Probes for INS

Ketamine has emerged as a widespread treatment for a variety of psychiatric disorders when used at sub-anesthetic doses, but the neural mechanisms underlying its acute action remain unclear. Here, we identified NMDA receptors containing the 2A subunit (GluN2A) on parvalbumin (PV)-expressing inhibitory interneurons as a pivotal target of low-dose ketamine. Genetically deleting GluN2A receptors globally or selectively from PV interneurons abolished the rapid enhancement of visual cortical responses and gamma-band oscillations by ketamine. Moreover, during the follicular phase of the estrous cycle in female mice, the ketamine response was transiently attenuated along with a concomitant decrease of grin2A mRNA expression within PV interneurons. Thus, GluN2A receptors on PV interneurons mediate the immediate actions of low-dose ketamine treatment, and fluctuations in receptor expression across the estrous cycle may underlie sex-differences in drug efficacy.

KCNQ2/3 channels, ubiquitously expressed neuronal potassium channels, have emerged as indispensable regulators of brain network activity. Despite their critical role in brain homeostasis, the mechanisms by which KCNQ2/3 dysfunction lead to hypersychrony are not fully known. Here, we show that deletion of KCNQ2/3 channels changed PV+ interneurons', but not SST+ interneurons', firing properties. We also find that deletion of either KCNQ2/3 or KCNQ2 channels from PV+ interneurons led to elevated homeostatic potentiation of fast excitatory transmission in pyramidal neurons. Pvalb-Kcnq2 null-mice showed increased seizure susceptibility, suggesting that decreases in interneuron KCNQ2/3 activity remodels excitatory networks, providing a new function for these channels.

A wealth of data has elucidated the mechanisms by which sensory inputs are encoded in the neocortex, but how these processes are regulated by the behavioral relevance of sensory information is less understood. Here, we focus on neocortical layer 1 (L1), a key location for processing of such top-down information. Using Neuron-Derived Neurotrophic Factor(NDNF) as a selective marker of L1 interneurons (INs) and in vivo 2-photon calcium imaging, electrophysiology, viral tracing, optogenetics, and associative memory, we find that L1 NDNF-INs mediate a prolonged form of inhibition in distal pyramidal neuron dendrites that correlates with the strength of the memory trace. Conversely, inhibition from Martinotti cells remains unchanged after conditioning but in turn tightly controls sensory responses in NDNF-INs. These results define a genetically addressable form of dendritic inhibition that is highly experience dependent and indicate that in addition to disinhibition, salient stimuli are encoded at elevated levels of distal dendritic inhibition.

Abstract

PURPOSE:

Understanding tumour heterogeneity is an important challenge in current cancer research. Transcription and epigenetic profiling of cultured melanoma cells have defined at least two distinct cell phenotypes characterised by distinctive gene expression signatures associated with high or low/absent expression of Microphthalmia-associated transcription factor (MITF). Nevertheless, heterogeneity of cellpopulations and gene expression in primary human tumours is much less well characterised.

EXPERIMENTAL DESIGN:

We performed single cell gene expression analyses on 472 cells isolated from needle biopsies of 5 primary human melanomas, 4 superficial spreading and one acral melanoma. The expression of MITF-high and MITF-low signature genes was assessed and compared to investigate intra and inter-tumoural heterogeneity and correlated gene expression profiles.

RESULTS:

Single cell gene expression analyses revealed varying degrees of intra and inter-tumour heterogeneity conferred by the variable expression of distinct sets of genes in different tumours. Expression of MITF partially correlated with that of its known target genes while SOX10 expression correlated best with PAX3 and ZEB2. Nevertheless, cells simultaneously expressing MITF-high and MITF-low signature genes were observed both by single cell analyses and RNAscope.

CONCLUSIONS:

Single cell analyses can be performed on limiting numbers of cells from primary human melanomas revealing their heterogeneity. While tumours comprised variable proportions of cells with the MITF-high and MITF-low gene expression signatures characteristic of melanoma cultures, primary tumours also comprised cells expressing markers of both signatures defining a novel cell state in tumours in vivo.

Background Aging is accompanied by altered thinking (cognition) and feeling (mood), functions that depend on information processing by brain cortical cell microcircuits. We hypothesized that age-associated long-term functional and biological changes are mediated by gene transcriptomic changes within neuronal cell-types forming cortical microcircuits, namely excitatory pyramidal cells (PYC) and inhibitory GABAergic neurons expressing vasoactive intestinal peptide (Vip), somatostatin (Sst) and parvalbumin (Pvalb). Methods To test this hypothesis, we assessed locomotor, anxiety-like and cognitive behavioral changes between young (2 months, n=9) and old (22 months, n=12) male C57BL/6 mice, and performed frontal cortex cell-type specific molecular profiling, using laser-capture microscopy and RNA sequencing. Results were analyzed by neuroinformatics and validated by fluorescent in situ hybridization. Results Old-mice displayed increased anxiety and reduced working memory. The four cell-types displayed distinct age-related transcriptomes and biological pathway profiles, affecting metabolic and cell signaling pathways, and selective markers of neuronal vulnerability (Ryr3), resilience (Oxr1), and mitochondrial dynamics (Opa1), suggesting high age-related vulnerability of PYCs, and variable degree of adaptation in GABAergic neurons. Correlations between gene expression and behaviors suggest that changes in cognition and anxiety associated with age are partly mediated by normal age-related cell changes, and that additional age-independent decreases in synaptic and signaling pathways, notably in PYC and SST-neurons further contribute to behavioral changes. Conclusions Our study demonstrates cell-dependent differential vulnerability and coordinated cell-specific cortical microcircuit molecular changes with age. Collectively, the results suggest intrinsic molecular links between aging, cognition and mood-related behaviors with SST-neurons contributing evenly to both behavioral conditions.

Abstract

BACKGROUND:

Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures.

RESULTS:

Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research.

CONCLUSIONS:

We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.