Focal macromolecule delivery in neuronal tissue using simultaneous pressure ejection and local electroporation

Abstract

Electroporation creates transient pores in the plasma membrane to introduce macromolecules within a cell or cell population. Generally, electrical pulses are delivered between two electrodes separated from each other, making electroporation less likely to be localised. We have developed a new device combining local pressure ejection with local electroporation through a double-barrelled glass micropipette to transfer impermeable macromolecules in brain slices or in cultured HEK293 cells. The design achieves better targeting of the site of pressure ejection with that of electroporation. With this technique, we have been able to limit the delivery of propidium iodide or dextran amine within areas of 100–200 μm diameter. We confirm that local electroporation is transient and show that when combined with pressure ejection, it allows local transfection of EGFP plasmids within HEK293 cells or within cerebellar and hippocampal slice cultures. We further show that local electroporation is less damaging when compared to global electroporation using two separate electrodes. Focal delivery of dextran amine dyes within trapezoid body fibres allowed tracing axonal tracts within brainstem slices, enabling the study of identified calyx of Held presynaptic terminals in living brain tissue. This labelling method can be used to target small nuclei in neuronal tissue and is generally applicable to the study of functional synaptic connectivity, or live axonal tracing in a variety of brain areas.

Acoustic Overexposure Increases the Expression of VGLUT-2 Mediated Projections from the Lateral Vestibular Nucleus to the Dorsal Cochlear Nucleus

“…Extracellular medium was then changed to an artificial cerebrospinal solution containing (in mM) 125 NaCl, 2.5 KCl, 10 glucose, 1.25 NaH 2 PO 4 , 26 NaHCO 3 , 2 sodium pyruvate, 3 myo-inositol, 0.5 ascorbic acid, 2 CaCl 2 , and 1 MgCl 2 (pH 7.4 when gassed with 95% O 2 ∶ 5% CO 2 ). Dextran amine dye (see below for composition) was delivered under binocular control, using a custom made combined pressure and electroporation system coupled to a double-barrelled glass pipette (TGC200-10, Clark Electromedical, Harvard Apparatus Ltd, Kent, UK) as described in Barker et al [39] ( Figure S1 ). Five ms square wave pulses were applied at 100 Hz for up to 10 s with 2 pressure ejection pulses of 10–20 psi, 10 ms occurring mid-way through the electroporation.…”

A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits

“…The development of electroporation techniques that allow for the delivery of marcromolecules through the creation of transient pores in the plasma membrane, have been used in several brain preparations such as the whole cerebellum (Yang, Appleby et al, 2004), cerebellar organotypic slice cultures (Murphy and Messer, 2001), the spinal cord (Bonnot, Mentis et al, 2005), the brainstem (Barker, Billups et al, 2009), and the thalamic reticular nucleus (Pinault, 1996). However, these techniques often require the use of complex electroporation/pressure ejection machinery (Barker, Billups et al, 2009) or the placing of the slice between electrode plates (Bonnot, Mentis et al, 2005), platinum wires (Murphy and Messer, 2001), or in an electroporation chamber (Bright, Kuo et al, 1996), all of which can affect neuronal health, and accessibility for subsequent recording.…”

“…However, these techniques often require the use of complex electroporation/pressure ejection machinery (Barker, Billups et al, 2009) or the placing of the slice between electrode plates (Bonnot, Mentis et al, 2005), platinum wires (Murphy and Messer, 2001), or in an electroporation chamber (Bright, Kuo et al, 1996), all of which can affect neuronal health, and accessibility for subsequent recording.…”

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage

“…Xenopus (Chernet and Levin, 2012;Falk et al, 2007), chick (Nakamura et al, 2004;Voiculescu et al, 2008), zebrafish (Bansal et al, 2009;Huang et al, 2007), insects (Ando and Fujiwara, 2013); organ explants, e.g. brain (Barker et al, 2009;del Rio and Soriano, 2010), retina (Matsuda and Cepko, 2004), cochlea (Driver and Kelley, 2010), gut (Abud et al, 2008), gonads (Gao et al, 2011); or in vitro reconstituted tissues, e.g. spheroids (Chopinet et al, 2011;Mellor et al, 2006).…”