Patch-sequencing (patch-seq) 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 (soma), 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 (scRNA-seq) 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 (iPSC), 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.
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