Zn-regulated GTPase metalloprotein activator 1 modulates vertebrate zinc homeostasis.

Zinc (Zn) is an essential micronutrient and cofactor for up to 10% of proteins in living organisms. During Zn limitation, specialized enzymes called metallochaperones are predicted to allocate Zn to specific metalloproteins. This function has been putatively assigned to G3E GTPase COG0523 proteins, yet no Zn metallochaperone has been experimentally identified in any organism. Here, we functionally characterize a family of COG0523 proteins that is conserved across vertebrates. We identify Zn metalloprotease methionine aminopeptidase 1 (METAP1) as a COG0523 client, leading to the redesignation of this group of COG0523 proteins as the Zn-regulated GTPase metalloprotein activator (ZNG1) family. Using biochemical, structural, genetic, and pharmacological approaches across evolutionarily divergent models, including zebrafish and mice, we demonstrate a critical role for ZNG1 proteins in regulating cellular Zn homeostasis. Collectively, these data reveal the existence of a family of Zn metallochaperones and assign ZNG1 an important role for intracellular Zn trafficking.

Oncogenic Fatty Acid Metabolism Rewires Energy Supply Chain in Gastric Carcinogenesis.

BACKGROUND & AIMS: Gastric carcinogenesis develops within a sequential carcinogenic cascade from precancerous metaplasia to dysplasia and adenocarcinoma, and oncogenic gene activation can drive the process. Metabolic reprogramming is considered a key mechanism for cancer cell growth and proliferation. However, how metabolic changes contribute to the progression of metaplasia to dysplasia remains unclear. We have examined metabolic dynamics during gastric carcinogenesis using a novel mouse model that induces Kras activation in zymogen-secreting chief cells. METHODS: We generated a Gif-rtTA;TetO-Cre;KrasG12D (GCK) mouse model that continuously induces active Kras expression in chief cells after doxycycline treatment. Histologic examination and imaging mass spectrometry were performed in the GCK mouse stomachs at 2 to 14 weeks after doxycycline treatment. Mouse and human gastric organoids were used for metabolic enzyme inhibitor treatment. The GCK mice were treated with a stearoyl- coenzyme A desaturase (SCD) inhibitor to inhibit the fatty acid desaturation. Tissue microarrays were used to assess the SCD expression in human gastrointestinal cancers. RESULTS: The GCK mice developed metaplasia and high-grade dysplasia within 4 months. Metabolic reprogramming from glycolysis to fatty acid metabolism occurred during metaplasia progression to dysplasia. Altered fatty acid desaturation through SCD produces a novel eicosenoic acid, which fuels dysplastic cell hyperproliferation and survival. The SCD inhibitor killed both mouse and human dysplastic organoids and selectively targeted dysplastic cells in vivo. SCD was up-regulated during carcinogenesis in human gastrointestinal cancers. CONCLUSIONS: Active Kras expression only in gastric chief cells drives the full spectrum of gastric carcinogenesis. Also, oncogenic metabolic rewiring is an essential adaptation for high-energy demand in dysplastic cells.

Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.

Tissues and organs are composed of distinct cell types that must operate in concert to perform physiological functions. Efforts to create high-dimensional biomarker catalogs of these cells have been largely based on single-cell sequencing approaches, which lack the spatial context required to understand critical cellular communication and correlated structural organization. To probe in situ biology with sufficient depth, several multiplexed protein imaging methods have been recently developed. Though these technologies differ in strategy and mode of immunolabeling and detection tags, they commonly utilize antibodies directed against protein biomarkers to provide detailed spatial and functional maps of complex tissues. As these promising antibody-based multiplexing approaches become more widely adopted, new frameworks and considerations are critical for training future users, generating molecular tools, validating antibody panels, and harmonizing datasets. In this Perspective, we provide essential resources, key considerations for obtaining robust and reproducible imaging data, and specialized knowledge from domain experts and technology developers.

Prospective on Imaging Mass Spectrometry in Clinical Diagnostics.

Imaging mass spectrometry (IMS) is a molecular technology utilized for spatially driven research, providing molecular maps from tissue sections. This article reviews matrix-assisted laser desorption ionization (MALDI) IMS and its progress as a primary tool in the clinical laboratory. MALDI mass spectrometry has been used to classify bacteria and perform other bulk analyses for plate-based assays for many years. However, the clinical application of spatial data within a tissue biopsy for diagnoses and prognoses is still an emerging opportunity in molecular diagnostics. This work considers spatially driven mass spectrometry approaches for clinical diagnostics and addresses aspects of new imaging-based assays that include analyte selection, quality control/assurance metrics, data reproducibility, data classification, and data scoring. It is necessary to implement these tasks for the rigorous translation of IMS to the clinical laboratory; however, this requires detailed standardized protocols for introducing IMS into the clinical laboratory to deliver reliable and reproducible results that inform and guide patient care.

Imaging Mass Spectrometry of Isotopically Resolved Intact Proteins on a Trapped Ion-Mobility Quadrupole Time-of-Flight Mass Spectrometer.

In this work, we demonstrate rapid, high spatial, and high spectral resolution imaging of intact proteins by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on a hybrid quadrupole-reflectron time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Historically, untargeted MALDI IMS of proteins has been performed on TOF mass spectrometers. While advances in TOF instrumentation have enabled rapid, high spatial resolution IMS of intact proteins, TOF mass spectrometers generate relatively low-resolution mass spectra with limited mass accuracy. Conversely, the implementation of MALDI sources on high-resolving power Fourier transform (FT) mass spectrometers has allowed IMS experiments to be conducted with high spectral resolution with the caveat of increasingly long data acquisition times. As illustrated here, qTOF mass spectrometers enable protein imaging with the combined advantages of TOF and FT mass spectrometers. Protein isotope distributions were resolved for both a protein standard mixture and proteins detected from a whole-body mouse pup tissue section. Rapid (∼10 pixels/s) 10 µm lateral spatial resolution IMS was performed on a rat brain tissue section while maintaining isotopic spectral resolution. Lastly, proof-of-concept MALDI-TIMS data was acquired from a protein mixture to demonstrate the ability to differentiate charge states by ion mobility. These experiments highlight the advantages of qTOF and timsTOF platforms for resolving and interpreting complex protein spectra generated from tissue by IMS.

Multidimensional mass profiles increase confidence in bacterial identification when using low-resolution mass spectrometers.

Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of Yersinia pestis. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical.

Rapid Multivariate Analysis Approach to Explore Differential Spatial Protein Profiles in Tissue.

Spatially targeted proteomics analyzes the proteome of specific cell types and functional regions within tissue. While spatial context is often essential to understanding biological processes, interpreting sub-region-specific protein profiles can pose a challenge due to the high-dimensional nature of the data. Here, we develop a multivariate approach for rapid exploration of differential protein profiles acquired from distinct tissue regions and apply it to analyze a published spatially targeted proteomics data set collected from Staphylococcus aureus-infected murine kidney, 4 and 10 days postinfection. The data analysis process rapidly filters high-dimensional proteomic data to reveal relevant differentiating species among hundreds to thousands of measured molecules. We employ principal component analysis (PCA) for dimensionality reduction of protein profiles measured by microliquid extraction surface analysis mass spectrometry. Subsequently, k-means clustering of the PCA-processed data groups samples by chemical similarity. Cluster center interpretation revealed a subset of proteins that differentiate between spatial regions of infection over two time points. These proteins appear involved in tricarboxylic acid metabolomic pathways, calcium-dependent processes, and cytoskeletal organization. Gene ontology analysis further uncovered relationships to tissue damage/repair and calcium-related defense mechanisms. Applying our analysis in infectious disease highlighted differential proteomic changes across abscess regions over time, reflecting the dynamic nature of host-pathogen interactions.

MALDI TIMS IMS of Disialoganglioside Isomers─GD1a and GD1b in Murine Brain Tissue.

Gangliosides are acidic glycosphingolipids, containing ceramide moieties and oligosaccharide chains with one or more sialic acid residue(s) and are highly diverse isomeric structures with distinct biological roles. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial analysis of gangliosides, among other biomolecules, directly from tissue sections. Integrating trapped ion mobility spectrometry with MALDI IMS allows for the analysis of isomeric lipid structures in situ. Here, we demonstrate the gas-phase separation and identification of disialoganglioside isomers GD1a and GD1b that differ in the position of a sialic acid residue, in multiple samples, including a standard mixture of both isomers, a biological extract, and directly from thin tissue sections. The unique spatial distributions of GD1a/b (d36:1) and GD1a/b (d38:1) isomers were determined in rat hippocampus and spinal cord tissue sections, demonstrating the ability to structurally characterize and spatially map gangliosides based on both the carbohydrate chain and ceramide moieties.

High-Resolution Imaging Mass Spectrometry of Human Donor Eye: Photoreceptors Cells and Basal Laminar Deposit of Age-Related Macular Degeneration.

Pathologies of the retina are clinically visualized in vivo with OCT and ex vivo with immunohistochemistry. Although both techniques provide valuable information on prognosis and disease state, a comprehensive method for fully elucidating molecular constituents present in locations of interest is desirable. The purpose of this work was to use multimodal imaging technologies to localize the vast number of molecular species observed with matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) in aged and diseased retinal tissues. Herein, MALDI IMS was utilized to observe molecular species that reside in photoreceptor cells and also a basal laminar deposit from two human donor eyes. The molecular species observed to accumulate in these discrete regions can be further identified and studied to attempt to gain a greater understanding of biological processes occurring in debilitating eye diseases such as age-related macular degeneration (AMD).

Highly multiplexed immunofluorescence of the human kidney using co-detection by indexing.

The human kidney is composed of many cell types that vary in their abundance and distribution from normal to diseased organ. As these cell types perform unique and essential functions, it is important to confidently label each within a single tissue to accurately assess tissue architecture and microenvironments. Towards this goal, we demonstrate the use of co-detection by indexing (CODEX) multiplexed immunofluorescence for visualizing 23 antigens within the human kidney. Using CODEX, many of the major cell types and substructures, such as collecting ducts, glomeruli, and thick ascending limb, were visualized within a single tissue section. Of these antibodies, 19 were conjugated in-house, demonstrating the flexibility and utility of this approach for studying the human kidney using custom and commercially available antibodies. We performed a pilot study that compared both fresh frozen and formalin-fixed paraffin-embedded healthy non-neoplastic and diabetic nephropathy kidney tissues. The largest cellular differences between the two groups was observed in cells labeled with aquaporin 1, cytokeratin 7, and α-smooth muscle actin. Thus, our data show the power of CODEX multiplexed immunofluorescence for surveying the cellular diversity of the human kidney and the potential for applications within pathology, histology, and building anatomical atlases.

Referenced Kendrick Mass Defect Annotation and Class-Based Filtering of Imaging MS Lipidomics Experiments.

Because of their diverse functionalities in cells, lipids are of primary importance when characterizing molecular profiles of physiological and disease states. Imaging mass spectrometry (IMS) provides the spatial distributions of lipid populations in tissues. Referenced Kendrick mass defect (RKMD) analysis is an effective mass spectrometry (MS) data analysis tool for classification and annotation of lipids. Herein, we extend the capabilities of RKMD analysis and demonstrate an integrated method for lipid annotation and chemical structure-based filtering for IMS datasets. Annotation of lipid features with lipid molecular class, radyl carbon chain length, and degree of unsaturation allows image reconstruction and visualization based on each structural characteristic. We show a proof-of-concept application of the method to a computationally generated IMS dataset and validate that the RKMD method is highly specific for lipid components in the presence of confounding background ions. Moreover, we demonstrate an application of the RKMD-based annotation and filtering to matrix-assisted laser desorption/ionization (MALDI) IMS lipidomic data from human kidney tissue analysis.

High Spatial Resolution MALDI Imaging Mass Spectrometry of Fresh-Frozen Bone.

Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis and osteomyelitis. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) facilitate the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of osseous tissue for MALDI IMS has been difficult due to its mineralized composition and heterogeneous morphology, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs, allowing the distribution of endogenous lipids to be linked to tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. High spatial resolution (10 µm) MALDI IMS was employed to characterize lipid distributions, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to the bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment.

Staphylococcus aureus exhibits heterogeneous siderophore production within the vertebrate host.

Siderophores, iron-scavenging small molecules, are fundamental to bacterial nutrient metal acquisition and enable pathogens to overcome challenges imposed by nutritional immunity. Multimodal imaging mass spectrometry allows visualization of host-pathogen iron competition, by mapping siderophores within infected tissue. We have observed heterogeneous distributions of Staphylococcus aureus siderophores across infectious foci, challenging the paradigm that the vertebrate host is a uniformly iron-depleted environment to invading microbes.

α-Cyano-4-hydroxycinnamic Acid and Tri-Potassium Citrate Salt Pre-Coated Silicon Nanopost Array Provides Enhanced Lipid Detection for High Spatial Resolution MALDI Imaging Mass Spectrometry.

We have developed a pre-coated substrate for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) that enables high spatial resolution mapping of both phospholipids and neutral lipid classes in positive ion mode as metal cation adducts. The MALDI substrates are constructed by depositing a layer of α-cyano-4-hydroxycinnamic acid (CHCA) and potassium salts onto silicon nanopost arrays (NAPA) prior to tissue mounting. The matrix/salt pre-coated NAPA substrate significantly enhances all detected lipid signals allowing lipids to be detected at lower laser energies than bare NAPA. The improved sensitivity at lower laser energy enabled ion images to be generated at 10 µm spatial resolution from rat retinal tissue. Optimization of matrix pre-coated NAPA consisted of testing lithium, sodium, and potassium salts along with various matrices to investigate the increased sensitivity toward lipids for MALDI IMS experiments. It was determined that pre-coating NAPA with CHCA and potassium salts before thaw-mounting of tissue resulted in a signal intensity increase of at least 5.8 ± 0.1-fold for phospholipids and 2.0 ± 0.1-fold for neutral lipids compared to bare NAPA. Pre-coating NAPA with matrix and salt also reduced the necessary laser power to achieve desorption/ionization by ∼35%. This reduced the effective diameter of the ablation area from 13 ± 2 µm down to 8 ± 1 µm, enabling high spatial resolution MALDI IMS. Using pre-coated NAPA with CHCA and potassium salts offers a MALDI IMS substrate with broad molecular coverage of lipids in a single polarity that eliminates the need for extensive sample preparation after sectioning.

Unsupervised machine learning for exploratory data analysis in imaging mass spectrometry.

Imaging mass spectrometry (IMS) is a rapidly advancing molecular imaging modality that can map the spatial distribution of molecules with high chemical specificity. IMS does not require prior tagging of molecular targets and is able to measure a large number of ions concurrently in a single experiment. While this makes it particularly suited for exploratory analysis, the large amount and high-dimensional nature of data generated by IMS techniques make automated computational analysis indispensable. Research into computational methods for IMS data has touched upon different aspects, including spectral preprocessing, data formats, dimensionality reduction, spatial registration, sample classification, differential analysis between IMS experiments, and data-driven fusion methods to extract patterns corroborated by both IMS and other imaging modalities. In this work, we review unsupervised machine learning methods for exploratory analysis of IMS data, with particular focus on (a) factorization, (b) clustering, and (c) manifold learning. To provide a view across the various IMS modalities, we have attempted to include examples from a range of approaches including matrix assisted laser desorption/ionization, desorption electrospray ionization, and secondary ion mass spectrometry-based IMS. This review aims to be an entry point for both (i) analytical chemists and mass spectrometry experts who want to explore computational techniques; and (ii) computer scientists and data mining specialists who want to enter the IMS field. © 2019 The Authors. Mass Spectrometry Reviews published by Wiley Periodicals, Inc. Mass SpecRev 00:1-47, 2019.

Diagnosis of melanoma by imaging mass spectrometry: Development and validation of a melanoma prediction model.

BACKGROUND: The definitive diagnosis of melanocytic neoplasia using solely histopathologic evaluation can be challenging. Novel techniques that objectively confirm diagnoses are needed. This study details the development and validation of a melanoma prediction model from spatially resolved multivariate protein expression profiles generated by imaging mass spectrometry (IMS). METHODS: Three board-certified dermatopathologists blindly evaluated 333 samples. Samples with triply concordant diagnoses were included in this study, divided into a training set (n = 241) and a test set (n = 92). Both the training and test sets included various representative subclasses of unambiguous nevi and melanomas. A prediction model was developed from the training set using a linear support vector machine classification model. RESULTS: We validated the prediction model on the independent test set of 92 specimens (75 classified correctly, 2 misclassified, and 15 indeterminate). IMS detects melanoma with a sensitivity of 97.6% and a specificity of 96.4% when evaluating each unique spot. IMS predicts melanoma at the sample level with a sensitivity of 97.3% and a specificity of 97.5%. Indeterminate results were excluded from sensitivity and specificity calculations. CONCLUSION: This study provides evidence that IMS-based proteomics results are highly concordant to diagnostic results obtained by careful histopathologic evaluation from a panel of expert dermatopathologists.

An orthogonal methods assessment of topical drug concentrations in skin and the impact for risk assessment in the viable epidermis.

Systemic toxicity assessments for oral or parenteral drugs often utilize the concentration of drug in plasma to enable safety margin calculations for human risk assessment. For topical drugs, there is no standard method for measuring drug concentrations in the stratum basale of the viable epidermis. This is particularly important since the superficial part of the epidermis, the stratum corneum (SC), is nonviable and where most of a topically applied drug remains, never penetrating deeper into the skin. We investigated the relative concentrations of a prototype kinase inhibitor using punch biopsy, laser capture microdissection, and imaging mass spectrometry methods in the SC, stratum basale, and dermis of minipig skin following topical application as a cream formulation. The results highlight the value of laser capture microdissection and mass spectrometry imaging in quantifying the large difference in drug concentration across the skin and even within the epidermis, and supports use of these methods for threshold-based toxicity risk assessments in specific anatomic locations of the skin, like of the stratum basale.

Pyruvate induces torpor in obese mice.

Mice subjected to cold or caloric deprivation can reduce body temperature and metabolic rate and enter a state of torpor. Here we show that administration of pyruvate, an energy-rich metabolic intermediate, can induce torpor in mice with diet-induced or genetic obesity. This is associated with marked hypothermia, decreased activity, and decreased metabolic rate. The drop in body temperature correlates with the degree of obesity and is blunted by housing mice at thermoneutrality. Induction of torpor by pyruvate in obese mice relies on adenosine signaling and is accompanied by changes in brain levels of hexose bisphosphate and GABA as detected by mass spectroscopy-based imaging. Pyruvate does not induce torpor in lean mice but results in the activation of brown adipose tissue (BAT) with an increase in the level of uncoupling protein-1 (UCP1). Denervation of BAT in lean mice blocks this increase in UCP1 and allows the pyruvate-induced torpor phenotype. Thus, pyruvate administration induces torpor in obese mice by pathways involving adenosine and GABA signaling and a failure of normal activation of BAT.

Spatial Metabolomics of the Human Kidney using MALDI Trapped Ion Mobility Imaging Mass Spectrometry.

Low molecular weight metabolites are essential for defining the molecular phenotypes of cells. However, spatial metabolomics tools often lack the sensitivity, specify, and spatial resolution to provide comprehensive descriptions of these species in tissue. MALDI imaging mass spectrometry (IMS) of low molecular weight ions is particularly challenging as MALDI matrix clusters are often nominally isobaric with multiple metabolite ions, requiring high resolving power instrumentation or derivatization to circumvent this issue. An alternative to this is to perform ion mobility separation before ion detection, enabling the visualization of metabolites without the interference of matrix ions. Additional difficulties surrounding low weight metabolite visualization include high resolution imaging, while maintaining sufficient ion numbers for broad and representative analysis of the tissue chemical complement. Here, we use MALDI timsTOF IMS to image low molecular weight metabolites at higher spatial resolution than most metabolite MALDI IMS experiments (20 µm) while maintaining broad coverage within the human kidney. We demonstrate that trapped ion mobility spectrometry (TIMS) can resolve matrix peaks from metabolite signal and separate both isobaric and isomeric metabolites with different distributions within the kidney. The added ion mobility data dimension dramatically increased the peak capacity for spatial metabolomics experiments. Through this improved sensitivity, we have found >40 low molecular weight metabolites in human kidney tissue, such as argininic acid, acetylcarnitine, and choline that localize to the cortex, medulla, and renal pelvis, respectively. Future work will involve further exploring metabolomic profiles of human kidneys as a function of age, sex, and race.

Dynamic Range Expansion by Gas-Phase Ion Fractionation and Enrichment for Imaging Mass Spectrometry.

In the analysis of biological tissue by imaging mass spectrometry (IMS), the limit of detection and dynamic range are of paramount importance in obtaining experimental results that provide insight into underlying biological processes. Many important biomolecules are present in the tissue milieu in low concentrations and in complex mixtures with other compounds of widely ranging abundances, challenging the limits of analytical technologies. In many IMS experiments, the ion signal can be dominated by a few highly abundant ion species. On trap-based instrument platforms that accumulate ions prior to mass analysis, these high abundance ions can diminish the detection and dynamic range of lower abundance ions. Herein, we describe two strategies for combating these challenges during IMS experiments on a hybrid QhFT-ICR MS. In one iteration, the mass resolving capabilities of a quadrupole mass filter are used to selectively enrich ions of interest via a technique previously termed continuous accumulation of selected ions. Second, we have introduced a supplemental dipolar AC waveform to the quadrupole mass filter of a commercial QhFT-ICR mass spectrometer to perform selected ion ejection prior to the ion accumulation region. This setup allows the selective ejection of the most abundant ion species prior to ion accumulation, thereby greatly improving the molecular depth with which IMS can probe tissue samples. The gain in sensitivity of both of these approaches roughly scales with the number of accumulated laser shots up to the charge capacity of the ion accumulation cell. The efficiencies of these two strategies are described here by performing lipid imaging mass spectrometry analyses of a rat brain.