Knock-in mouse models for studying somatostatin and cholecystokinin expressing cells

doi:10.21203/rs.3.rs-1598371/v1

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Knock-in mouse models for studying somatostatin and cholecystokinin expressing cells

https://doi.org/10.21203/rs.3.rs-1598371/v1

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Somatostatin (SST) and Cholecystokinin (CCK) are peptide hormones that regulate the endocrine system, cell proliferation and neurotransmission. Knock-in mouse models to study cell types that produce these critically important molecules are limited to date. To address this, we utilized the novel Easi-CRISPR system to generate two knock-in mouse strains with Cre recombinase in SST- and CCK-expressing cells and validated their utility in the developing and adult brain tissues. Developmental and adult SST and CCK expressions were preserved and showed an appropriate expression pattern in both models. These two mouse models serve as valuable tools for research studies related to SST and CCK biology. Specifically, they can be used as reporters to study development, physiology, or pathophysiology of SST and CCK expressing cells. In addition, they can be used as Cre driver models to conditionally delete floxed genes in SST and CCK expressing cells across various tissues.

somatostatin

cholecystokinin

CRISPR/Cas9

Cre

ZsGreen1

Easi-CRISPR

Peptide hormones are critical, multifunctional molecules that are expressed in multiple organ systems, including brain¹. They are secreted into the circulatory system following specific stimuli, or locally released in a communication between cells and are important for fine-tuning of many homeostatic mechanisms².

Somatostatin (SST) is a peptide hormone that regulates the endocrine system, cell proliferation and neurotransmission^3–5. It binds to G protein-coupled somatostatin receptors⁶. SST is expressed in the highest quantity in the gastrointestinal tract including the pancreas, but also the central nervous system^{7, 8}. In the digestive system it inhibits both insulin and glucagon secretion, reduces the activity of parietal cells of the stomach and gastrin-producing cells, thereby slowing down the motility of the small bowel^{9, 10}. However, SST is also a critical molecule in the brain^{7, 11}. Widespread, but spatially selective axonal and somatodendritic SST immunostaining can be observed throughout the entire brain of all studied mammalian species. In the mammalian forebrain, SST is almost exclusively localized in GABA-ergic interneurons^{12, 13}. In the neocortex, SST + interneurons represent ~ 30% of all GABAergic interneurons^14–16. A recent high-throughput analysis of neocortical interneurons suggested that SST-containing neurons in L2 and L5 could be divided into three primary types: Martinotti cells, which are defined by a multipolar appearance and a prominent axonal arbor in L1; neurons with a bitufted appearance; and a small population of basket cells with a fast-spiking phenotype^{12, 17}.

Cholecystokinin (CCK) is produced by I-cells in the lining of the duodenum and is also released by neurons in the brain¹⁸. It acts on two types of receptors found throughout the gut and central nervous system¹⁹. The most recognized functions of this hormone are in digestion and regulation of appetite. In the gut system, it is synthesized and released by enteroendocrine cells in the mucosal lining of the small intestine (I cells) and neurons of the enteric nervous system^{20, 21}. It regulates digestion by inducing contraction of the gallbladder, relaxation of the sphincter of Oddi, increasing bile acid production in the liver, delaying gastric emptying, and stimulation of digestive enzyme production in the pancreas^{22, 23}. In addition, CCK also plays a crucial role in the CNS. As a neuropeptide, CCK is involved in the neurobiology of anxiety, depression, psychosis, cognition, nociception, and feeding behavior^{24, 25}. CCK immunoreactivity is present in the neocortex, hippocampus, amygdala, septum, and thalamus of all studied mammalian species ^{26, 27}. Notably, CCK immunoreactivity is present in both principal cells and interneurons of the brain^{27, 28}. It is expressed in dopaminergic, glutamatergic and GABA-ergic neurons, modulating region- and cell-specific functions^{29, 30}. Most CCK expressing interneurons belong to the basket cell class which are predominantly synapsing with the cell soma of pyramidal neurons and double bouquet cells that are targeting dendrites^{31, 32}. Electrophysiological features of CCK interneurons include regular-spiking activity and feedforward inhibition of pyramidal cells³³.

Importantly, both of these peptides play a critical role in neurotransmission, acting as auxiliary messenger molecules that are co-released in nerve cells with one or more small molecule (classic) neurotransmitters^{19, 34}. Knock-in mouse models of SST and CCK are relatively limited, but there is a great need to understand their function, especially in the brain. To facilitate such studies, we utilized the novel Easi-CRISPR system^{35, 36} to generate mouse strains with Cre recombinase in SST- and CCK-expressing cells. We validated the utility of our models in the developing and adult brain tissue.

Generation of Sst-Cre and Cck-Cre knock-in mice

Due to the experimental need for Sst and Cck animal models, these two genes were chosen to create unique Cre knock-in mice. The full nomenclature for the newly generated strains are C57BL/6-Sst^{em1(P2A−iCre−T2A−mCherry)Mirn} and C57BL/6-Cck^{em1(iCre−T2A−mCherry−P2A)Mirn}. The short abbreviations of these mice, used in the text, are Sst-Cre and Cck-Cre. The improved Cre (iCre)³⁷ coding sequence was inserted along with a mCherry reporter, and the three coding sequences were separated with viral proteases (P2A and T2A)³⁸. Schematics of CRISPR targeting for Sst and Cck loci are shown in Figs. 1 and 2. For the Sst locus, a P2A-iCre-T2A-mCherry cassette was inserted immediately upstream of the stop codon (C terminus fusion). For the Cck locus, iCre-P2A-mCherry-T2A cassette was inserted at the start codon (N terminus fusion). Knock-in mice were generated using the Easi-CRISPR method, which is briefly described in materials and methods section. Two correctly targeted and sequence verified founders were obtained for the Sst-Cre knock-in model. One correctly targeted founder was obtained for the Cck-Cre knock-in model.

A duplex PCR genotyping assay (containing two flanking primers and a primer binding to the insert sequence) was able to differentiate all three genotypes (wild type, heterozygous and homozygous knock-in) (Figure 3).

Validation of SST and CCK expression in Sst-Cre and Cck-Cre knock-in animals

Visualization of the targeted neurons is extremely important in the majority of research studies. However, the constructs by themselves should not interfere with the expression of the genes. To confirm the expression of mCherry protein in the brain, and to validate intact SST protein expression, we performed double labeling using anti-mCherry and anti-SST antibodies (Figure 4A). The presented coronal section through hippocampal area CA1 and adjacent cortex revealed a complete overlap between mCherry and SST expression. Similarly, double labeling using anti-mCherry and anti-CCK antibodies in hippocampal area CA1 and cortex showed complete and exclusive overlap (Figure 4B). This suggests a correct expression pattern of the reporters without interference with the expression of endogenous peptides. The purpose of including the mCherry cDNA in the constructs was to use it as a marker for Cre expression. However, the fluorescence signals were relatively weak (data not shown) and thus they might have not represented all of the brain cells in which Sst or Cck promoters are thought to be active. Nevertheless, the use of anti-mCherry antibody was helpful in accurately identifying all the SST and CCK positive cells.

To further corroborate these findings, Sst-Cre mice were crossed with Ai6 (RCL-ZsGreen) reporter mice³⁹. As expected, the progeny of Sst-Cre and Ai6 breeding and Cck-Cre and Ai6 breeding showed very strong ZsGreen1 fluorescence in the brain. We analyzed the expression of ZsGreen1 throughout the whole brain in male and female mice at P0, P7, P14 and adult mice. Images of coronal brain sections of Sst-Cre mice are shown in Fig. 5, while examples of micrographs of Cck-Cre mice brains are depicted in Fig. 6. These images revealed that the overall pattern of SST and CCK expression from birth through the adulthood was consistent with those reported in the Allen brain atlas⁴⁰. Inserts illustrate correct localization of Cre recombinase in SST + and CCK + neurons observable with ZsGreen1 fluorescence throughout the brain, including the hippocampus and neocortex. Additional rostro-caudal brain sections of SST and CCK expression in adult mice can be found in Supplemental Figs. 1 and 2.

Primary neuronal cultures

Next, we tested the utility of Sst-Cre and Cck-Cre mice for in vitro studies. We utilized cortical neuronal cultures, prepared from the cross of Sst-Cre and Ai6(RCL-ZsGreen) mice or Cck-Cre and Ai6(RCL-ZsGreen). Dissociated cortical neurons retained ZsGreen1 fluorescence, remained viable, and we could continuously monitor these cells in in vitro cultures. Different batches of cultures were grown for different amount of time, allowing us to follow the development of SST and CCK expressing neurons for 5 days (Fig. 7). These cultures retained stable ZsGreen1 fluorescence up to 9 days in culture (data not shown). In addition to labeling neuronal somata, fluorescence revealed the development of extensive neuronal arborizations. In addition, we were able to observe different morphological classes of neurons and growth cones.

In a separate set of experiments, Sst-Cre mice were crossed with Ai6(RCL-ZsGreen) mice and Cck-Cre with Ai14 (tdTomato) mice. Embryonic day 14 cortical neuronal cultures were prepared independently from these two mice crosses and grown either separately or combined. An example of combined culture is shown in Figure 8. The ZsGreen and tdTomato fluorescence were present and visible at the time dissociated embryonic neurons are put into culture and the endogenous fluorescence progressively strengthened in the SST+ and CCK+ neurons as they differentiated in the culture. This method allows simultaneous studies of growth and arborization, growth cone extension and synapse formation of SST and CCK neurons in vitro.

These are the first SST and CCK transgenic animals generated with the novel, Easi-CRISPR technology. This method solves a major problem of animal genome engineering, namely the inefficient insertion of the targeted DNA cassette. It is based on a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. Unlike the traditional embryonic stem (ES) based homologous recombination approach, that rely on additional genetic elements in the knock-in cassettes (such as positive and negative selection markers), the Easi-CRISPR method can generate models containing only the desired sequences^{35, 36}. For example, in this case, only the coding sequences for the iCre and mCherry along with 2A peptidases between them were precisely inserted in-frame next to start or stop codons of the Cck or Sst genes, respectively. In direct comparison, the previously generated Cre driver lines (for the same two loci) contain extra sequences than just the Cre cDNA⁴¹. In some loci, the additional sequences in the knock-in alleles can affect the normal expression of the endogenous gene.

For example, there are three Cre driver lines generated for the Sst locus, and they all seem to have compromised expression of the Sst gene, that could be attributable to the additional sequences inserted at the targeted site. The Sst tm2.1(cre)Zjh/J line shows significantly diminished endogenous SST expression, while the 129S-Sst tm1Ute/J line is a null mutant⁴¹. In addition, the B6(Cg)-Sst tm1(cre/ERT2)Zjh/J mice both abolishes Sst gene function and expresses a CreERT2 fusion protein from the Sst promoter/enhancer elements. There are also Flp recombinase knock-in (Ssttm3.1(flpo)Zjh/J are Sst-ires-Flp or SOM-IRES-Cre)⁴² and tTA transactivator knock-in models (Cg-Ssttm1.2(tTA2)Hze/J) generated previously and both of these mice appear to interfere with the endogenous expression of SST in the targeted neurons. Thus, our SST mice occupy a unique niche among all SST-engineered knock-in strains.

Similarly, our Cck knock-in mice also represent a unique experimental model in comparison to the publicly available knock-in rodents. For example, the C57BL/6N-Tg(Cck-EGFP/RNAi:Gad1)2Mirn/J transgenic mice mouse line is a hybrid reporter/miRNA knockdown model, where CCK interneuronal expression of a synthetic Gad1 miRNA precursor results in Gad1 transcript downregulation (silencing of GAD1 expression) via endogenous miRNA processing mechanisms^{43, 44}. These animals, while of utility in interneuronal inactivation studies, have very limited use as reporter mice. Working with other Cck transgenic mouse strains has also limitations. B6N.Cg-Cck^{tm1.1(cre)Zjh}/J mice have an internal ribosome entry site and Cre recombinase in the 3' UTR of the Cck locus with yet unknown expression from the Cck-IRES-Cre allele⁴⁵, while the C57BL/6-Tg(Cck-cre)CKres/J strain expresses Cre at high levels in the neocortex with no expression observed in subcortical regions⁴⁶. In addition, B6.129-Cck^tm1Lcs/J is a knock-in/knockout strain where Cck gene is disrupted with a β-geo fusion cassette and the homozygous phenotype manifests metabolic and behavioral disturbances⁴⁷. The validation studies we performed suggest that the construct insertion is both appropriate and functional. CCK and SST expression are consistent with previously reported data in the neocortex, hippocampus, and midbrain, as well as in the peripheral organs. Furthermore, it is also consistent with pervious immunohistochemistry studies of the developmental timeline of these neurons, as well as laminar distribution of the labeled cells.

Utilization of our SST and CCK knock-in animals opens the door to a host of studies, with two broad purposes. First, they can be used as reporters to study development, physiology, or pathophysiology of SST + and CCK + cells. When bred with reporter mice, they allow to study neuronal migration and wiring of the developing brain or determine how different manipulations/insults affect the CCK and SST expressing cells throughout the lifespan. This can encompass both in vivo and in vitro studies, as demonstrated by our data. After crossing with different reporter mice, these SST and CCK knock-in mice can be also used in intersectional approaches (i.e., breeding Cre and Flp with appropriate reporters), visualizing both neuronal populations in the brain with different fluorophores.

Our knock-in mice can also be used in a second, even more important set of studies related to gene function. By breeding the Cre-driver SST and CCK mice with various floxed mice, we can obtain new knowledge on the roles of genes and cells in physiology and behavior. For example, crossing these mice with the floxed Gad1 mice will lead to elimination of the GAD67 protein, the main GABA synthesizing enzyme⁴⁸. This, and crossing with Gad 2 floxed mice, is particularly important for interneuronal studies, as Gad1 or Gad2 would be only deleted in SST + or CCK + interneurons and would not interfere with the function of the rest of SST + and CCK + cells in the brain or the body. This approach would also allow a functional differentiation between the roles of inhibited CCK + interneurons and the preserved CCK + glutamatergic projection neurons. In addition, the utilization of our models opens the door to a host of behavioral studies, gaining insights into the specific behaviors that arise form inhibition of SST + and CCK + neurons. This is pivotal for understanding disease processes, as interneuronal dysfunction has been observed in a host of neuropsychiatric disorders, including schizophrenia, major depression, bipolar disorder, epilepsy, and many others^49–53.

Notably, the Sst-Cre and Cck-Cre knock-in mice we generated can be used also as tools for studies wherever these genes are expressed. As both peptides have a well-defined, specific role outside the brain, these mice can be utilized in gastrointestinal and endocrine studies^{4, 54–56}. The only limit would be the availability of murine models that contain floxed genes of interest. Upon publication, our knock-in animals will be shared without any restriction with the broader scientific community via the Jackson Laboratories repository.

Mice. Sst^em1(^{P2A−iCre−T2A−mCherry})^Mirn and Cck^{em1(iCre−T2A−mCherry−P2A)Mirn} mouse lines were generated in this study. Additional strains used were obtained from the Jackson Laboratories: C57Bl/6J (stock number 000664), B6.Cg-Gt(ROSA)26Sor^{tm6(CAG−ZsGreen1)Hze}/J (stock number 007906) (common name Ai6 or Ai6(RCL-ZsGreen) and B6.Cg-Gt(ROSA)26Sor^{tm14(CAG−tdTomato)Hze}/J (common name Ai14)³⁹. Mice were bred and group-housed in the pathogen-free animal facility at the University of Nebraska Medical Center (Omaha, NE). Mice were housed in individually ventilated cages that are changed once every two weeks within a laminar flow workstation using micro-isolator technique with ClidoxR-S sterilant. All caging, bedding (cob), and nesting materials were autoclaved prior to use. Water is R/O filtered, chlorinated, and supplied by the HydropacR system. Water and food (Teklad LM-485 Mouse/Rat autoclaved Diet 7012 or Teklad Global 19% Protein Extruded Rodent Diet) were provided ad libitum during the 12-h light/dark cycle. Crinkle paper and social housing were provided in every cage as an environmental enrichment.

Ethical statement. All experimental procedures were approved by the University of Nebraska Medical Center’s Institutional Animal Care and Use Committee and the guidelines for proper animal use and care were strictly followed according to the Guide for the Care and Use of Laboratory animals of the National Institute of Health.

CRISPR reagents, mice generation, and genotyping. Knock-in mice were generated using the Easi-CRISPR method ^{35, 36}. Knock-in models containing new sequences of up to 2kb can be created using Easi-CRISPR with high efficiency^57-60. The Easi-CRISPR approach utilizes three components: guide RNA, Cas9 protein to create a dsDNA break in the genome, and a long ssDNA donor to serve as a template to repair the dsDNA break. CRISPR guide RNAs were designed using CRISPOR⁶¹, Two-part guide (crRNA and tracrRNA) and Cas9 proteins were purchased from Alt-R^TM CRISPR guide RNAs, Integrated DNA Technologies, Inc. (IDT)⁶², Coralville, IA, USA), long ssDNA donors were obtained from Azenta Life Sciences NJ, USA. CRISPR RiboNucleoProtein (RNP) complexes were microinjected into one cell staged zygotes isolated from super-ovulated C57Bl/6J females (Jackson laboratories stock number 000664). The mouse knock-in protocols that include isolation and microinjection of zygotes, their implantation into pseudo-pregnant mice followed by genotyping of founder offspring were as described previously^{35, 63}. Genomic DNAs from the liveborn offspring were extracted from tail biopsies for genotyping PCRs. Genotyping strategies, primer sequences, and the expected amplicon sizes are shown in Figures 1 and 2. The knock-in regions, including the homology arms, were sequenced using multiple overlapping PCR fragments. Heterozygous founder mice were bred back to the parental strain to establish germ line transmission. Further heterozygous breeding was used to generate mice used for a homozygous colony to breed with the Ai6 and Ai14 reporter strains to generate experimental mice for final analysis. The Sst-Cre knock-in mice were generated in 2018 and transferred to our laboratory in February 2019 and we have bred these mice for 8 generations. The Cck-Cre knock-in mice were generated in 2019 and transferred to our laboratory in February 2020 and we have bread them for 4 generations. Each generation was genotyped and expression of mCherry-Cre verified with anti-mCherry antibody using IHC in brain sections in both male and female mice (n>4M + n>4F).

Immunohistochemistry. Mice were anesthetized with isoflurane and transcardially perfused with saline followed by 4% paraformaldehyde. Brains were harvested and postfixed in 4% paraformaldehyde overnight at 4°C. After fixation brains were incubated in 10%, 20%, and 30% sucrose, 24 hours at 4°C in each solution. Next, brains were frozen and cut on cryostat as 50 µm coronal sections for P0 and 35µm for other animal ages. Floating sections were collected and either processed for immunohistochemistry or mounted on glass slides. To aid in visualizing brain structures, nuclei were counterstained with Hoechst dye (0.5 μg/mL final concentration, Thermo Fisher Scientific, Waltham, MA). The primary antibodies used include anti-mCherry (Cell signaling, cat. no: 43590, host: rabbit, dilution used: 1:500), anti-SST (Genetex, cat. no: GTX71935, host: mouse, dilution used: 1:500), anti-CCK (Immuno Star, cat. no: 20078, host: rabbit, dilution used: 1:500). The secondary antibodies include donkey-anti-rabbit CY3 (Jackson Immuno Research, cat.no: 711-165-152, dilution used: 1:500), donkey-anti-rabbit Alexa Fluor 488 (Jackson Immuno Research, cat. no: 711-485-152, dilution used: 1:500) and donkey-anti-mouse 488 Alexa Fluor 488 (Jackson Immuno Research, cat. no: 715-547-003, dilution used: 1:500). Slides were imaged on a Nikon Eclipse FM inverted confocal laser-scanning microscope. Images were acquired using a 40x (1.4 oil Plan APO OFN25 DIC, 0.17 WD, 0.13) oil objective and LU-NV laser unit measuring wavelengths: 405 nm, 488 nm, and 561 nm. One set of sections was imaged with ImageXpress Pico instrument using 4x, 10x, or 20x objectives. Control staining examples are shown in Supplemental Figure 3. The IHC analysis of Sst-Cre and Cck-Cre mice was observational, and it included comparison of observed spatial staining within all brain regions in our mouse models with the SST and CCK staining in C57Bl adult mice in the Allen Brain Atlas. Every Sst-Cre and Cck-Cre mouse had consistent expression across all brain regions and across different mouse generations. For the analysis of Sst-Cre and Cck-Cre at least 4 females and 4 males were analyzed with anti-mCherry, anti-Sst and anti-Cck antibodies over the period of two years. For figure generation in the manuscript, one litter was used for P0, P7 and P14 and four adults (2 female plus 2 males) from each Sst-Cre x ZsGreen1 and Cck-Cre x ZsGreen1 breedings. Sst^Cre/+: Rosa^ZsGreen1/+1 litter with 8 pups (4F+4M) P0; 1 litter with 6 pups (2F+4M) P7; 1 litter with 8 pups (3F+5M) P14; adult 2F+2M. Cck^Cre/+: Rosa^ZsGreen1/+1 litter with 8 pups (4F+4M) P0; 1 litter with 3 pups (2F+1M) P7; 1 litter with 3 pups (2F+1M) P14; adult 2F+2M.

Primary neuronal cultures. Primary cortical neuronal cultures were prepared from E14 mouse embryos as previously described⁶⁴. Briefly, the brain was placed in pre-chilled HBSS solution (without Ca²⁺ or Mg²⁺), and two cortices were dissected and cut with scissors into small tissue pieces of similar sizes and transferred to Trypsin/EDTA (0.25%) for 15 min at 37°C. Trypsin was inactivated by adding Trypsin Inhibitor (Sigma T6522) for 5 min. After incubation, the solutions were removed, and the tissue resuspended in Neurobasal medium with B-27 supplement (Gibco #17504-044) and triturated with a fire-polished Pasteur pipette. The cells were pelleted by centrifugation for 10 min at 90 g. The cell pellet was resuspended in Neurobasal medium with B-27 supplement and the cells counted. The cells were plated on poly-D-lysine coated 96-well plates at 70,000 cells/well. The growth medium was Neurobasal medium with B-27 supplement, and Glutamax. Cells were incubated at 37°C, 5% CO₂ for up to 10 days. At the endpoint of the incubation, cultures were either imaged live or fixed with 4% PFA. In some experiment, 10 μL of diluted Hoechst dye was added to the wells in the 96-well plate to visualize the nuclei. The cells were imaged using an ImageXpress Pico at 4x, 10x or 20x magnification or EVOS microscope. Primary neuronal cultures were prepared from three independent Sst-Cre X ZsGreen1 Reporter breeding and from two independent Cck-Cre x ZsGreen1 Reporter breeding and from one Cck-Cre x dtTomato Reporter breeding. Cortical cells were pooled from the whole embryonic litter, thus the embryonic neuronal cultures contained mix of neurons derived from unknown number of males and females. Multiple 96-well plates were prepared every time and one plate was taken for analysis at 3, 6 or 9 days to confirm that endogenous ZsGreen1 or dtTomato were present in neuronal cultures for the whole duration of the experiment.

Conflict of interest: The authors declare no conflict of interest.

Acknowledgements

We are thankful to the UNMC DNA Sequencing Core Facility and the UNMC Comparative Medicine Department for their outstanding technical support of our experiments.

Funding

This work was supported by NIH grant R01 MH067234 (KM). CBG would like to acknowledge funding support from the NIH grants R35HG010719 and R21GM129559. The funding source had no influence on the study design, data collection or analyses, the decision to publish, or the preparation of this manuscript.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author within reasonable limits. Sst-Cre and Cck-Cre mice will be deposited into the Jackson Laboratories animal model repository immediately upon publication.

Authors’ contributions

ZK and KM conceived and designed the study. CBG and RQ generated the mice. MB performed IHC and imaging. AA maintained the mouse colony, performed the genotyping, and assisted with tissue preparation for IHC. MB, AA, CG, RQ, ZK and KM interpreted the results and wrote the manuscript. All authors have read and given approval of the final version of the manuscript.

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There is NO Competing Interest.

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Supplemental Figure 1. Examples of rostro-caudal brain sections of Sst^mCh-Cre/+/Ai6^ZsGreen/1+endogenous fluorescence in the brain of an adult male mice. Low magnification (4x) of the major brain coronal rostro-caudal regions is shown. All images were acquired with ImageXpress Pico Instrument. Abbreviations: SST = somatostatin.
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Supplemental figure 2. Examples of rostro-caudal brain sections of Cck^mCh-Cre/+/Ai6^ZsGreen1/+endogenous fluorescence in the brain of an adult male mice. Low magnification (4x) of the major brain coronal rostro-caudal regions is shown. All images were acquired with ImageXpress Pico Instrument. Abbreviations: CCK = cholecystokinin.
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Supplemental figure 3. A) Control staining for double labeling of anti-mCherry (red) and anti-SST (green) antibodies in the brain of Sst^{mCh-Cre/mCh-Cre}adult male mouse. B) Control staining for double labeling of anti-mCherry (red) and anti-Cck (green) antibodies in the brain of Cck^{mCh-Cre/mCh-Cre}adult male mouse. For both stainings cell nuclei were stained with Hoechst dye (white), primary antibody was omitted, while all the other steps were performed equally as for the experimental staining. Details are shown for hippocampal area CA1 (upper panel) and cortex (lower panel). Magnification of the images is 40x. Abbreviations: CA1 = Cornu ammonis subfield 1, CCK = cholecystokinin, CTX = cortex, HIP = hippocampus, SST = somatostatin.

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Knock-in mouse models for studying somatostatin and cholecystokinin expressing cells

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Generation of Sst-Cre and Cck-Cre knock-in mice

Primary neuronal cultures

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