Morphological Subpopulations Research Articles

Evaluation of a Self-Supervised Machine Learning Method for Screening of Particulate Samples: A Case Study in Liquid Formulations

Imaging is commonly used as a characterization method in the pharmaceuticals industry, including for quantifying subvisible particles in solid and liquid formulations. Extracting information beyond particle size, such as classifying morphological subpopulations, requires some type of image analysis method. Suggested methods to classify particles have been based on pre-determined morphological features or use supervised training of convolutional neural networks to learn image representations in relation to ground truth labels. Complications arising from highly complex morphologies, unforeseen classes, and time-consuming preparation of ground truth labels, are some of the challenges faced by these methods. In this work, we evaluate the application of a self-supervised contrastive learning method in studying particle images from therapeutic solutions. Unlike with supervised training, this approach does not require ground truth labels and representations are learned by comparing particle images and their augmentations. This method provides a fast and easily implementable tool of coarse screening for morphological attribute assessment. Furthermore, our analysis shows that in cases with relatively balanced datasets, a small subset of an image dataset is sufficient to train a convolutional neural network encoder capable of extracting useful image representations. It is also demonstrated that particle classes typically observed in protein solutions administered by pre-filled syringes emerge as separated clusters in the encoder's embedding space, facilitating performing tasks such as training weakly-supervised classifiers or identifying the presence of new subpopulations.

Single-cell RNA-seq uncovered hemocyte functional subtypes and their differentiational characteristics and connectivity with morphological subpopulations in Litopenaeus vannamei.

Hemocytes play central roles in shrimp immune system, whereas whose subclasses have not yet been completely defined. At present, the morphological classification of hemocytes is inadequate to classify the complete hemocyte repertoire and elucidate the functions and differentiation and maturation processes. Based on single-cell RNA sequencing (scRNA-seq) of hemocytes in healthy Litopenaeus vannamei, combined with RNA-FISH and flow cytometric sorting, we identified three hemocyte clusters including TGase+ cells, CTL+ cells and Crustin+ cells, and further determined their functional properties, potential differentiation trajectory and correspondence with morphological subpopulations. The TGase+ cells were mainly responsible for the coagulation, exhibiting distinguishable characteristics of hyalinocyte, and appeared to be developmentally arrested at an early stage of hemocyte differentiation. The CTL+ cells and Crustin+ cells arrested at terminal stages of differentiation mainly participated in recognizing foreign pathogens and initiating immune defense responses, owning distinctive features of granule-containing hemocytes. Furthermore, we have revealed the functional sub-clusters of three hemocyte clusters and their potential differentiation pathways according to the expression of genes involved in cell cycle, cell differentiation and immune response, and the successive differentiation and maturation of hyalinocytes to granule-containing hemocytes have also mapped. The results revealed the diversity of shrimp hemocytes and provide new theoretical rationale for hemocyte classification, which also facilitate systematic research on crustacean immunity.

Label-free morphological sub-population cytometry for sensitive phenotypic screening of heterogenous neural disease model cells

Label-free image analysis has several advantages with respect to the development of drug screening platforms. However, the evaluation of drug-responsive cells based exclusively on morphological information is challenging, especially in cases of morphologically heterogeneous cells or a small subset of drug-responsive cells. We developed a novel label-free cell sub-population analysis method called “in silico FOCUS (in silico analysis of featured-objects concentrated by anomaly discrimination from unit space)” to enable robust phenotypic screening of morphologically heterogeneous spinal and bulbar muscular atrophy (SBMA) model cells. This method with the anomaly discrimination concept can sensitively evaluate drug-responsive cells as morphologically anomalous cells through in silico cytometric analysis. As this algorithm requires only morphological information of control cells for training, no labeling or drug administration experiments are needed. The responses of SBMA model cells to dihydrotestosterone revealed that in silico FOCUS can identify the characteristics of a small sub-population with drug-responsive phenotypes to facilitate robust drug response profiling. The phenotype classification model confirmed with high accuracy the SBMA-rescuing effect of pioglitazone using morphological information alone. In silico FOCUS enables the evaluation of delicate quality transitions in cells that are difficult to profile experimentally, including primary cells or cells with no known markers.

Unveiling the Native Morphology of Extracellular Vesicles from Human Cerebrospinal Fluid by Atomic Force and Cryogenic Electron Microscopy.

Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 1010 particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 109 particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.

A machine learning pipeline revealing heterogeneous responses to drug perturbations on vascular smooth muscle cell spheroid morphology and formation

Machine learning approaches have shown great promise in biology and medicine discovering hidden information to further understand complex biological and pathological processes. In this study, we developed a deep learning-based machine learning algorithm to meaningfully process image data and facilitate studies in vascular biology and pathology. Vascular injury and atherosclerosis are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMC behaviors would promote the development of therapeutic targets to treat vascular diseases. However, the response to drug treatments among VSMCs with the same diseased vascular condition is often heterogeneous. Here, to identify the heterogeneous responses of drug treatments, we created an in vitro experimental model system using VSMC spheroids and developed a machine learning-based computational method called HETEROID (heterogeneous spheroid). First, we established a VSMC spheroid model that mimics neointima-like formation and the structure of arteries. Then, to identify the morphological subpopulations of drug-treated VSMC spheroids, we used a machine learning framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our machine learning approach successfully showed that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect spheroid morphology, suggesting that multiple drug responses of VSMC spheroid formation exist. Overall, our HETEROID pipeline enables detailed quantitative drug characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis.

Derivation and Molecular Characterization of a Morphological Subpopulation of Human iPSC Astrocytes Reveal a Potential Role in Schizophrenia and Clozapine Response.

Astrocytes are the most abundant cell type in the human brain and are important regulators of several critical cellular functions, including synaptic transmission. Although astrocytes are known to play a central role in the etiology and pathophysiology of schizophrenia, little is known about their potential involvement in clinical response to the antipsychotic clozapine. Moreover, astrocytes display a remarkable degree of morphological diversity, but the potential contribution of astrocytic subtypes to disease biology and drug response has received little attention. Here, we used state-of-the-art human induced pluripotent stem cell (hiPSC) technology to derive a morphological subtype of astrocytes from healthy individuals and individuals with schizophrenia, including responders and nonresponders to clozapine. Using functional assays and transcriptional profiling, we identified a distinct gene expression signature highly specific to schizophrenia as shown by disease association analysis of more than 10 000 diseases. We further found reduced levels of both glutamate and the NMDA receptor coagonist d-serine in subtype astrocytes derived from schizophrenia patients, and that exposure to clozapine only rescued this deficiency in cells from clozapine responders, providing further evidence that d-serine in particular, and NMDA receptor-mediated glutamatergic neurotransmission in general, could play an important role in disease pathophysiology and clozapine action. Our study represents a first attempt to explore the potential contribution of astrocyte diversity to schizophrenia pathophysiology using a human cellular model. Our findings suggest that specialized subtypes of astrocytes could be important modulators of disease pathophysiology and clinical drug response, and warrant further investigations.

Abstract 4039: Simultaneous quantification of activated immune cells and PD-L1 expressing circulating tumor cells (CTCs) in peripheral blood of cancer patients receiving checkpoint inhibitor therapy

Abstract Purpose: Activated immune cell subpopulations and circulating tumor cells in patients receiving checkpoint inhibitor therapy were characterized with the aim of developing more sensitive diagnostic tools for prediction of immunotherapy response or resistance. Methods: Blood samples from bladder, kidney, and prostate cancer patients undergoing checkpoint inhibitor therapy were collected at baseline and on-therapy at convenience and sent to Epic Sciences for processing. Red blood cells were lysed and nucleated cells were plated onto glass slides and stained with CTC (pan-CK, CD45, PD-L1 and DAPI) and immune panels (CD4, CD8, Ki-67, and DAPI). 2-3 million cells per patient were imaged using high throughput digital pathology pipelines and changes in immune cell sub-populations and changes in circulating tumor cell (CTC) burden with PD1/PD-L1 immunotherapy were assessed. Results: A method to profile activated (CD8+Ki-67+ and CD4+Ki-67+) leukocyte subpopulations and PD-L1+ CTCs in a single blood sample was developed and feasibility was demonstrated in cell lines, healthy donor (HD), and patient blood. CTCs were detected in 73% (24/33) of patients tested. Of the 33 baseline samples tested, 12% (4/33) had PD-L1+ CTCs detected. No PD-L1+ CTCs were detected in on-therapy samples. Of 14 patients with matched samples, 57% (8/14) patients had an increase in activated CD4+ leukocytes and 36% (5/14) patients had an increase in activated CD8+ leukocytes in on-therapy samples compared to baseline. A decrease in activated CD4+ leukocytes was observed in 21% (3/14) patients and a decrease in activated CD8+ leukocytes was also observed in 7% (1/14) patients with therapy. Conclusions: We developed a liquid biopsy-based platform that can simultaneously measure immune related biomarkers in CTCs and leukocytes at single cell resolution from ~1 mL of blood. The platform has extreme sensitivity over conventional methodologies and immune cell populations existing in less than 1% of the total are readily quantified. Efforts to identify morphological sub-populations within each canonical leukocyte category and comparison of leukocyte and CTC biomarker panels to patient outcomes are ongoing. Citation Format: Rachel Krupa, Robin Richardson, Priscilla Ontiveros, Joseph Schonhoft, Jiyun Byun, David Lu, Sean Nisperos, Yipeng Wang, Mark Landers, Ryan Dittamore. Simultaneous quantification of activated immune cells and PD-L1 expressing circulating tumor cells (CTCs) in peripheral blood of cancer patients receiving checkpoint inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4039.

Morphological profiling using machine learning reveals emergent subpopulations of interferon-γ–stimulated mesenchymal stromal cells that predict immunosuppression

BackgroundAlthough a preponderance of pre-clinical data demonstrates the immunosuppressive potential of mesenchymal stromal cells (MSCs), significant heterogeneity and lack of critical quality attributes (CQAs) based on immunosuppressive capacity likely have contributed to inconsistent clinical outcomes. This heterogeneity exists not only between MSC lots derived from different donors, tissues and manufacturing conditions, but also within a given MSC lot in the form of functional subpopulations. We therefore explored the potential of functionally relevant morphological profiling (FRMP) to identify morphological subpopulations predictive of the immunosuppressive capacity of MSCs derived from multiple donors, manufacturers and passages. MethodsWe profiled the single-cell morphological response of MSCs from different donors and passages to the functionally relevant inflammatory cytokine interferon (IFN)-γ. We used the machine learning approach visual stochastic neighbor embedding (viSNE) to identify distinct morphological subpopulations that could predict suppression of activated CD4+ and CD8+ T cells in a multiplexed quantitative assay. ResultsMultiple IFN-γ–stimulated subpopulations significantly correlated with the ability of MSCs to inhibit CD4+ and CD8+ T-cell activation and served as effective CQAs to predict the immunosuppressive capacity of additional manufactured MSC lots. We further characterized the emergence of morphological heterogeneity following IFN-γ stimulation, which provides a strategy for identifying functional subpopulations for future single-cell characterization and enrichment techniques. DiscussionThis work provides a generalizable analytical platform for assessing functional heterogeneity based on single-cell morphological responses that could be used to identify novel CQAs and inform cell manufacturing decisions.

Morphological analysis of the filamentous fungus Penicillium chrysogenum using flow cytometry\u2014the fast alternative to microscopic image analysis

An important parameter in filamentous bioreactor cultivations is the morphology of the fungi, due to its interlink to productivity and its dependency on process conditions. Filamentous fungi show a large variety of morphological forms in submerged cultures. These range from dispersed hyphae, to interwoven mycelial aggregates, to denser hyphal aggregates, the so-called pellets. Depending on the objective function of the bioprocess, different characteristics of the morphology are favorable and need to be quantified accurately. The most common method to quantitatively characterize morphology is image analysis based on microscopy. This method is work intensive and time consuming. Therefore, we developed a faster, at-line applicable, alternative method based on flow cytometry. Within this contribution, this novel method is compared to microscopy for a penicillin production process. Both methods yielded in comparable distinction of morphological sub-populations and described their morphology in more detail. In addition to the appropriate quantification of size parameters and the description of the hyphal region around pellets, the flow cytometry method even revealed a novel compactness parameter for fungal pellets which is not accessible via light microscopy. Hence, the here presented flow cytometry method for morphological analysis is a fast and reliable alternative to common tools with some new insights in the pellet morphology, enabling at-line use in production environments.

Nicotine-induced and D1-receptor-dependent dendritic remodeling in a subset of dorsolateral striatum medium spiny neurons

Nicotine is one of the most addictive substances known, targeting multiple memory systems, including the ventral and dorsal striatum. One form of neuroplasticity commonly associated with nicotine is dendrite remodeling. Nicotine-induced dendritic remodeling of ventral striatal medium spiny neurons (MSNs) is well-documented. Whether MSN dendrites in the dorsal striatum undergo a similar pattern of nicotine-induced structural remodeling is unknown. A morphometric analysis of Golgi-stained MSNs in rat revealed a natural asymmetry in dendritic morphology across the mediolateral axis, with larger, more complex MSNs found in the dorsolateral striatum (DLS). Chronic nicotine produced a lasting (at least 21day) expansion in the dendritic complexity of MSNs in the DLS, but not dorsomedial striatum (DMS). Given prior evidence that MSN subtypes can be distinguished based on dendritic morphology, MSNs were segregated into morphological subpopulations based on the number of primary dendrites. Analysis of these subpopulations revealed that DLS MSNs with more primary dendrites were selectively remodeled by chronic nicotine exposure and remodeling was specific to the distal-most portions of the dendritic arbor. Co-administration of the dopamine D1 receptor (D1R) antagonist SCH23390 completely reversed the selective effects of nicotine on DLS MSN dendrite morphology, supporting a causal role for dopamine signaling at D1 receptors in nicotine-induced dendrite restructuring. Considering the functional importance of the DLS in shaping and expressing habitual behavior, these data support a model in which nicotine induces persistent and selective changes in the circuit connectivity of the DLS that may promote and sustain addiction-related behavior.

Automated High-Throughput Quantification Reveals Morphological Heterogeneity in Mammalian Cell Populations

Non-genetic heterogeneities such as transcriptional noise and epigenetic modifications can lead to genetically clonal cells exhibiting heterogeneous responses to a uniform input. Heterogeneities in cellular and organelle morphology represent an additional type of non-genetic heterogeneity that may be capable of producing phenotypic variability, given structure-function relationships demonstrated at the biochemical level as well as morphological dysregulations observed in disease contexts. Until now, morphological heterogeneity in genetically clonal populations has remained largely unexplored due to a lack of appropriate tools. In this study, we develop an automated high-throughput system to quantitatively map morphological heterogeneity in a population of genetically clonal mouse embryonic fibroblasts (MEFs). We show that the morphological landscape is non-smooth, with cells resting in distinct morphological subpopulations. We additionally demonstrate tools to determine the relationship between cell cycle stage and morphological space state, as well as tools to map changes to the morphology space upon drug treatment. In total, we demonstrate a robust, high-throughput automated system for mapping the morphological landscape of a population of mammalian cells as well as tools to probe changes to this landscape upon external perturbation. This work has potential applicability in a variety of settings ranging from morphology-based drug and siRNA screens to improved quantitative screening tools for cytopathological applications.

Structure and function of bistratified intrinsically photosensitive retinal ganglion cells in the mouse.

A subpopulation of retinal ganglion cells (RGCs) expresses the photopigment melanopsin, rendering these cells intrinsically photosensitive (ipRGCs). These cells are critical for competent circadian entrainment, pupillary light reflex, and other non-imaging-forming photic responses. Research has now demonstrated the presence of multiple subpopulations of ipRGC based on the dendritic stratification in the inner plexiform layer (IPL), those monostratified in the Off sublamina (M1), those monostratified in the On sublamina (M2,4,5), and those bistratified in both the On and the Off sublaminae (M3). Despite evidence that M1 and M2 cells are distinct subpopulations of ipRGC based on distinct morphological and physiological properties, the inclusion of M3 cells as a distinct subtype has remained controversial. Aside from the identification of M3 cells as a morphological subpopulation of ipRGC, to date there have been no functional descriptions of M3 cell physiology or synaptic inputs. Our data provide the first in-depth description of M3 cell structural and functional properties. We report that M3 cells form a morphologically heterogeneous population but one that is physiologically homogeneous with properties similar to those of M2 cells.

Differential Cone Pathway Influence on Intrinsically Photosensitive Retinal Ganglion Cell Subtypes

A small subset of ganglion cells in the mammalian retina express the photopigment melanopsin and are intrinsically photosensitive (ipRGCs). These cells are the primary conduits through which photic information is relayed to non-image-forming visual centers that mediate behaviors such as the pupillary light reflex and circadian entrainment. M1 and M2 cells comprise distinct morphological subpopulations of ipRGC, and possess physiological diversity in their intrinsic membrane properties and intrinsic light responses. Additionally, evidence now indicates that all ipRGCs receive photic information from rods/cones via synaptic signaling. It has recently been reported that Off-stratifying M1 cells paradoxically receive input from the On pathway within the Off sublamina of the inner plexiform layer. The purpose of the current study was to examine the functional consequences of cone pathway signaling to M1 and M2 cells. Using pharmacological tools and single-cell recordings of synaptic responses in wild-type and melanopsin-null mice, we found that the On pathway forms the primary excitatory synaptic input to both M1 and M2 cells. This input was much more influential in shaping the light-evoked responses and resting membrane properties of M2 cells than M1 cells. These findings indicate a surprising differential reliance upon cone-mediated phototransduction by ipRGC subpopulations. These findings also suggest that ipRGC subtypes signal diverse photic information to various non-image-forming visual centers.

Organization of endogenous opioids in the rostral agranular insular cortex of the rat

The rostral agranular insular cortex (RAIC) of rats has opioid receptors and has been implicated in the analgesic and reinforcing effects of opiates. To help in understanding the function of endogenous opioids in this structure, we sought to identify and describe the opioid peptides intrinsic to the RAIC by using immunohistochemical methods. Immunolabeling for proopiomelanocortin (POMC), the precursor to beta-endorphin, and endomorphin 1 and 2 on sectioned rat forebrain revealed limited labeling consisting of individual varicose fibers. Immunolabeling for prodynorphin and enkephalin revealed numerous immunopositive cell bodies and fibers with distribution and morphology unique to each. Prodynorphin-immunopositive cell bodies consisted of two types: large, lightly labeled, pyramidal-shaped cell bodies in lamina V and more intensely labeled, small, ovoid cell bodies scattered in other lamina. Axonal fibers immunolabeled for prodynorphin varied in size and were found in all lamina. Immunolabeling for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) was rarely found in dynorphin-containing cell bodies (6%, 10/167) but was visible within a subpopulation of axons. Enkephalin immunolabeling was detected within a single morphological subpopulation of nonpyramidal neurons located predominantly in lamina II/III, 30% (33/109) of which were also GABA immunopositive. Axons immunolabeled for enkephalin were also abundant in lamina II/III. These results suggest that dynorphin and enkephalin peptides are the predominant endogenous opioids in the RAIC and their distinct distributions suggest divergent functional roles. The localization of prodynorphin immunoreactivity to pyramidal cells suggests the possibility that this neuropeptide may be used in RAIC projection neurons, whereas enkephalin distribution was more characteristic of a role in local networks.

Fiber diameter distributions in the chinchilla’s ampullary nerves

A morphometric study of the chinchilla’s ampullary nerves was conducted to produce an unbiased accounting of the diameter distribution of their constituent fibers. Diameter analyses were determined from 1 μm plastic-embedded nerve sections taken at a plane immediately proximal to the sensory epithelium. We found these nerves to be composed of 2094±573 fibers, having diameters that ranged from 0.5 to 8 μm. The distributions of diameters were positively skewed, where approximately 75% of the fibers were found to have diameters less than 3.5 μm. An analysis of the spatial distribution of diameters within the nerve section revealed that the lateralmost areas of the nerve contained larger fractions of fibers within the smallest diameter quintiles, and the central area harbored greater proportions of the larger diameter quintiles. However, significant fractions of all quintiles were found in all areas. These data were integrated with available data of Fernandez et al. (1998) to produce diameter estimates of calyx, dimorphic, and bouton morphology subpopulations. In view of a general relationship between diameter, innervation locus, and an afferent’s physiologic characteristics, these data provide the basis for developing a perspective for the in situ distribution of afferent response dynamics.

Morphometric multivariate analysis of GABAergic neurons containing calretinin and neuronal nitric oxide synthase in the mouse hippocampus

Several studies reported the morphology of calretinin-positive (CR+) neurons and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) labeled or neuronal nitric oxide synthase-positive (nNOS+) neurons in the rodent hippocampus, where these neurons showed similar morphological features. In addition, a previous study reported the frequent colocalization of CR and NADPH-d in the rat hippocampus. In this study, we aimed to examine whether CR+ neurons and nNOS+ neurons belong to a same morphological subpopulation of GABAergic neurons in the mouse hippocampus. Neurons were immunocytochemically classified into three groups, i.e., CR+/nNOS−, CR−/nNOS+ and CR+/nNOS+ groups. The present morphometric analysis was performed in the mouse Ammon’s horn, because CR+/nNOS+ neurons were rarely found in the mouse dentate gyrus. We selected three morphometric parameters, i.e., soma area, soma form factor (FF) and number of primary dendrites. Dunnett’s post-hoc analysis revealed that soma area, soma FF and number of primary dendrites were significantly larger in CR−/nNOS+ group than in CR+/nNOS− and CR+/nNOS+ groups. The morphometric data of CR+/nNOS+ group were quite similar to those of CR+/nNOS− group. The morphometric multivariate logistic regression analysis revealed that these three morphometric parameters were independent significant variables to discriminate between CR+/nNOS− and CR−/nNOS+ groups, and the majority of CR+/nNOS− and CR−/nNOS+ groups were correctly classified from the morphometric features. The present results clearly indicate that CR+/nNOS− neurons and CR−/nNOS+ neurons belong to different morphological subpopulations, and lead us to speculate that they might play different functional roles in the hippocampal circuit. The further application of morphometric multivariate analysis would be valuable to understand the functional roles of chemically defined neurons in the various brain regions.

Spindle neurons of the human anterior cingulate cortex.

The human anterior cingulate cortex is distinguished by the presence of an unusual cell type, a large spindle neuron in layer Vb. This cell has been noted numerous times in the historical literature but has not been studied with modern neuroanatomic techniques. For instance, details regarding the neuronal class to which these cells belong and regarding their precise distribution along both ventrodorsal and anteroposterior axes of the cingulate gyrus are still lacking. In the present study, morphological features and the anatomic distribution of this cell type were studied using computer-assisted mapping and immunocytochemical techniques. Spindle neurons are restricted to the subfields of the anterior cingulate cortex (Brodmann's area 24), exhibiting a greater density in anterior portions of this area than in posterior portions, and tapering off in the transition zone between anterior and posterior cingulate cortex. Furthermore, a majority of the spindle cells at any level is located in subarea 24b on the gyral surface. Immunocytochemical analysis revealed that the neurofilament protein triple was present in a large percentage of these neurons and that they did not contain calcium-binding proteins. Injections of the carbocyanine dye DiI into the cingulum bundle revealed that these cells are projection neurons. Finally, spindle cells were consistently affected in Alzheimer's disease cases, with an overall loss of about 60%. Taken together, these observations indicate that the spindle cells of the human cingulate cortex represent a morphological subpopulation of pyramidal neurons whose restricted distribution may be associated with functionally distinct areas.

Na + current kinetics are not the determinants of the action potential duration in neurons of the rat ventral tegmental area

Na + currents were recorded from two morphological subpopulations of neurons acutely dissociated from the rat ventral tegmental area (VTA). About 45% of 56 VTA cells examined possessed only the ordinary type of Na + current which was blocked by a low concentration (0.2 μM) of TTX. However, the remaining 55% had the Na + current which contained a small fraction of the TTX-insensitive component, irrespective of morphological variations and action potential durations of VTA cells. The peak amplitude of the TTX-insensitive component was less than 10% of the peak amplitude of the total Na + current. The activation and inactivation kinetics of the TTX-insensitive component were much the same as those of the overwhelming TTX-sensitive component of the Na + current in VTA cells but differed from those of the TTX-insensitive Na + current reported in peripheral sensory neurons. Thus, it was concluded that the well-known different action potential durations found for subpopulations of VTA cells are not due to multiplicity of Na + channel kinetics.

Localization of acetylcholine receptors on isolated CNS neurons: cellular and subcellular differentiation

Significant progress has been made in determining regional expression of neurotransmitter receptors within the CNS, but little information is available at the neuronal level. In the current study, to begin characterizing cellular and subcellular aspects of receptor differentiation, we have localized ACh receptors on neurons isolated from the chicken CNS. Localization was determined autoradiographically using 2 cholinergic receptor probes: 3H-propylbenzilylcholine mustard (PrBCM) a muscarinic antagonist, and 125I-alpha-bungarotoxin (BTX), a putative nicotinic ligand in the avian CNS. To isolate neurons, we incubated embryonic chicken retinas (E13-E19) in Ca2+/Mg2+-free buffer containing 8 units/ml papain for 20 min and then gently agitated the tissue by trituration. Large numbers of dendrite-rich neurons, belonging to recognizable morphological subpopulations (e.g., multipolar neurons of various sizes, small bipolar and unipolar neurons), were collected on slides for autoradiography. Cell isolation had no effect on ligand binding levels, and a high ratio of specific to nonspecific binding allowed us to associate silver grains with receptor position. Muscarinic-receptor-positive cells comprised a recognizable subpopulation that had small rounded cell bodies (6-7 micron) and a single emergent arbor. The cells had an axial or elongated appearance. Muscarinic receptors were abundant over dendrites but absent from cell bodies. Segregation to dendrites was complete by E13, the age when synapses reportedly first begin to appear. Cells labeled with 125I-BTX were more heterogeneous in morphology. The most striking BTX-positive cells comprised neurons with large cell bodies (approximately 15 micron) and multiple processes. Dendrites were profusely labeled, but only sporadic labeling was seen on cell bodies, and often this was at sites crossed by labeled dendrites. Maximum labeling occurred in the distal, smallest-caliber ends of the dendritic arbors. All limbs of BTX-positive multipolar neurons expressed abundant receptors. Occasionally, a thin uniform-caliber process was seen branching from a primary dendrite, and such processes, which may have been axons, were never labeled. Very small neurons with bipolar morphology also showed minimal or no labeling on one process, despite dense labeling on the other. Photoreceptors and Mueller cells were never labeled with BTX or PrBCM. The data show that, within a discrete CNS region, specific subpopulations of neurons independently regulate expression of ACh receptors and that, even early in development, control mechanisms segregate receptors to physiologically appropriate regions of the cell surface.

The immunologic and immunotherapeutic sequelae of intraperitoneal BCG. II: Serial changes in spleen weight, morphology, cell populations and in vitro mitogen responses.

FOLLOWING the IP administration of an immunotherapeutic dose of BCG, sequential analysis of spleen weight, morphology, and cell subpopulations was performed over a sixty day period.3H-thymidine uptake, by basal and optimally stimulated spleen cell cultures, was also measured over the same period. Despite a prolonged increase in spleen weight, little perturbation of the percentage distribution of the cell subpopulations and no alteration inin vitro responses were found. The results are discussed in relation to previous reports of immunosuppression following the administration of BCG.