We would like to acknowledge financial support from the MRC UK (G0800498). We would also like to thank Professor J-M Fritschy, University of Zurich, for providing the GABAA-R subunit-specific &#947;2 antibody and Professor R. Harvey, UCL School of Pharmacy, for providing the pcDNA&#160;3.1(+) expression vectors containing antibiotic resistance genes for production of stably transfected HEK293 cell lines.

<ol>
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The authors declare that they have no competing financial interests.

Although this protocol is not technically difficult to perform, there are several critical steps that must be followed to achieve the most accurate and repeatable co-culture assays. Firstly, cultured medium spiny neurons must be seeded at an optimal density. If seeded too sparsely, neurons tend to develop very slowly and survival is greatly reduced. On the other hand, if seeded too densely, neurons tend to aggregate which compromises the analysis of contacts with HEK293 cells. Secondly, it is recommended to transiently express a fluorescent reporter, GFP or mCherry in HEK293 cells stably expressing GABAARs, prior to platting them into the co-culture. This allows reliable recognition of HEK293 cells, which can be compromised by similarity in shape and size between these cells and rare surviving glia cell in neuronal cultures. To achieve the efficient transfection with GFP or mCherry cDNA, HEK293 cell lines have to be in the exponential growth phase and seeded at the appropriate density in 6-well plates. Sparse seeding followed by transfection will cause cells to grow poorly, while over-seeding will prevent the cells from taking up the cDNA. Ideally, cells should be seeded so that they are between 70 - 90% confluent on the day of transfection. Thirdly, transfection must be optimized for each cell line used, as some cell lines are more sensitive than the others. This is because constitutive GABAAR expression in HEK293 cells reduces cell survival and the ability of cells to recover after transfection. Moreover, survival depends on the type of GABAARs expressed in HEK293 cells, with some cell lines being significantly more sensitive than the others. Transfection using liposomal reagent is an optimal method for expressing foreign proteins in fast growing cell lines, providing both the high transfection efficiency and level of expression. However, this reagent causes too much damage to slowly growing cell lines, for which we regularly use a non-liposomal transfection reagent. This works in a similar way to the liposomal reagent but the amount of DNA required for efficient transfection is significantly reduced. This allows greater cell survival (roughly 80 - 90% compared with 60% using liposomal reagent) but with lower transfection efficiency (60%). Lastly, the number of control HEK293 or &#945;1/&#946;2/&#947;2 HEK293 expressing cells added to neuronal cultures needs to be optimized. Adding too few cells compromises the successful analysis of contacts between HEK293 cells and neurons, because they become very rare. Conversely, adding too many HEK293 cells causes neuronal cell death within few hours.
Embryonic medium spiny neuron cultures should ideally be prepared using striatal tissue dissected from embryonic age 15 - 17. However, it often happens that embryos are slightly younger or older than the optimal age. In this case, the number of neurons seeded in culture will need to be varied. Tissue that is younger than E15 may need to be seeded at a slightly lower density, whilst tissue that is older than E17 may need to be seeded at a higher density, to allow optimal cell survival. Furthermore, cytosine arabinoside (Ara-C) may need to be added to older cultures to prevent growth of glia, which is more abundant in older tissue.
When creating co-cultures, it is important to plate the optimized number of transfected HEK293 or &#945;1/&#946;2/&#947;2 HEK293 expressing cells, as mentioned above. However, it may be necessary to determine this for each individual cell line, because of differences in their survival. Typically 30,000 cells in a maximum volume of 50 &#956;l should be added to each well of a 24-well dish, which already contains 500 &#956;l of neuronal culture medium, as this ensures that the conditioned neuronal medium is not diluted too much and that the conditions within each well remain fairly constant, e.g. the concentration of growth factors. Adding volumes greater than 50 &#956;l to each well would generally kill the neurons.
One of the major disadvantages of the co-culture technique is that the neuronal cultures are created from dissociated cells grown as a monolayer, which means that the neurons have been removed from their normal microenvironment and are unable to establish their normal anatomical organization. Therefore they lack the appropriate connections, inputs and secreted molecules from other cells that may influence the initial stages of synapse development. For example, in vivo medium spiny neurons are densely innervated by glutamatergic inputs from the cortex, thalamus and other brain regions34, however, in our neuronal cultures glutamatergic synapses do not form because these inputs are damaged during dissection of the striatal tissue. How the absence of functional glutamatergic synapses in cultured medium spiny neurons affects their ability to form GABAergic synapses with each other and/or HEK293 cells expressing GABAARs remains an open question. This question could be easily addressed by culturing medium spiny neurons together with cortical glutamatergic neurons thereby allowing them to form functional synapses35 prior to the addition of HEK293 cells. An alternative approach would be to design a co-culture model system based on organotypic slice cultures, which maintain some of the cytoarchitecture which may be important for maturation and synapse formation. However, organotypic slice cultures have dense and heterogeneous neuropil which can compromise the analysis performed here. Another important disadvantage of using co-culture assays is that GABAARs expressed at the surface of HEK293 cells are not clustered as they are in neurons, although this appears not to be necessary for synapse formation given a high enough surface expression30. For example, in the rodent brain and in hippocampal cultures, the &#945;1 GABAAR subunit is found in most GABAergic synapses on all postsynaptic domains of pyramidal cells. However, the &#945;2 is specifically located in a subset of synapses on the somata and dendrites but is highly enriched in the axon initial segment, as revealed by immunofluorescence and electron microscopy36. Given that synapse formation in the co-cultures can still be reliably detected and analyzed30, this suggests that the density of GABAARs at the cell surface of HEK293 cells may be similar to, or even higher than the density of these receptors within synaptic clusters in neurons. This can explain, at least in part, why synaptic adhesion proteins, such as neuroligin, and postsynaptic density proteins, such as gephyrin, are not necessary for synapse formation in the co-cultures, if the appropriately assembled GABAARs are present at sufficient density.
It is well documented that GABAARs are structurally and functionally heterogeneous, and that the receptor subunit composition determines their subcellular localization and pharmacological properties. For example, incorporation of the 2 subunits is known to be a prerequisite for the synaptic localization of GABAARs while the subunit is almost exclusively present in extrasynaptic GABAARs. The receptors that incorporate only &#945;&#946; combinations are also thought to be predominantly localized to the extrasynaptic domains12-14. Whether this specificity is maintained in our co-culture system can be easily tested by transiently transfecting 2 or subunit cDNAs into HEK293 cell lines stably expressing &#945; and &#946; subunits, before adding them to neuronal cultures. Our preliminary experiments using this approach have suggested that synaptic contacts are readily formed only in the presence of the 2 subunit, indicating that the specificity observed in vivo is likely to be preserved in vitro (data not shown).
Furthermore, GABAARs incorporating different &#945; subunits are selectively localized to synaptic contacts formed with specific types of presynaptic neurons. For example, in the globus pallidus, the &#945;1-GABAARs are generally found at striatopallidal (str-GP) and palliopallidal (GP-GP) synapses, which are located on dendrites and somatic regions of the medium spiny neurons, respectively. The &#945;3-GABAARs are located in perisomatic regions of medium spiny neurons and are contacted by local GP axon collaterals, whilst the &#945;2-GABAARs are located on distal dendrites of these neurons and contacted primarily by inputs from the striatum32. Expression of specific &#945; subunits in different types of synapses and in different neuronal compartments has also been demonstrated in other brain areas such as hippocampus21 and neocortex18,20. These findings raise the question as to how the specific inhibitory synapses are formed in the brain. Does the adhesion of a specific type of presynaptic terminal induce the insertion of specific GABAAR subtypes into the points of contact? Are the receptors trafficked to specific subcellular locations according to their subunit composition, where their plasma membrane insertion is a prerequisite for the adhesion of axonal terminals of specific origin? To date, these questions remain unanswered. The use of reduced model systems such as the co-culture model system, allow us to start answering this complex questions because the system is easily amenable to transfection of DNA constructs and application of reagents and, importantly, it is suitable for live cell imaging analysis30. Thus, using this model system we can start testing the role of individual molecules, including different types of GABAARs, known to be present at synaptic contacts. Another advantage is that synapses in this model system form rapidly, within minutes to hours, reducing the duration of experiments. Similar co-culture model systems were successfully employed in the past to screen for the novel synaptogenic molecules27,37,38.
Understanding how the central nervous system develops, matures and forms connections between neurons so intricately to control, for example, behavior or cognition, is of fundamental importance. This distant aim will only be achieved by delineating the molecular mechanisms that govern the individual steps of recognition and cell-to-cell communication during development. Due to sheer complexity, the molecular details of these multiple cellular interactions can currently be studied with precision only in reduced systems. However, the ability to increase the complexity of these systems by expressing multiple combinations of proteins and studying how they interact has some advantages in comparison with, for example, genetic deletion approaches. This is because the accurate interpretation of the effects of a single gene deletion is often compromised by changes associated with compensatory mechanisms masking the effects of original lesions, particularly in the developing brain. The simple yet informative co-culture technique described here has allowed a discovery of the structural role of GABAARs in synapse formation and opened a possibility to investigate how GABAARs and other cell adhesion molecules and/or synaptic matrix proteins interact with each other during synaptogenesis. Synaptic matrix proteins are of particular interest given that they have recently been demonstrated to play a key role in glutamatergic synapse formation39. Further development of co-culture models is important because they have the potential to advance our knowledge of the molecular mechanisms which guide the &#8216;normal&#8217; brain development and thus increase our understanding of how these mechanisms are altered in many neurodevelopmental diseases, such as epilepsy, schizophrenia, autism spectrum disorders and many others.

GABA is one of the earliest neurotransmitters found in the embryonic brain, preceding the most abundant excitatory neurotransmitter glutamate1. During development, GABA depolarizes and excites immature neurons, playing a key role in regulating cell proliferation, migration and formation of neuronal networks without inducing excitotoxicity. In the adult brain, the reversal potential for GABAA receptor channels is shifted to more negative potentials due to a decrease in the intracellular concentration of chloride. This shift is caused by up-regulation of the potassium-chloride co-transporter (KCC2), which transports chloride out of the cell, and, in parallel, down-regulation of the sodium-potassium-chloride transporter (NKCC1), which has the opposite effect2.
In the brain, GABA primarily binds to either GABAA or GABAB receptors to mediate fast or slow synaptic inhibition, respectively. GABAARs are a class of receptors also known as heteropentameric ionotropic or ligand-gated Cys-loop ion channels. Two molecules of GABA are required for activation of the receptor, which is permeable to chloride ions and to a lesser degree, bicarbonate ions. The increase in chloride conductance decreases the effectiveness of depolarizing, excitatory events in activating the postsynaptic neuron3.
Structural diversity of GABAARs has long been recognized as a key factor in determining their wide range of functional and pharmacological properties. Native GABAARs are hetero-pentamers composed of subunits with multiple isoforms classified as: &#945;(1-6), &#946;(1-3), &#947;(1-3), &#948;, &#949;, &#960; and &#952;3, with a common transmembrane topology comprising a large N-terminal extracellular domain, four transmembrane domains (TMs), and a major intracellular domain between TMs 3 and 44. The &#946;3 and &#947;2 subunits are essential for synaptic inhibition and organism survival, because mice bearing genetic deletion of these subunits die after birth5,6. In contrast, individual isoforms of &#945; subunit are important for the function of specific synaptic connections in the brain associated with different behaviors such as anxiety, sedation, arousal, and others, but are not, individually, essential for life7-9. GABAARs are the main sites of action for a variety of drugs with potent sedative, hypnotic, anxiolytic and anticonvulsant effects, such as benzodiazepines, barbiturates, neurosteroids and anesthetics7,10,11.
Synaptic GABAARs typically contain a &#947;2 subunit, two &#946; subunits (most commonly &#946;2 or &#946;3) and two &#945; subunits (&#945;1, &#945;2, &#945;3 or &#945;5)12,13. The predominant class of extra-synaptic receptors contains the &#948; subunit in combination with two &#945; subunits (&#945;4 or &#945;6), and two &#946; subunits (&#946;2 or &#946;3)14. Subcellular localization of GABAARs to axons, dendrites or soma, and insertion into the plasma membrane are dependent on the presence of &#946;-subunits15,16. However, selective incorporation of different GABAAR subtypes into distinct types of synapses correlates well with the presence of specific &#945; subunits (&#945;1, &#945;2, &#945;3 or &#945;5)7,17,18. Importantly, deletion of &#945;1 or &#945;2 subunit in mice causes ultrastructural changes at inhibitory synapses19. This suggests that GABAARs themselves may play a direct role in regulating synapse formation.
Evidence indicates that GABAergic synapse development is a precisely co-ordinated sequence of events, in which both the neuronal targets contacted by different types of inhibitory axons and the receptors that are clustered at each class of inhibitory synapse are selective and functionally attuned17,20-22. This fundamental principle of specificity at GABAergic synapses raises the question as to how the pre- and postsynaptic partners recognize each other during the initiation of synaptic contacts.
In vitro co-culture assays have been applied successfully to study some of the mechanisms of synapse formation and to test the role of individual synaptic cleft-spanning proteins in this process. One of the common trans-synaptic interacting protein combinations that function bi-directionally to mediate synapse formation and maturation, are the Neurexins (Nrxns) and Neuroligins (NLs). Nrxns are presynaptic proteins that exhibit alternative splicing within their laminin-neurexin-sex hormone-binding protein domains, giving rise to many different isoforms23. While the Nrxns also interact with other proteins, NLs are thought to be their ubiquitous postsynaptic partners24. Together these proteins contribute to holding the presynaptic and postsynaptic membranes in close and rigid apposition25. The two most abundant isoforms are NL-1 and NL-2 which are present at excitatory and inhibitory synapses, respectively26. One of the earliest co-culture model systems, designed to investigate trans-synaptic protein interactions, employed different types of non-neuronal cells, most commonly immortal cell lines such as Human Embryonic Kidney (HEK) 293 cells, to over-express NL-2. When these cells were cultured with pontine neurons, an accumulation of presynaptic proteins in close proximity to the surface of the HEK cells was observed, indicating formation of synapse-like contacts. Addition of soluble &#946;-neurexin to these co-cultures inhibited the formation of contacts, suggesting that trans-synaptic interactions between Nrxns and NLs are necessary for synaptic contact formation27. Moreover, transient expression of &#946;-neurexin in COS (CV-1 (simian) in Origin, and carrying the SV40 genetic material) cells co-cultured with dissociated hippocampal glutamatergic and GABAergic neurons induced expression of the postsynaptic protein gephryin and of GABAAR subunits &#947;2 and &#945;2 at points of contact between these two cells types28. Another example of a co-culture model used to study synapse formation involved HEK293 cells, transiently transfected with GABAAR subunits &#945;2/&#946;3/&#947;2 and NL-2, and a mixed population of hypothalamic neurons29. This study concluded that the expression of NL-2 is an absolute requirement for formation of inhibitory synapses.
However, in the recent co-culture study, stably transfected &#945;1/&#946;2/&#947;2 GABAARs in HEK293 cells were found to be sufficient to induce functional synapses when co-cultured with GABAergic medium spiny neurons, without the need for additional trans-synaptic or postsynaptic adhesion proteins. However, a prominent increase in synapse formation and strength was observed when NL-2 was co-expressed with GABAARs30. This indicates that this co-culture model system has advantages over previously described model systems, most evidently an increased sensitivity and reliability of synaptic contact detection. Two important factors contributing to the overall improvement in detection of synaptic contacts are: i) The use of stably transfected HEK293 cell lines with high and consistent expression of GABAAR subunits at the surface of individual cells. This consistency facilitates quantitative comparisons between different co-culture conditions. ii) The use of a pure population of GABAergic medium spiny neurons cultured from the embryonic striatum31 removes complications and ambiguities resulting from the use of mixed neuronal populations and allows, for example, selection of the most appropriate postsynaptic GABAAR types that can be compared with each other during synapse formation.
Formation of synapses is thought to involve many trans-synaptic signals within pre- and postsynaptic cell adhesion complexes. Due to the bi-directional nature of synaptic signaling and the sheer numbers of cell adhesion molecules, it is difficult to identify key components involved in synapse formation. Thus, transfecting a single cell adhesion protein into a non-neuronal cell (in this case, the two most prevalent postsynaptic targets for GABAergic medium spiny neurons in vivo,&#945;1/&#946;2/&#947;2 or &#945;1/&#946;3/&#947;2 GABAARs32) greatly reduces the complexity of trans-synaptic signals available at the postsynaptic surface and allows precise quantitative analysis of the efficacy of this protein in promoting synapse formation.

<table border="0" cellpadding="0" cellspacing="0" width="1601">
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			<td height="22" style="height:22px;width:469px;">Stable HEK&#945;1&#946;2&#947;2 line</td>
			<td style="width:627px;">Sanofi-Synthelabo, Paris</td>
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			<td style="width:111px;">A3294</td>
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			<td height="64" rowspan="3" style="height:64px;width:469px;">Guinea pig anti-&#947;2 GABAA receptor antibody</td>
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			<td rowspan="3" style="width:111px;">N/A</td>
			<td rowspan="3" style="width:396px;"></td>
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			<td height="21" style="height:21px;width:627px;">(Institute of Zurich, Switzerland) Fritschy, J.M and Mohler, H.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:627px;">J. Comp. Neurol. 359 (1) 154&#8211;194 (1995).</td>
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			<td height="22" style="height:22px;width:469px;">Triton X-100</td>
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			<td style="width:111px;">H5141</td>
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			<td height="22" style="height:22px;width:469px;">Mouse anti-Glutamate Decarboxylase (GAD)65 antibody</td>
			<td style="width:627px;">Merck Millipore</td>
			<td style="width:111px;">MAB351</td>
			<td style="width:396px;"></td>
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			<td height="22" style="height:22px;width:469px;">Mouse anti-synapsin antibody</td>
			<td style="width:627px;">Synaptic Systems</td>
			<td style="width:111px;">106-011</td>
			<td style="width:396px;"></td>
		</tr>
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			<td height="22" style="height:22px;width:469px;">Mouse anti-&#946;2/3 antibody (BD17)</td>
			<td style="width:627px;">Merck Millipore</td>
			<td style="width:111px;">MAB341</td>
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			<td height="43" rowspan="2" style="height:43px;width:469px;">Rabbit anti-&#945;1 GABAA receptor antibody</td>
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			<td rowspan="2" style="width:396px;"></td>
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			<td height="22" style="height:22px;width:627px;">(UCL School of Pharmacy, London) FA Stephenson et al., J. Comp. Neurol.416 (2) 158-172.</td>
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			<td height="22" style="height:22px;width:469px;"><a name="RANGE!A49">Mounting reagent (Prolong Gold)</a></td>
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			<td style="width:111px;">P36930</td>
			<td style="width:396px;">Use at room temperature</td>
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	</tbody>
</table>

inhibitory synapse formation in a co-culture model incorporating gabaergic medium spiny neurons and hek293 cells stably expressing gabaa receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (&#947;-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. 
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. 
To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

The protocol for this neuron-HEK293 cell co-culture model system has been finely tuned to allow optimal cell survival. In this system, formation of synapse-like contacts and their analysis relies on stable and consistent expression of all three GABAAR subunits which assemble into a functional receptor. It is therefore important to use immunocytochemical analysis to test for subunit expression at the surface of HEK293 cell before adding them to neuronal cultures. In these experiments, cell surface expression of &#945;1, &#946;2 and &#947;2 subunits (Figure 1A), or &#945;1, &#946;3 and &#947;2 subunits (Figure 1B), was detected using subunit-specific antibodies which bind to the extracellular epitopes of these subunits. A high degree of co-localization between these subunits at the HEK293 surface was demonstrated.
After confirming the surface expression and co-localization of GABAAR subunits in HEK293 cells, co-cultures were prepared using HEK293 cells expressing &#945;1/&#946;2/&#947;2 GABAAR subunits and medium spiny neurons cultured for 14 days (14 days in vitro (DIV)). Cells in co-culture were incubated for 24 hr, fixed and analyzed using immunocytochemistry and confocal microscopy. Analysis of contacts indicated that GAD65-positive GABAergic axon terminals formed only sporadic contacts with the control HEK293 cells (Figure 2A, 2B). The number of contacts detected at 4 hr was 7.3 &#177; 0.9 per HEK293 cell, and this number was reduced to 5.5 &#177; 0.5 connections (mean &#177; SEM) per HEK293 cell at 24 hr after adding HEK293 cells to the cultured neurons. In contrast, GAD65-positive GABAergic axon terminals formed numerous synapse-like contacts with HEK293 cells expressing GABAARs. The number of contacts obtained at 4 hr after adding HEK293 cells was 28.3 &#177; 4.7 per HEK293 cell, and this number was further increased to 52.1 &#177; 6.3 (mean &#177; SEM) per HEK293 cell at 24 hr in co-culture (Figure 2A, 2B).
To determine whether these synapse-like contacts were &#8216;active,&#8217; i.e. supported vesicular transmitter release, a vesicle-luminal domain-specific anti-synaptotagmin Cy5-conjugated antibody was added to the co-culture medium after 23 hours of incubation. This antibody is only incorporated into presynaptic nerve terminals when a pore forms between the synaptic vesicle lumen and the extracellular fluid in the synaptic cleft during neurotransmitter release. Following release, the pore closes, leaving the synaptotagmin Cy5-conjugated fluorescent antibody attached to synaptotagmin inside the vesicle. In this way, only the vesicles actively engaged in neurotransmitter release are labeled with the antibody. In these experiments few if any contacts between the control HEK293 cells and the medium spiny neuron terminals were &#8216;active&#8217; as shown by the lack of co-localization between the presynaptic GAD65/synaptotagmin fluorescence and mCherry fluorescence in HEK293 cells (Figure 3A). In contrast, many &#8216;active&#8217; contacts were formed between the medium spiny neuron terminals and &#945;1/&#946;2/&#947;2-expressing HEK293 cells, as revealed by a high degree of co-localization between GAD65/synaptotagmin and mCherry, expressed specifically in HEK293 cells (Figure 3B).
To test whether a different subtype of GABAAR can also promote synapse-like formation in vitro, we have co-cultured &#945;1/&#946;3/&#947;2 expressing HEK293 cells with medium spiny neurons. Again, control HEK293 cells rarely received contacts with synapsin-positive presynaptic terminals reaching 10.8 &#177; 0.48 (mean &#177; SEM) contacts per HEK293 cell after 24 hr in co-culture (Figure 4A left, 4B). However, HEK293 cells expressing &#945;1/&#946;3/&#947;2 GABAARs form significantly more synapse-like contacts with synapsin-positive presynaptic terminals of medium spiny neurons reaching 25.3 &#177; 0.27 (mean &#177; SEM) contacts per HEK293 cell after 24 hr in co-culture (Figure 4A right, 4B). This indicates that &#945;1/&#946;3/&#947;2 GABAARs expressed in HEK293 cells are also able to promote synaptic contact formation, albeit their potency is lower than the potency of &#945;1/&#946;2/&#947;2-containing GABAARs.
These experiments indicate that the co-culture model system developed in our laboratory permits quantitative analysis of synaptic contact formation in vitro as well as evaluation of the efficacy of different subtypes of GABAARs in this process. These experiments further demonstrate that GABAARs, in addition to being critical functional components of GABAergic synapses, may play a key role in the process of recognition and formation of synaptic contacts between inhibitory neurons and the appropriate neuronal target cells, independently of other synaptic adhesion proteins.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/52115/52115fig1highres.jpg" width="600" /> 
Figure 1. Immunocytochemical analysis of expression of GABAAR &#945;1/&#946;2/&#947;2 or &#945;1/&#946;3/&#947;2 in stable HEK293 cell lines. Antibodies recognizing the extracellular domains of GABAAR subunits were used to label receptors expressed at the cell surface. (A) HEK293 cell line expressing &#945;1 (Alexa Fluor 488), &#946;2 (Alexa Fluor 555) and &#947;2 (Cy5) at high levels. (B) HEK293 cell line expressing &#945;1 (Alexa Fluor 488), &#946;2 (Alexa Fluor 555) and &#947;2 (Cy5) subunits at high levels. Scale bar: 10 &#956;m. <a href="https://www.jove.com/files/ftp_upload/52115/52115fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/52115/52115fig2highres.jpg" width="600" /> 
Figure 2. GABAergic medium spiny neurons form synapse-like contacts with &#945;1/&#946;2/&#947;2- expressing HEK293 cells in co-culture. (A) Fluorescent labeling of presynaptic terminals with anti-GAD65 antibodies (in green) and HEK293 cells with mCherry (in red, left) or the GABAAR &#947;2 subunit (in blue, right), revealed points of co-localization between these markers indicating formation of synapse-like contacts after 4 or 24 hr in co-culture. Scale bar: 10 &#956;m. (B) Quantitative analysis of synapse-like contacts. HEK293 cells were identified based on their shape as revealed by DIC imaging and/or mCherry expression, and the number of contacts between GAD-65 positive puncta (in green) and the surface of HEK293 cells was counted by eye in each optical section of a Z-stack series (8 - 10) per cell using the imaging software, and expressed as the number of contacts/cell. The graph shows the number of contacts between medium spiny neurons and control HEK293 cells (light grey) or &#945;1/&#946;2/&#947;2-HEK293 cells(black) after 4 and 24 hr in co-culture (mean &#177; SEM, n = 8 in each condition from two independent experiments). This figure has been modified from Fuchs et al. (2013)30. <a href="https://www.jove.com/files/ftp_upload/52115/52115fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/52115/52115fig3highres.jpg" width="600" /> 
Figure 3. GABAARs promote formation of active synaptic contacts. Immunolabeling of synapse-like contacts formed after 24 hours in co-culture between medium spiny neuron terminals positive for GAD65 (Alexa Fluor 405 cyan) and (A) control HEK293 cells, or (B) HEK293-&#945;1/&#946;2/&#947;2 cells, both transiently transfected with mCherry construct (red). Active contacts are identified by co-localization between the vesicle luminal domain-specific anti-synaptotagmin antibody (Cy5) and GAD65-specific antibody both in presynaptic terminals, and mCherry expressed in HEK293 cells. Scale bar: 10 &#956;m. <a href="https://www.jove.com/files/ftp_upload/52115/52115fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/52115/52115fig4highres.jpg" width="600" /> 
Figure 4. GABAergic medium spiny neurons form synapse-like contacts with &#945;1/&#946;3/&#947;2- subunit expressing HEK293 cells in co-culture. (A) Fluorescent labeling of presynaptic terminals with anti-synapsin I antibodies (in green), and control HEK293 cells (left), or &#945;1/&#946;3/&#947;2 expressing HEK293 cells, both transiently transfected with mCherry (in red), revealed points of co-localization between these markers indicating the formation of synapse-like contacts after 24 hr in co-culture. Scale bar: 10 &#956;m. (B) Quantitative analysis of synapse-like contacts. HEK293 cells were identified by mCherry expression, and the number of contacts between synapsin I-positive puncta (in green) and the surface of HEK293 cells was counted by eye in each optical section of a Z-stack series (8 - 10) per cell using imaging software, and expressed as the number of contacts/cell. The graph shows the number of contacts between medium spiny neurons and control HEK293 cells (light grey) or &#945;1/&#946;3/&#947;2-HEK293 cells(black) after 24 hr in co-culture (mean &#177; SEM, n = 8-12 cells in each condition, from two independent experiments). <a href="https://www.jove.com/files/ftp_upload/52115/52115fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors at JoVE.com

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

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17.3K Views.  University College London. The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

Video: Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

A Functional Motor Unit in the Culture Dish: Co-culture of Spinal Cord Explants and Muscle Cells

Human primary muscle cells cultured aneurally in monolayer rarely contract spontaneously because, in the absence of a nerve component, cell differentiation is limited and motor neuron stimulation is missing1. These limitations hamper the in vitro study of many neuromuscular diseases in cultured muscle cells. Importantly, the experimental constraints of monolayered, cultured muscle cells can be overcome by functional innervation of myofibers with spinal cord explants in co-cultures.
Here, we show the different steps required to achieve an efficient, proper innervation of human primary muscle cells, leading to complete differentiation and fiber contraction according to the method developed by Askanas2. To do so, muscle cells are co-cultured with spinal cord explants of rat embryos at ED 13.5, with the dorsal root ganglia still attached to the spinal cord slices. After a few days, the muscle fibers start to contract and eventually become cross-striated through innervation by functional neurites projecting from the spinal cord explants that connecting to the muscle cells. This structure can be maintained for many months, simply by regular exchange of the culture medium.
The applications of this invaluable tool are numerous, as it represents a functional model for multidisciplinary analyses of human muscle development and innervation. In fact, a complete de novo neuromuscular junction installation occurs in a culture dish, allowing an easy measurement of many parameters at each step, in a fundamental and physiological context.
Just to cite a few examples, genomic and/or proteomic studies can be performed directly on the co-cultures. Furthermore, pre- and post-synaptic effects can be specifically and separately assessed at the neuromuscular junction, because both components come from different species, rat and human, respectively. The nerve-muscle co-culture can also be performed with human muscle cells isolated from patients suffering from muscle or neuromuscular diseases3, and thus can be used as a screening tool for candidate drugs. Finally, no special equipment but a regular BSL2 facility is needed to reproduce a functional motor unit in a culture dish. This method thus is valuable for both the muscle as well as the neuromuscular research communities for physiological and mechanistic studies of neuromuscular function, in a normal and disease context.

Cultured muscle cells are an inadequate model to recapitulate innervated muscle in vivo. A functional motor unit can be reproduced in vitro by innervation of differentiated human primary muscle cells using rat embryo spinal cord explants. This article describes how co-cultures of spinal cord explants and muscle cells are established.

Human primary muscle cells cultured  aneurally in monolayer rarely contract spontaneously because, in the absence of  a nerve component, cell ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials through saltatory conduction. Moreover, an increasing number of reports suggest that oligodendrocytes interact with neurons beyond myelination, notably through the secretion of soluble factors. Here, we present a detailed protocol allowing purification of oligodendroglial lineage cells from glial cell cultures also containing astrocytes and microglial cells. The method relies on overnight shaking at 37 &#176;C, which allows selective detachment of the overlying oligodendroglial cells and microglial cells, and the elimination of microglia by differential adhesion. We then describe the culture of oligodendrocytes and production of oligodendrocyte-conditioned medium (OCM). We also provide the kinetics of OCM treatment or oligodendrocytes addition to purified hippocampal neurons in co-culture experiments, studying oligodendrocyte-neuron interactions.

Herein, we display an efficient method for the purification of oligodendrocytes and production of oligodendrocyte-conditioned medium that can be used for co-culture experiments.

In the central nervous system, oligodendrocytes are well-known for their role in axon myelination, that accelerates the propagation of action potentials ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...