Patch-sequencing
Patch-sequencing is a modification of patch-clamp technique that combines electrophysiological, transcriptomic and morphological characterization of individual neurons. In this approach, the neuron's cytoplasm is collected and processed for RNAseq after electrophysiological recordings are performed on it. The cell is simultaneously filled with a dye that allows for subsequent morphological reconstruction.
Background
Neuronal cell-typing requires simultaneous capturing of multiple data modalities
While a neuron's electrical properties are important when defining a cell type its morphology, types of neurotransmitters released, neurotransmitter receptors expressed at synapses, as well as the neuron's location in the nervous system and its local circuit are equally important. Neurons come in a huge diversity of shapes with many differences in cell bodies, dendrites, and axons. The position of the dendrites determines which other neurons a cell receives its input from and their shape can have massive impacts on how a neuron responds to this input. Likewise the targets of a neuron's axon determine its outputs. The types of synapses formed between neurons' axons and dendrites are equally important as well. For instance in the cortical microcircuit of the mammalian cortex, portrayed to the right, cells have highly specific projection patterns both within the local circuit as well as across cortical and non-cortical regions. Dendritic geometry influences the electrical behavior of neurons as well, having a massive influence on how dendrites process input in the form of postsynaptic potentials. Disordered geometry and projection patterns has been linked to a diverse set of psychiatric and neurological conditions including autism and schizophrenia though the behavioral relevance of these phenotypes is not yet understood. Neuronal cell types appeared to often vary continuously between each other. Previous attempts at neuronal classification by morpho-electric properties have been limited by the use of incompatible methodologies and different cell line selection.With the advent of single-cell RNA-sequencing it was hoped that there would exist genes that would be consistently expressed only in neurons with specific classically defined properties. These genes would serve as cell markers. This would provide a better means to delineate neuron types quickly and easily using only mRNA sequencing. However it appeared that scRNA-seq only served to reinforce the fact that overly rigid cell type definitions are not always the best way to characterize neurons. Furthermore gene expression is dynamically regulated, varying over various time scales in response to activity in cell type specific ways to allow for neuronal plasticity. Like other tissues, developmental processes also need to be considered. Matching results from scRNA-seq to classically defined neuronal cell types is very challenging for all these reasons and additionally single-cell RNA-seq has its own drawbacks for neuronal classification. While scRNA-seq enables the study of gene expression patterns from individual neurons, it disrupts the tissue for individual cell isolation and thus it is difficult to infer a neuron's original position in the tissue or observe its morphology. Linking the sequencing information to a neuronal subtype, defined previously by electrophysiological and morphological characteristics is a slow and complicated process. The simultaneous capture and integration of multiple data types by patch-seq makes it ideal for neuronal classification, uncovering new correlations between gene expression, electrophysiological and morphological properties and neuronal function. This makes patch-seq a truly interdisciplinary method, requiring collaboration between specialists in electrophysiology, sequencing, and imaging.
Preparation and model system choice
Patch-seq can be done in any model system including cell culture for neurons. Neurons for culture may be collected from neuronal tissue then disassociated or made from induced pluripotent stem cells, neurons that have been grown out of human stem cell lines. Cell culture preparation is the easiest to apply patch clamp to and give the experimenter control over what ligands the neuron is exposed to, for instance hormones or neurotransmitters. The benefit of total experimental control however also means the neurons are not subject to the natural environment they would be exposed to during development. As mention previously the position their dendrites and axons extend into as well as the neuron's position with a brain structure is incredibly important for understanding its role within a circuit. Many preparations exist for brain slices from different animal species. Owing to the presence of cell or debris in the way of the pipette and a target cell the preparation will need to be slightly modified, often slight positive pressure is applied to the pipette to prevent any unintended seals from forming. If understanding how behavior is tied to the dynamics of the neuronal events is of interest it is possible to record in vivo as well. Though adapting patch clamp for in vivo studies can be very difficult for mechanical reasons especially during a behavioral task but has been done. Automated in vivo patch clamp methods have been developed. Very little difference exists between preparations for mammalian species though the greater diversity of neuronal sizes in non-human primate and primate cortex may necessitate using different tip diameters and pressures for forming seals without killing target neurons. Patch-seq is also applied to non-neuronal studies such as pancreatic or cardiac cells.Patch-sequencing workflow
After choosing a model system and preparation type patch-seq experiments have a similar workflow. First a seal between the cell and the pipette is established so that recording and collection may take place. Cells can be filled with a fluorescent label for imaging during recording. Following recording negative pressure is applied to capture the cytosol and often the nucleus for sequencing. This process is repeated until cells in the preparation have degraded and are not worth collecting data from. Post-hoc analysis of imaging data allows for morphological reconstruction. Like wise complex post-hoc processing of transcriptomic data is often required as well in order to handle a large number of confounds when collecting cytosolic contents from cells via the pipette. In the initial stages, before forming the seal between the pipette and the cell, the tissue slices are prepared using a compresstome vibratome.To obtain thin sections of tissue, these devices are used. This device ensures that the target cells are accessible for patch clamping. The quality and precision of the tissue slicing are important for the success of the patch-seq experiment. The thickness and condition of these tissue slices influence the efficiency of cell targeting and the quality of the patch seal. An appropriate slice preparation is essential to the overall success of the patch-seq workflow.Forming the seal between pipette and cell
The patch pipette is designed for whole cell recording so its opening diameter is larger than experiments done to examine single ion-channels. For the most part standard patch clamp protocols may be used although there are some small situation dependent modifications to the pipette and the internal solution. An even wider diameter may be used to facilitate the aspiration of the inside of the cell into the pipette but it may need to be adjusted based on the target cell type. Negative pressure is applied to enhance the seal which will be better for recording as well as prevent intra-cellular fluid leakage and contamination after or during collection of cell contents for sequencing. During recording biotin can be diffused into the cell via the pipette for imaging and later morphological reconstruction.Electrophysiological recording
Once a seal has been established cells are subject to different stimulation regimes using the voltage clamp, such as ramps, square pulses and noisy current injections. Features of the cell body's membrane are recorded including resting membrane potential and threshold potential. Features observed from generated action potentials such as AP width, AP amplitude, after-depolarization and after-hyperpolarization amplitude are also recorded. Whole-cell recordings are performed using patch recoding pipettes filled with a small volume of intracellular solution, calcium chelators, RNA carriers and RNase inhibitors. The addition of RNase inhibitors, such as EGTA, enhances transcriptome analysis by preserving higher quality RNA from the samples. Recording time can take between 1 and 15 minutes without affecting the neuron structure due to swelling, with lower values increasing throughput of the technique. The data is recorded and analyzed with commercial or open-source software such as MIES, PATCHMASTER, pCLAMP, WaveSurfer, among others.Neurons are pre-stimulated to verify their resting membrane potential and stabilize their baseline across and within experiments. Cells are then stimulated by ramp and square currents, their electrophysiological properties are recorded and measured. After stimulus the membrane potential must return to the baseline value for recordings to be consistent and robust. Negative pressure is used at the end of the recordings to return the membrane stability. Measurements need to satisfy these conditions to be considered for further analysis. During recording cell viability needs to be maintained as being patch-clamped is stressful for the cell.
It is crucial to have healthy acute or live brain slices for electrophysiological recordings, as the health of the neuron significantly impacts the quality of the data obtained. Healthy brain slices are typically prepared using tools such as the Compresstome vibratome, ensuring optimal conditions for accurate and reliable recordings.