Background

In situ hybridization (ISH) is a technique utilized in histological analyses that involves using antisense nucleic acid probes containing complementary bases to the target mRNA for the detection of mRNA transcripts [1]. Historically, ISH has utilized radioactive probes (using sulfur 35 as a radioisotope) to target mRNA molecules within a cell population in tissue sections [2]. Non-radioactive methods were also developed by utilizing digoxigenin RNA probes [3]. However, these methods, while allowing for the detection of a single species of mRNA, are not suited to simultaneously visualize protein and RNA species in individual cells. More recently, fluorescent probes were developed to allow for the analysis of multiple mRNA species using a distinct fluorophore. This technique is similar to immunohistochemistry (IHC), which involves the utilization of antibodies that recognize specific epitopes on proteins, thus allowing for the detection and visualization of these proteins within a certain cell [4]. Both techniques have their limitations and advantages. ISH allows for temporal and spatial analysis of gene transcription within a given cell and can be visualized as individual puncta, although more advanced methods exist that can provide comprehensive information about mRNA translation within a given cell population or tissue (RNA sequencing). IHC is a common, robust method to visualize proteins, reflecting the cumulative results of translation. Although there is no temporal resolution, it is a useful method for analyzing cell quantities, cell morphology, and cell subtype identification within tissues. However, IHC analysis can be subjective and difficult to replicate. When utilizing ISH and IHC together, it allows for simultaneous measurements of transcription and translation, allowing for the identification of the same or different markers. In addition, the visual properties of each method complement each other, such that IHC immunoreactivity results in diffuse labeling throughout the cell or cell processes in the case of a neuron, while ISH labeling results in puncta-like positivity. The combination of ISH and IHC allows for straightforward co-localization of mRNA transcripts within a cell subtype. This is particularly important because neurons can be identified and classified based on their protein expression.

Within the central nervous system (CNS), there is a large diversity of neuronal subtypes, including excitatory and inhibitory interneurons, specialized neurons containing neuropeptides, and non-neuronal cells such as astrocytes, microglia, and oligodendrocytes. While region-level analyses provide valuable information about overall gene expression, deeper insight can be gained by studying changes at the level of specific neuronal subtypes due to diseases or CNS activity. This is particularly important as transcript distribution can differ dramatically between neighboring cells and drive unique physiological roles [5]. Since each subtype contributes in distinct ways to network dynamics, analyzing cell type–specific processes provides a more accurate link between molecular regulation, circuit activity, and, ultimately, behavior, yielding a clearer understanding of the target under study. Neuronal subtypes can be easily identified by proteins that are uniquely expressed in the subpopulation. For example, tyrosine hydroxylase can be used to label neurons that produce catecholamines (i.e., dopamine and norepinephrine) because it is an enzyme involved in the synthesis of these neurotransmitters. Additional neurotransmitters can be detected by other unique proteins. Examples include parvalbumin, which is expressed in GABAergic interneurons, tryptophan hydroxylase, which denotes serotonergic neurons, and vesicular glutamate transporter, which is highly expressed in glutamate neurons.

The protocol detailed below has been optimized to detect and quantify GPR75 mRNA within the mouse brain. GPR75 is an orphan G-protein-coupled receptor (Gαq subtype), highly expressed within the brain as compared to the periphery [6]. Recent works have suggested GPR75 to have a large role in the regulation of metabolism [7]. This protocol was developed to further the understanding of GPR75 expression patterns in an effort to elucidate the function of this orphan receptor. This was achieved by combining ISH and IHC to co-localize GPR75 mRNA within different neuronal subtypes using several key neuronal markers. The examples provided within this protocol are not representative of all neuronal subtypes; see Table 1 for more examples of antibodies that can be used with IHC to detect different neuronal subtypes, as well as other brain cell types, such as microglia and astrocytes.

This method of combining ISH with IHC within the same tissue is not uncommon in the field of neuroscience. Moreover, many researchers may use this system to analyze the localization of mRNA differences with protein expression of the same marker [8]. Research using this method has been published [9–11]. However, there is no clear protocol outlining the steps involved for analysis and preparation. Such a protocol, if similarly utilized by researchers, will provide a better avenue to share discoveries.