7.1. Microinjection

Microinjection is a versatile transfection method, suitable for almost all cells. The technique involves direct insertion of a hollow microneedle into a subcellular location of the membrane and delivers a precise amount of biomolecules into cells irrespective of their size, shape, and chemical nature [136]. The approach is quite labor-intensive and occasionally causes substantial stresses due to disruption of the plasma membrane, resulting in decreased survival rates of transfected neurons. Despite these drawbacks, microinjection has successfully delivered exogenous proteins, cDNA constructs, peptides, drugs, and particles into transfection-challenged individual neurons. One such example is the delivery of active recombinant enzymes (caspase-3, -6, -7, and -8) into individual primary neurons. The neurons displayed caspase-specific responses, including prolonged time-dependent apoptosis by caspase-6 (>0.5 pg/cell) [137]. The selectively toxic of Aβ_1–42 via activation of the p53 and Bax proapoptotic pathway to only neurons was also proved by microinjecting Aβ_1–40, Aβ_1–42, and control reverse peptides Aβ_40–1 and Aβ_42–1 or cDNAs expressing cytosolic or secreted Aβ_1–40 and Aβ_1–42 in primary human neuron cultures, neuronal, and non-neuronal cell lines [138]. The mechanistic dissection of single-neural stem cell behavior in tissue was further evaluated by microinjection. The microinjection set-up consisted of a phase-contrast microscope with epifluorescence, trajectory, and micromanipulator [139]. Although current imaging techniques are equipped to monitor such behavior, the genetic manipulation tools are still devoid of achieving a balance between the gene expression and timescale for the singular gene product. Microinjection in mouse embryonic brain organotypic slice culture targeting individual neuroepithelial/radial glial cells (apical progenitors) avoided these shortcomings. The apical progenitor microinjection acutely manipulated the single-neural stem, and progenitor cells within the tissue and the cell cycle parameters otherwise indecipherable to apical progenitors in utero, go-through self-renewing divisions and neurons were produced. The microinjection of recombinant proteins, single genes, or complex RNA blends stimulated acute and distinct modifications in the behavior of apical progenitor cells and also changed the destiny of progeny [140]. Further, the role of two essential genes in mammalian neocortex expansion, namely the human-specific gene ARHGAP11B [141] and Insm1 [142] was assessed via microinjection.

Another study highlighted the fast and efficient CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats- associated protein 9) technology for the disruption of gene expression involved in neurodevelopment [143,144,145,146]. The technology eradicates the restrictions of transgenic knockouts and RNAi-mediated knockdowns. A radial glial cell (RGCs) in telencephalon slice of heterozygous E14.5 Tis21:: GFP mice were microinjected as shown in Figure 10a, to distinguish the progeny cells from the microinjected aRGCs. The microinjection cargo included recombinant Cas9 protein with either gRNA (gLacZ) or gGFP control. In this experiment, dextran 10,000-Alexa 555 (Dx-A555) acted as a fluorescent tracer for the aforementioned identification. Microinjection mainly aims single aRGCs in the G1 phase of the cell cycle, and therefore facilitates the monitoring of the CRISPR/Cas9-mediated disruption effect of gene (under observation) expression in the same cell cycle of the microinjected neural stem cell, as depicted in Figure 10b–d [147]. The microinjection mediated CRISPR technology provides new prospects for functional screenings and to determine the loss-of-function in the individual cell.

CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats- associated protein 9) -induced disruption of green fluorescence protein (GFP) expression in the daughter cells of single microinjected aRGCs in organotypic slices of the telencephalon of Tis21::GFP mouse embryos. (a) Scheme of the Cas9/gRNA complex microinjection. (b) Reconstruction of optical sections with maximum intensity projections for daughter cells of single aRGCs microinjected with either Cas9/control gRNA (top) or Cas9/gGFP (bottom) revealed by Dx-A555 immunofluorescence (magenta); cell 1, aRGC daughter; cell 2, BP daughter. Dashed lines depict ventricular surface. Scale bars, 20 μm. (c) Single optical sections of cells 1 and 2 shown in (b), showing the effects of Cas9 and control gRNA (top) or gGFP (bottom) on GFP expression. Scale bars, 5 μm. (d) Quantification of the proportion of daughter cells (Dx-A555⁺) of microinjected cells showing GFP expression 24 h after control (Con, white) or gGFP (black) microinjection. (* p < 0.05, Fisher’s test) Reprinted with the permission of [147].

Kohara et al. performed simultaneous injection of DNAs of green fluorescence protein tagged with brain-derived neurotrophic factor (BDNF) and red fluorescence protein (RFP) into a single-neuron (Figure 11). Thereafter, they visualized the expression, localization, and transport of BDNF in the injected single-neuron. This co-expression of two fluorescent proteins revealed the activity-dependent trans-neuronal delivery of BDNF [148]. Shull et al. recently developed a robotic platform for image-guided microinjection of desired volumes of biomolecules into single-cell. In this study, they delivered exogenous mRNA into apical progenitors of the neurons in the fetal human brain tissue. For the autoinjector, the injection pressure was set between 75 and 125 m bar, and it was microinjected from the ventricular surface to the depths of 10, 15, and 25 µm with the efficiency of 68%, 22%, and 11%, respectively. Thus, the autoinjector can deliver exogenous materials into targeted cells to the cluster of cells with high control and at single-cell resolution [119].

Cortical neurons expressing brain-derived neurotrophic factor (BDNF): (a) with green fluorescence protein after 24 h of delivery; (b) stained with anti-BDNF antibody; (c) merge image of both green fluorescence protein and anti-BDNF antibody. Reprinted with permission from [148].

A variant of microinjections has been formulated combining electrophysiology recordings, electrical micro-stimulation, and pharmacological alterations in local neural activity, most commonly used in monkey. The combination of the above-mentioned activities helps in providing a better way of explaining neural mechanisms [149]. Therefore, targeting simultaneous drug delivery, neurophysiological recording, and electrical microstimulation, various groups have developed “microinjectrode” systems. Sommer et al. established the primary connection between corollary discharge and visual processing via injectrode and segregating single cortical neurons. The results showed that spatial visual processing impairs if the corollary discharge from the thalamus is disturbed [150]. Crist et al. developed a microinjectrode which contains a recording electrode in addition to an injection cannula, facilitating simultaneous drug delivery and extracellular neural recording in monkeys. But the recording wire of the syringe typically recorded multi-unit activity, with frequent single-cell isolation [151]. Subsequently, modified injectrodes were introduced to achieve better recording quality and the ability to alter both neuronal activity and behavior in animals, an example being shown in Figure 12 with single-neuron recording, electrical microstimulation and microinjection in the frontal eye field (FEF), along with recorded single-neuron waveforms [84,149,152,153].

Microinjectrode system and its application. Briefly, a thin microelectrode passes through a 32 G cannula (OD: 236 m) which is connected to a T-junction via a ferrule. The electrode goes into a T-junction and a polyimide-coated glass tube with the terminal soldered to a gold pin. The polyimide tubing, gold pin, and ferrule are all pasted together. The middle part shows cross-sections through different parts of microinjectrode, i.e., the top ferrule, middle T-junction and bottom the cannula. An enlarged view of the microelectrode and cannula tips shows their relative position and size. A sample experiment is also displayed with single-neuron recording, electrical microstimulation and microinjection being performed in the frontal eye field (FEF). The single-neuron waveforms (black traces) segregated from background (gray traces) are also presented. Reprinted with the permission of [149].