Huck, N;Donovan, L;Shen, H;Jordan, C;Muwanga, G;Bridges, C;Forman, T;Cordonnier, S;Haight, E;Dale-Huang, F;Takemura, Y;Tawfik, V;
| DOI: 10.1016/j.ynpai.2022.100106
Chronic pain is a common and often debilitating problem that affects 100 million Americans. A better understanding of pain’s molecular mechanisms is necessary for developing safe and effective therapeutics. Microglial activation has been implicated as a mediator of chronic pain in numerous preclinical studies; unfortunately, translational efforts using known glial modulators have largely failed, perhaps at least in part due to poor specificity of the compounds pursued, or an incomplete understanding of microglial reactivity. In order to achieve a more granular understanding of the role of microglia in chronic pain as a means of optimizing translational efforts, we utilized a clinically-informed mouse model of complex regional pain syndrome (CRPS), and monitored microglial activation throughout pain progression. We discovered that while both males and females exhibit spinal cord microglial activation as evidenced by increases in Iba1, activation is attenuated and delayed in females. We further evaluated the expression of the newly identified microglia-specific marker, TMEM119, and identified two distinct populations in the spinal cord parenchyma after peripheral injury: TMEM119+ microglia and TMEM119- infiltrating myeloid lineage cells, which are comprised of Ly6G + neutrophils and Ly6G- macrophages/monocytes. Neurons are sensitized by inflammatory mediators released in the CNS after injury; however, the cellular source of these cytokines remains somewhat unclear. Using multiplex in situ hybridization in combination with immunohistochemistry, we demonstrate that spinal cord TMEM119+ microglia are the cellular source of cytokines IL6 and IL1β after peripheral injury. Taken together, these data have important implications for translational studies: 1) microglia remain a viable analgesic target for males and females, so long as duration after injury is considered; 2) the analgesic properties of microglial modulators are likely at least in part related to their suppression of microglial-released cytokines, and 3) a limited number of neutrophils and macrophages/monocytes infiltrate the spinal cord after peripheral injury but have unknown impact on pain persistence or resolution. Further studies to uncover glial-targeted therapeutic interventions will need to consider sex, timing after injury, and the exact target population of interest to have the specificity necessary for translation.
Topilko, T;Diaz, SL;Pacheco, CM;Verny, F;Rousseau, CV;Kirst, C;Deleuze, C;Gaspar, P;Renier, N;
PMID: 35123655 | DOI: 10.1016/j.neuron.2022.01.012
Optimizing reproductive fitness in mammalians requires behavioral adaptations during pregnancy. Maternal preparatory nesting is an essential behavior for the survival of the upcoming litter. Brain-wide immediate early gene mapping in mice evoked by nesting sequences revealed that phases of nest construction strongly activate peptidergic neurons of the Edinger-Westphal nucleus in pregnant mice. Genetic ablation, bidirectional neuromodulation, and in vitro and in vivo activity recordings demonstrated that these neurons are essential to modulate arousal before sleep to promote nesting specifically. We show that these neurons enable the behavioral effects of progesterone on preparatory nesting by modulating a broad network of downstream targets. Our study deciphers the role of midbrain CART+ neurons in behavioral adaptations during pregnancy vital for reproductive fitness.
Kim, S;Oh, H;Choi, SH;Yoo, YE;Noh, YW;Cho, Y;Im, GH;Lee, C;Oh, Y;Yang, E;Kim, G;Chung, WS;Kim, H;Kang, H;Bae, Y;Kim, SG;Kim, E;
PMID: 36130507 | DOI: 10.1016/j.celrep.2022.111398
Myelin transcription factor 1 like (Myt1l), a zinc-finger transcription factor, promotes neuronal differentiation and is implicated in autism spectrum disorder (ASD) and intellectual disability. However, it remains unclear whether Myt1l promotes neuronal differentiation in vivo and its deficiency in mice leads to disease-related phenotypes. Here, we report that Myt1l-heterozygous mutant (Myt1l-HT) mice display postnatal age-differential ASD-related phenotypes: newborn Myt1l-HT mice, with strong Myt1l expression, show ASD-like transcriptomic changes involving decreased synaptic gene expression and prefrontal excitatory synaptic transmission and altered righting reflex. Juvenile Myt1l-HT mice, with markedly decreased Myt1l expression, display reverse ASD-like transcriptomes, increased prefrontal excitatory transmission, and largely normal behaviors. Adult Myt1l-HT mice show ASD-like transcriptomes involving astrocytic and microglial gene upregulation, increased prefrontal inhibitory transmission, and behavioral deficits. Therefore, Myt1l haploinsufficiency leads to ASD-related phenotypes in newborn mice, which are temporarily normalized in juveniles but re-appear in adults, pointing to continuing phenotypic changes long after a marked decrease of Myt1l expression in juveniles.
Jin, S;Maddern, XJ;Campbell, EJ;Lawrence, AJ;
PMID: 36038028 | DOI: 10.1016/j.neulet.2022.136858
Projections to the striatum are well-identified. For example, in the ventral striatum, two major inputs to the medial nucleus accumbens shell include the ventral subiculum and basolateral amygdala. However, the chemical phenotype(s) of these projection neurons remain unclear. In this study, we examined amygdalostriatal and corticostriatal connectivity in rats using injections of the retrograde tracer cholera toxin b into the nucleus accumbens shell. To determine the neurotransmitter identity of projection neurons, we combined retrograde tracing with RNAscope in-situ hybridization, using mRNA probes against vesicular transporters associated with glutamatergic (VGluT1 - Slc17a7, VGluT2 - Slc17a6) or GABAergic (VGaT - Slc32a1) neurotransmission. Confocal imaging was used to examine vesicular transporter mRNA expression in the ventral subiculum and basolateral amygdala inputs to the nucleus accumbens shell. Both projections contained mostly VGluT1-expressing neurons. Interestingly, almost a quarter of ventral subiculum to nucleus accumbens shell projections co-expressed VGluT1 and VGluT2 compared to a relatively small number (∼3%) that were co-expressed in basolateral amygdala to nucleus accumbens shell afferents. However, almost a quarter of basolateral amygdala to nucleus accumbens shell projections were VGaT-positive. These findings highlight the diverse proportions of glutamatergic and GABAergic afferents in two major projections to the nucleus accumbens shell and raise important questions for functional studies.
Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-on-chip model
Thacker, VV;Sharma, K;Dhar, N;Mancini, GF;Sordet-Dessimoz, J;McKinney, JD;
PMID: 33908688 | DOI: 10.15252/embr.202152744
Severe cases of SARS-CoV-2 infection are characterized by hypercoagulopathies and systemic endotheliitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of an immune cell-mediated cytokine storm remain unknown. Using a vascularized lung-on-chip model, we find that infection of alveolar epithelial cells leads to limited apical release of virions, consistent with reports of monoculture infection. However, viral RNA and proteins are rapidly detected in underlying endothelial cells, which are themselves refractory to apical infection in monocultures. Although endothelial infection is unproductive, it leads to the formation of cell clusters with low CD31 expression, a progressive loss of barrier integrity and a pro-coagulatory microenvironment. Viral RNA persists in individual cells generating an inflammatory response, which is transient in epithelial cells but persistent in endothelial cells and typified by IL-6 secretion even in the absence of immune cells. Inhibition of IL-6 signalling with tocilizumab reduces but does not prevent loss of barrier integrity. SARS-CoV-2-mediated endothelial cell damage thus occurs independently of cytokine storm.
Mathys H, Davila-Velderrain J, Peng Z, Gao F, Mohammadi S, Young JZ, Menon M, He L, Abdurrob F, Jiang X, Martorell AJ, Ransohoff RM, Hafler BP, Bennett DA, Kellis M, Tsai LH.
PMID: 31042697 | DOI: 10.1038/s41586-019-1195-2
Alzheimer's disease (AD) is a pervasive neurodegenerative disorder, the molecular and cellular complexity of which remains poorly understood. Here, we profiled and analysed 80,660 single-nucleus transcriptomes from prefrontal cortex of 48 individuals with varying degrees of AD pathology. We identified transcriptionally-distinct subpopulations across six major brain cell-types, including those associated with pathology and characterized by regulators of myelination, inflammation, and neuron survival. The strongest AD-associated changes appeared early in pathological progression and were highly cell-type-specific, whereas genes upregulated in late-stage were common across cell types and primarily involved in global stress response. Surprisingly, we found an overrepresentation of female cells in AD-associated subpopulations, and substantially different transcriptional responses between sexes in multiple cell types, including oligodendrocytes. Overall, myelination-related processes were recurrently perturbed in multiple cell types, suggesting a key role in AD pathophysiology. Our single-celltranscriptomic resource provides a first blueprint for interrogating the molecular underpinnings and cellular basis of AD.
Warren BL, Mendoza MP, Cruz FC, Leao RM, Caprioli D, Rubio FJ, Whitaker LR, McPherson KB, Bossert JM, Shaham Y, Hope BT.
PMID: 27335401 | DOI: 10.1523/JNEUROSCI.0140-16.2016
Abstract
In operant learning, initial reward-associated memories are thought to be distinct from subsequent extinction-associated memories. Memories formed during operant learning are thought to be stored in "neuronal ensembles." Thus, we hypothesize that different neuronal ensembles encode reward- and extinction-associated memories. Here, we examined prefrontal cortex neuronal ensembles involved in the recall of reward and extinction memories of food self-administration. We first trained rats to lever press for palatable food pellets for 7 d (1 h/d) and then exposed them to 0, 2, or 7 daily extinction sessions in which lever presses were not reinforced. Twenty-four hours after the last training or extinction session, we exposed the rats to either a short 15 min extinction test session or left them in their homecage (a control condition). We found maximal Fos (a neuronal activity marker) immunoreactivity in the ventral medial prefrontal cortex of rats that previously received 2 extinction sessions, suggesting that neuronal ensembles in this area encode extinction memories. We then used the Daun02 inactivation procedure to selectively disrupt ventral medial prefrontal cortex neuronal ensembles that were activated during the 15 min extinction session following 0 (no extinction) or 2 prior extinction sessions to determine the effects of inactivating the putative food reward and extinction ensembles, respectively, on subsequent nonreinforced food seeking 2 d later. Inactivation of the food reward ensembles decreased food seeking, whereas inactivation of the extinction ensembles increased food seeking. Our results indicate that distinct neuronal ensembles encoding operant reward and extinction memories intermingle within the same cortical area.
SIGNIFICANCE STATEMENT:
A current popular hypothesis is that neuronal ensembles in different prefrontal cortex areas control reward-associated versus extinction-associated memories: the dorsal medial prefrontal cortex (mPFC) promotes reward seeking, whereas the ventral mPFC inhibits reward seeking. In this paper, we use the Daun02 chemogenetic inactivation procedure to demonstrate that Fos-expressing neuronal ensembles mediating both food reward and extinction memories intermingle within the same ventral mPFC area.
Lee S, Lee E, Kim R, Kim J, Lee S, Park H, Yang E, Kim H, Kim E.
PMID: 29970987 | DOI: 10.3389/fnmol.2018.00209
Shank2 is an abundant postsynaptic scaffolding protein implicated in neurodevelopmental and psychiatric disorders, including autism spectrum disorders (ASD). Deletion of Shank2 in mice has been shown to induce social deficits, repetitive behaviors, and hyperactivity, but the identity of the cell types that contribute to these phenotypes has remained unclear. Here, we report a conditional mouse line with a Shank2 deletion restricted to parvalbumin (PV)-positive neurons (Pv-Cre;Shank2fl/fl mice). These mice display moderate hyperactivity in both novel and familiar environments and enhanced self-grooming in novel, but not familiar, environments. In contrast, they showed normal levels of social interaction, anxiety-like behavior, and learning and memory. Basal brain rhythms in Pv-Cre;Shank2fl/fl mice, measured by electroencephalography, were normal, but susceptibility to pentylenetetrazole (PTZ)-induced seizures was decreased. These results suggest that Shank2 deletion in PV-positive neurons leads to hyperactivity, enhanced self-grooming and suppressed brain excitation.
Wu, Y;Xu, W;Ma, L;Yu, Z;Wang, Y;Yu, CR;
PMID: 35497500 | DOI: 10.1016/j.crmeth.2022.100201
We describe a cost-effective, highly sensitive, and quantitative method for in situ detection of RNA molecules in tissue sections. This method, dubbed Yn-situ, standing for Y-branched probe in situ hybridization, uses a single-strand DNA preamplifier with multiple initiation sites that trigger a hybridization chain reaction (HCR) to detect polynucleotides. By characterizing the performance of this method, we show that the Yn-situ method, in conjunction with an improved fixation step, is sensitive enough to allow detection of RNA molecules using fewer probes targeting short nucleotide sequences than existing methods. A set of five probes can produce quantitative results with smaller puncta and higher signal-to-noise ratio than the 20-probe sets commonly required for HCR and RNAscope. We show that the high sensitivity and wide dynamic range allow quantification of genes expressed at different levels in the olfactory sensory neurons. We describe key steps of this method to enable broad utility by individual laboratories.
Cheng, S;Butrus, S;Tan, L;Xu, R;Sagireddy, S;Trachtenberg, JT;Shekhar, K;Zipursky, SL;
PMID: 35063073 | DOI: 10.1016/j.cell.2021.12.022
The role of postnatal experience in sculpting cortical circuitry, while long appreciated, is poorly understood at the level of cell types. We explore this in the mouse primary visual cortex (V1) using single-nucleus RNA sequencing, visual deprivation, genetics, and functional imaging. We find that vision selectively drives the specification of glutamatergic cell types in upper layers (L) (L2/3/4), while deeper-layer glutamatergic, GABAergic, and non-neuronal cell types are established prior to eye opening. L2/3 cell types form an experience-dependent spatial continuum defined by the graded expression of ∼200 genes, including regulators of cell adhesion and synapse formation. One of these genes, Igsf9b, a vision-dependent gene encoding an inhibitory synaptic cell adhesion molecule, is required for the normal development of binocular responses in L2/3. In summary, vision preferentially regulates the development of upper-layer glutamatergic cell types through the regulation of cell-type-specific gene expression programs.
La Manno G, Soldatov R, Zeisel A, Braun E, Hochgerner H, Petukhov V, Lidschreiber K, Kastriti ME, Lönnerberg P, Furlan A, Fan J, Borm LE, Liu Z, van Bruggen D, Guo J, He X, Barker R, Sundström E, Castelo-Branco G, Cramer P, Adameyko I, Linnarsson S, Kharc
PMID: 30089906 | DOI: 10.1038/s41586-018-0414-6
RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput1. However, this approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity-the time derivative of the gene expression state-can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols. RNA velocity is a high-dimensional vector that predicts the future state of individual cells on a timescale of hours. We validate its accuracy in the neural crest lineage, demonstrate its use on multiple published datasets and technical platforms, reveal the branching lineage tree of the developing mouse hippocampus, and examine the kinetics of transcription in human embryonic brain. We expect RNA velocity to greatly aid the analysis of developmental lineages and cellular dynamics, particularly in humans.
Advanced science (Weinheim, Baden-Wurttemberg, Germany)
Yi, T;Wang, N;Huang, J;Wang, Y;Ren, S;Hu, Y;Xia, J;Liao, Y;Li, X;Luo, F;Ouyang, Q;Li, Y;Zheng, Z;Xiao, Q;Ren, R;Yao, Z;Tang, X;Wang, Y;Chen, X;He, C;Li, H;Hu, Z;
PMID: 36961096 | DOI: 10.1002/advs.202300189
Sevoflurane has been the most widely used inhaled anesthetics with a favorable recovery profile; however, the precise mechanisms underlying its anesthetic action are still not completely understood. Here the authors show that sevoflurane activates a cluster of urocortin 1 (UCN1+ )/cocaine- and amphetamine-regulated transcript (CART+ ) neurons in the midbrain involved in its anesthesia. Furthermore, growth hormone secretagogue receptor (GHSR) is highly enriched in sevoflurane-activated UCN1+ /CART+ cells and is necessary for sleep induction. Blockade of GHSR abolishes the excitatory effect of sevoflurane on UCN1+ /CART+ neurons and attenuates its anesthetic effect. Collectively, their data suggest that anesthetic action of sevoflurane necessitates the GHSR activation in midbrain UCN1+ /CART+ neurons, which provides a novel target including the nucleus and receptor in the field of anesthesia.
