebrospinal fluid (ACSF) and subjected to electrophysiological measurement after recovery. Recently, the introduction of genetically encoded calcium indicators (GECIs) has offered a less invasive strategy to label neurons with fluorescence markers and to record calcium signals simultaneously. GECIs are based on chimeric fluorescent proteins, and can be used to monitor calcium transients in living cells and organisms (Tian et al., 2012). Because they are encoded by DNA, GECIs can be delivered to the intact brain and targeted to defined populations of neurons and specific subcellular compartments for repeated long-term, measurements in vivo (Fosque et al., 2015). Alternatively, these genetic indicators can be transfected in vitro (Hamad et al., 2011; Jack et al., 2018) to study receptor pharmacology. When compared to the electrophysiological approach, calcium imaging has the advantage to simultaneously allow imaging of a large neuronal network population when loaded with a chemical calcium indicator (e.g. OGB-1AM (Göbel and Helmchen, 2007; Hamad et al., 2015), or single cell recording (Akerboom et al., 2012; Hamad et al., 2011; Hires et al., 2008; Tian et al., 2009) without significant photo-bleaching and with a good signal-to-noise ratio. However, calcium imaging has a low temporal resolution in comparison to standard electrophysiological techniques, but it minimizes cellular damage since it does not require electrodes that penetrate into the tissue.

Biolistic transfection is an effective method to transfect brain slices in vitro and ex-vivo, which is based on blasting gold particles coated with the desired DNA plasmid into the cells. Biolistic transfection offers the possibility of transfection with different DNAs (Ma et al., 2007), and it is highly efficient in transfecting neurons. As a major drawback, the gold particles usually do not penetrate deep into thick slices, and, due to the particle bombardment, dendritic injury leading to cell death can be caused (Alasia et al., 2015). Alternatively, many non-viral nanosystems can be employed (O’Mahony et al., 2013) (e.g. cationic and stealth liposomes, cationic polymers, dendrimers, cyclodextrin, and cell-penetrating peptides (CPPs)). However, they do not target a specific cell type and they could elicit cytotoxicity.

The current study aimed at testing the possibility to transfect acute slices shortly after preparation and to process these slices for physiological studies without a need to cultivate the acute slices in vitro. As a GMM model, we exemplarily used a Reelin conditional knockout mouse (Reln^cKO) and their control littermate to show that physiological and pharmacological readouts can be obtained and compared between different experimental groups by means of calcium imaging in a mouse model whithout a GECI transgene. Our results demonstrate that acute slices transfected with the GECI GCaMP6s exhibit a remarkable fluorescent protein expression 4 h post-transfection, allow to detect spontaneous activity and to compare receptor pharmacology in such a mouse model. The possibility to record acute slices in a short time-window has the advantage to reduce any structural modifications that acute slices undergo over the time.