Labeling Of Neurons Research Articles

Responsiveness of afferent renal nerve units in renovascular hypertension in rats

Previous data suggest that renal afferent nerve activity is increased in hypertension exerting sympathoexcitatory effects. Hence, we wanted to test the hypothesis that in renovascular hypertension, the activity of dorsal root ganglion (DRG) neurons with afferent projections from the kidneys is augmented depending on the degree of intrarenal inflammation. For comparison, a nonhypertensive model of mesangioproliferative nephritis was investigated. Renovascular hypertension (2-kidney, 1-clip [2K1C]) was induced by unilateral clipping of the left renal artery and mesangioproliferative glomerulonephritis (anti-Thy1.1) by IV injection of a 1.75-mg/kg BW OX-7 antibody. Neuronal labeling (dicarbocyanine dye [DiI]) in all rats allowed identification of renal afferent dorsal root ganglion (DRG) neurons. A current clamp was used to characterize neurons as tonic (sustained action potential [AP] firing) or phasic (1–4 AP) upon stimulation by current injection. All kidneys were investigated using standard morphological techniques. DRG neurons exhibited less often tonic response if in vivo axonal input from clipped kidneys was received (30.4% vs. 61.2% control, p < 0.05). However, if the nerves to the left clipped kidneys were cut 7 days prior to investigation, the number of tonic renal neurons completely recovered to well above control levels. Interestingly, electrophysiological properties of neurons that had in vivo axons from the right non-clipped kidneys were not distinguishable from controls. Renal DRG neurons from nephritic rats also showed less often tonic activity upon current injection (43.4% vs. 64.8% control, p < 0.05). Putative sympathoexcitatory and impaired sympathoinhibitory renal afferent nerve fibers probably contribute to increased sympathetic activity in 2K1C hypertension.

Topographical Differences in Orexin Activation after Pain Assessments in Hb AA and HbSS Sickle Mice

The orexins are neuropeptides that are synthesized in the perifornical area (PFA), lateral hypothalamus (LH) and dorsomedial hypothalamus (DMH) and mediate arousal, sleep, energy balance, food and drug reward. The LH region is associated with reward, while the PFA and DMH are more associated with stress and arousal. Data suggest that orexin-A has an analgesic effect on inflammatory pain and it may affect mechanisms underlying the maintenance of neuropathic pain. To date, there have been no published studies to investigate this system as a potential target to treat neuropathic pain in a mouse model of sickle cell disease (SCD). Therefore, we seek to determine activational and topographical differences in neuronal activation of the orexin system in a mouse model of SCD by using an array of behavioral measures to characterize pain in transgenic mice expressing human hemoglobin.Female transgenic sickle mice that express exclusively (99%) human sickle hemoglobin (HbSS) and age-/gender-matched controls (HbAA mice; n = 5/group, 20-30g) expressing normal human hemoglobin A are used in the study. All behavioral tests are performed in a quiet room maintained at a constant temperature of 23- 25°C. All mice are habituated to each test protocol and environment for at least three consecutive days before collecting baseline measurements and testing. Three measures are used to assess pain-related behaviors: thermal/heat hyperalgesia (test for heat sensitivity), cold hyperalgesia (for cold sensitivity), and mechanical hyperalgesia (sensitivity to mechanical stimulus). Ninety minutes after the pain assessment testing, brains from HbAA and HbSS mice are quick frozen and hypothalamic brain sections from the mice are processed to visualize orexin-A and c-Fos using double label immunohistochemistry.Preliminary analyses show that significant topographical differences are observed in LH versus PFA for HbAA and HbSS mice (p<0.05) and LH versus DMH for HbAA and HbSS mice (p<0.05). This indicates that the number of double labeled neurons in the LH is significantly lower than the number of double labeled neurons in the PFA and DMH of HbAA and HbSS mice. Additionally, a significant topographical difference is observed in LH versus DMH for only HbSS mice (p<0.05). These data demonstrate that the number of activated orexin neurons is greater after pain testing in the DMH (a region more associated with stress and arousal) versus LH in HbSS mice. There is a trend towards a significant increase in activated LH orexin neurons in HbAA versus HbSS mice. Additional studies are underway to determine whether fewer reward-associated, orexin neurons in the LH are activated in HbSS versus HbAA mice after pain testing, and whether this decrease in orexin activation may provide a possible explanation for the increased hyperalgesia in HbSS sickle mice, which contributes to neuropathic pain observed in SCD. DisclosuresGupta:Tautona Group: Consultancy, Honoraria; Fera Pharmaceuticals LLC: Consultancy, Honoraria.

Functionally distinct POMC-expressing neuron subpopulations in hypothalamus revealed by intersectional targeting

Pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus represent key regulators of metabolic homeostasis. Electrophysiological and single-cell sequencing experiments have revealed a remarkable degree of heterogeneity of these neurons. However, the exact molecular basis and functional consequences of this heterogeneity have not yet been addressed. Here, we have developed new mouse models in which intersectional Cre/Dre-dependent recombination allowed for successful labeling, translational profiling and functional characterization of distinct POMC neurons expressing the leptin receptor (Lepr) and glucagon like peptide 1 receptor (Glp1r). Our experiments reveal that POMCLepr+ and POMCGlp1r+ neurons represent largely nonoverlapping subpopulations with distinct basic electrophysiological properties. They exhibit a specific anatomical distribution within the arcuate nucleus and differentially express receptors for energy-state communicating hormones and neurotransmitters. Finally, we identify a differential ability of these subpopulations to suppress feeding. Collectively, we reveal a notably distinct functional microarchitecture of critical metabolism-regulatory neurons.

Immunohistochemical and Genetic Labeling of Hairy and Glabrous Skin Innervation.

Cutaneous innervation is an essential component of the mammalian sensory nervous system. During development, genetically and morphologically diverse subtypes of sensory neurons use distinct molecular pathways to innervate end organs or form free nerve endings in glabrous and hairy skin. Peripheral neurons can be damaged by acute injury or degenerate due to chronic conditions including diabetes and chemotherapy, leading to peripheral neuropathy. The analysis of skin and cutaneous innervation can be applied to many research endeavors, from developmental neuroscience to pharmaceutical testing. Due to the natural hydrophobicity and heterogenous makeup of the skin (dense, keratinized cells as well as sparse, extracellular-matrix-bound cells), its histological analysis presents unique challenges compared to that of many other tissues. This series of protocols describes histological methods for generalized immunohistochemistry and subtype-specific genetic labeling of sensory neurons in mouse skin in both whole-mount and section formats. We provide detailed methodology of tissue preparation for hairy and glabrous skin, several types of labeling, and counting of hair follicles in flat-mounted mouse skin. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Cryosectioning and immunostaining of mouse hairy skin Alternate Protocol 1: Alternate method for preparation and fixation of mouse hairy skin Basic Protocol 2: Sectioning of mouse paw glabrous skin Basic Protocol 3: Whole-mount immunolabeling of mouse skin Basic Protocol 4: Sparse labeling of skin-innervating neurons with a Cre-dependent membrane-bound alkaline phosphatase reporter Alternate Protocol 2: Sparse labeling of skin-innervating neurons with a Cre-dependent fluorescent reporter Basic Protocol 5: Oil Red O staining of skin.

Distinct Nicotinic Responses Map onto Electrophysiologically‐ and Morphologically‐Distinct Pyramidal Neurons Within Layer 5 of The Mouse Prefrontal Cortex

Neurotransmission mediated by acetylcholine at its nicotinic receptors (nAChRs) within the medial prefrontal cortex (mPFC) is critical for higher-order cognitive tasks. Pyramidal neurons within layer 5 of the mPFC express nAChRs and form the primary signalling outputs from this brain region. These neurons may be categorized as either regular-firing or initial burst-firing, based on threshold responses to positive current injection. The objective of this study was to use whole-cell electrophysiology and biocytin neuron labelling in young postnatal mice (age 15-20 days) of both sexes to characterize the morphology and nAChR function within each of these neuron categories. When comparing the neuron categories, we first determined that regular-firing neurons have a greater membrane resistance and spike adaptation ratio, and a lower magnitude post-firing hyperpolarization. Regular-firing neurons also exhibited a lower frequency of spontaneous excitatory postsynaptic potentials. Detailed morphological analysis determined that regular-firing neurons have more apical dendrite matter distal to the soma and more basal dendrite matter proximal to the soma. Pharmacological experiments using receptor-selective antagonists revealed that nicotinic responses are mediated by distinct nAChR isoforms that are expressed selectively on each of the two pyramidal neuron categories. Specifically, these experiments demonstrate that heteromeric α4β2* nAChRs are present on regular-firing pyramidal neurons and homomeric α7 nAChRs are present on burst-firing pyramidal neurons. Ongoing experiments aim to determine the projection targets for each type of pyramidal neuron within each sex. These novel findings suggest a detailed receptor-based mechanism by which acetylcholine neurotransmission is regulated within output neurons of mPFC layer 5, which may impact the development of cognitive circuits involving this brain region.

Effects of ARC-hippocampus obestatin neural pathway on gastric motility and gastric emptying in diabetic rats

Objective: To investigate the obestatin neural projections from arcuate nucleus (ARC) to hippocampus in diabetic rats, and its effects on gastric motility and gastric emptying of rats. Methods: Diabetic model was established by fructose intake combined with streptozotocin injected intraperitoneally in healthy male Wistar rats. Diabetic rats were randomly divided into five groups: control group (NS group), 0.1, 1 and 10 pmol obestatin group, and obestatin + NBI27914 group, with 7 rats in each group. 0.5 μl saline (NS), obestatin (0.1 pmol, 1 pmol, 10 pmol) or the mixture (10 pmol obestatin + 60 pmol NBI27914) was injected into the hippocampus respectively, the gastric motility was recorded immediately after administration, and the gastric emptying was studied 15 min later. ARC-hippocampus obestatin neural pathway and ARC obestatin mRNA expression were compared between normal and diabetic rats with fluorogold (FG) retrograde tracing and immunofluorescence histochemical staining. Results: Compared with normal rats, the number of ARC FG/obestatin double labeled neurons and the expression level of ARC obestatin mRNA were decreased significantly in diabetic rats (P＜0.05); Obestatin could inhibit gastric motility and gastric emptying in a dose-dependent manner (P＜0.05~0.01) and the effects of obestatin could be partially blocked by NBI27914, an antagonist of corticotropin releasing factor receptor 1 (CRFR1) (P＜0.05). Compared with normal rats, the inhibitory effects of obestatin on gastric motility and gastric emptying were significantly decreased in diabetic rats (P＜0.05). Conclusion: There is an obestatin neural pathway between ARC and hippocampus, which participates in the regulation of gastric motility and gastric emptying in diabetic rats, and CRFR1 signal pathway is involved in this process. The damage of this neural pathway may participate in gastric motility dysfunction in early stage of diabetes.

Optical Imaging of Pancreatic Innervation.

At the time of Ivan Pavlov, pancreatic innervation was studied by looking at pancreas secretions in response to electrical stimulation of nerves. Nowadays we have ways to visualize neuronal activity in real time thanks to advances in fluorescent reporters and imaging techniques. We also have very precise optogenetic and pharmacogenetic approaches that allow neuronal manipulations in a very specific manner. These technological advances have been extensively employed for studying the central nervous system and are just beginning to be incorporated for studying visceral innervation. Pancreatic innervation is complex, and the role it plays in physiology and pathophysiology of the organ is still not fully understood. In this review we highlight anatomical aspects of pancreatic innervation, techniques for pancreatic neuronal labeling, and approaches for imaging pancreatic innervation in vitro and in vivo.

In vivo uptake of a fluorescent conjugate of melanin-concentrating hormone in the rat brain

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide synthesized by posterior hypothalamic and incerto-hypothalamic neurons that project throughout the central nervous system. The MCHergic system modulates several important functions such as feeding behavior, mood and sleep. MCH exerts its biological functions through interaction with the MCHR-1 receptor, the only functional receptor present in rodents. The internalization process of MCHR-1 triggered by MCH binding was described in vitro in non-neuronal heterologous systems with over-expression of MCHR-1. Reports of in vivo MCHR-1 internalization dynamics are scarce, however, this is an important process to explore based on the critical functions of the MCHergic system. We had previously determined that 60 min after intracerebroventricular (i.c.v.) microinjections of MCH conjugated with fluorophore rhodamine (R-MCH), the dorsal and median raphe nucleus presented R-MCH positive labeled neurons. In the present work, we further studied the in vivo uptake process focusing on the distribution and time-dependent pattern of R-MCH positive cells 10, 20 and 60 min (T10, T20 and T60, respectively) after i.c.v. microinjection of R-MCH. We also explored this uptake process to see whether it was receptor- and clathrin-dependent and examined the phenotype of R-MCH positive cells and their proximity to MCHergic fibers. We found a great number of R-MCH positive cells with high fluorescence intensity in the lateral septum, nucleus accumbens and hippocampus at T20 and T60 (but not at T10), while a lower number with low intensity was observed in the dorsal raphe nucleus. At T20, in rats pre-treated with a MCHR-1 antagonist (ATC-0175) or with phenylarsine oxide (PAO), a clathrin endocytosis inhibitor, a robust decrease (> 50 %) of R-MCH uptake occurred in these structures. The R-MCH positive cells were identified as neurons (NeuN positive, GFAP negative) and some MCHergic fibers run in the vicinities of them. We concluded that neurons localized at structures that were close to the ventricular surfaces could uptake R-MCH in vivo through a receptor-dependent and clathrin-mediated process. Our results support volume transmission of MCH through the cerebrospinal fluid to reach distant targets. Finally, we propose that R-MCH would be an effective tool to study MCH-uptake in vivo.

Expression and Cell Type-specific Localization of Inflammasome Sensors in the Spinal Cord of SOD1(G93A) Mice and Sporadic Amyotrophic lateral sclerosis Patients

Inflammasomes are key components of the innate immune system and activation of these multiprotein platforms is a crucial event in the etiopathology of amyotrophic lateral sclerosis (ALS). Inflammasomes consist of a pattern recognition receptor (PRR), the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and caspase 1. Exogenous or endogenous “danger signals” can trigger inflammasome assembly and promote maturation and release of pro-inflammatory cytokines, including interleukin 1β. Previous studies have demonstrated presence and activation of NLRP3 in spinal cord tissue from SOD1(G93A) mice and human sporadic ALS (sALS) patients. However, regulation and cell type-specific localization of other well-known PRRs has not yet been analysed in ALS. Here, we explored gene expression, protein concentration and cell type-specific localization of the NLRP1, NLRC4 and AIM2 inflammasomes in spinal cord samples from SOD1(G93A) mice and sALS patients. Transcription levels of NLRP1 and NLRC4, but not AIM2, were elevated in symptomatic SOD1(G93A) animals. Immunoblotting revealed elevated protein levels of NLRC4, which were significantly increased in sALS vs. control patients. Immunofluorescence studies revealed neuronal labelling of all investigated PRRs. Staining of AIM2 was detected in all types of glia, whereas glial type-specific labelling was observed for NLRP1 and NLRC4. Our findings revealed pathology-related and cell type-specific differences in the expression of subsets of PRRs. Besides NLRP3, NLRC4 appears to be linked more closely to ALS pathogenesis.

Local FK506 drug delivery enhances nerve regeneration through fresh, unprocessed peripheral nerve allografts

ObjectiveNerve allografts offer many advantages in the reconstruction of peripheral nerve gaps: they retain their native microstructure, contain pro-regenerative Schwann cells, are widely available, and avoid donor site morbidity. Unfortunately, clinical use of nerve allografts is limited by the need for systemic immunosuppression and its adverse effects. To eliminate the toxicity of the systemic immunosuppressant FK506, we developed a local FK506 drug delivery system (DDS) to provide drug release over 28 days. The study objective was to investigate if the local FK506 DDS enhances nerve regeneration in a rodent model of nerve gap defect reconstruction with immunologically-disparate nerve allografts. MethodsIn male Lewis rats, a common peroneal nerve gap defect was reconstructed with either a 20 mm nerve isograft from a donor Lewis rat or a 20 mm fresh, unprocessed nerve allograft from an immunologically incompatible donor ACI rat. After 4 weeks of survival, nerve regeneration was evaluated using retrograde neuronal labelling, quantitative histomorphometry, and serum cytokine profile. ResultsTreatment with both systemic FK506 and the local FK506 DDS significantly improved motor and sensory neuronal regeneration, as well as histomorphometric indices including myelinated axon number. Rats with nerve allografts treated with either systemic or local FK506 had significantly reduced serum concentrations of the pro-inflammatory cytokine IL-12 compared to untreated vehicle control rats with nerve allografts. Serum FK506 levels were undetectable in rats with local FK506 DDS. InterpretationThe local FK506 DDS improved motor and sensory nerve regeneration through fresh nerve allografts to a level equal to that of either systemic FK506 or nerve isografting. This treatment may be clinically translatable in peripheral nerve reconstruction or vascularized composite allotransplantation.

Glial cell type-specific gene expression in the mouse cerebrum using the piggyBac system and in utero electroporation

Glial cells such as astrocytes and oligodendrocytes play crucial roles in the central nervous system. To investigate the molecular mechanisms underlying the development and the biological functions of glial cells, simple and rapid techniques for glial cell-specific genetic manipulation in the mouse cerebrum would be valuable. Here we uncovered that the Gfa2 promoter is suitable for selective gene expression in astrocytes when used with the piggyBac system and in utero electroporation. In contrast, the Blbp promoter, which has been used to induce astrocyte-specific gene expression in transgenic mice, did not result in astrocyte-specific gene expression. We also identified the Plp1 and Mbp promoters could be used with the piggyBac system and in utero electroporation to induce selective gene expression in oligodendrocytes. Furthermore, using our technique, neuron-astrocyte or neuron-oligodendrocyte interactions can be visualized by labeling neurons, astrocytes and oligodendrocytes differentially. Our study provides a fundamental basis for specific transgene expression in astrocytes and/or oligodendrocytes in the mouse cerebrum.

Spinal Excitatory Dynorphinergic Interneurons Contribute to Burn Injury-Induced Nociception Mediated by Phosphorylated Histone 3 at Serine 10 in Rodents

The phosphorylation of serine 10 in histone 3 (p-S10H3) has recently been demonstrated to participate in spinal nociceptive processing. However, superficial dorsal horn (SDH) neurons involved in p-S10H3-mediated nociception have not been fully characterized. In the present work, we combined immunohistochemistry, in situ hybridization with the retrograde labeling of projection neurons to reveal the subset of dorsal horn neurons presenting an elevated level of p-S10H3 in response to noxious heat (60 °C), causing burn injury. Projection neurons only represented a small percentage (5%) of p-S10H3-positive cells, while the greater part of them belonged to excitatory SDH interneurons. The combined immunolabeling of p-S10H3 with markers of already established interneuronal classes of the SDH revealed that the largest subset of neurons with burn injury-induced p-S10H3 expression was dynorphin immunopositive in mice. Furthermore, the majority of p-S10H3-expressing dynorphinergic neurons proved to be excitatory, as they lacked Pax-2 and showed Lmx1b-immunopositivity. Thus, we showed that neurochemically heterogeneous SDH neurons exhibit the upregulation of p-S10H3 shortly after noxious heat-induced burn injury and consequential tissue damage, and that a dedicated subset of excitatory dynorphinergic neurons is likely a key player in the development of central sensitization via the p-S10H3 mediated pathway.

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

Widespread corticopetal projections from the oval paracentral nucleus of the intralaminar thalamic nuclei conveying orofacial proprioception in rats.

The oval paracentral nucleus (OPC) was initially isolated from the paracentral nucleus (PC) within the intralaminar thalamic nuclei in rats. We have recently shown that the rat OPC receives proprioceptive inputs from jaw-closing muscle spindles (JCMSs). However, it remains unknown which cortical areas receive thalamic inputs from the OPC, and whether the cortical areas receiving the OPC inputs are distinct from those receiving inputs from the other intralaminar nuclei and sensory thalamic nuclei. To address this issue, we injected an anterograde tracer, biotinylated dextranamine (BDA), into the OPC, which was electrophysiologically identified by recording of proprioceptive inputs from the JCMSs. Many BDA-labeled axonal fibers and terminals from the OPC were ipsilaterally observed in the rostral and rostroventral regions of the primary somatosensory cortex (S1), the rostral region of the secondary somatosensory cortex (S2), and the most rostrocaudal levels of the granular insular cortex (GI). In contrast, a BDA injection into the caudal PC, which was located slightly rostral to the OPC, resulted in ipsilateral labeling of axonal fibers and terminals in the rostrolateral region of the medial agranular cortex and the rostromedial region of the lateral agranular cortex. Furthermore, injections of a retrograde tracer, Fluorogold, into these S1, S2, and GI regions, resulted in preferential labeling of neurons in the ipsilateral OPC among the intralaminar and sensory thalamic nuclei. These findings reveal that the rat OPC has widespread, but strong corticopetal projections, indicating that there exist divergent corticopetal pathways from the intralaminar thalamic nucleus, which process JCMS proprioceptive sensation.

Mass Generation, Neuron Labeling, and 3D Imaging of Minibrains.

Minibrain is a 3D brain in vitro spheroid model, composed of a mixed population of neurons and glial cells, generated from human iPSC derived neural stem cells. Despite the advances in human 3D in vitro models such as aggregates, spheroids and organoids, there is a lack of labeling and imaging methodologies to characterize these models. In this study, we present a step-by-step methodology to generate human minibrain nurseries and novel strategies to subsequently label projection neurons, perform immunohistochemistry and 3D imaging of the minibrains at large multiplexable scales. To visualize projection neurons, we adapt viral transduction and to visualize the organization of cell types we implement immunohistochemistry. To facilitate 3D imaging of minibrains, we present here pipelines and accessories for one step mounting and clearing suitable for confocal microscopy. The pipelines are specifically designed in such a way that the assays can be multiplexed with ease for large-scale screenings using minibrains and other organoid models. Using the pipeline, we present (i) dendrite morphometric properties obtained from 3D neuron morphology reconstructions, (ii) diversity in neuron morphology, and (iii) quantified distribution of progenitors and POU3F2 positive neurons in human minibrains.

Presynaptic Inhibition of Pain and Touch in the Spinal Cord: From Receptors to Circuits.

Sensory primary afferent fibers, conveying touch, pain, itch, and proprioception, synapse onto spinal cord dorsal horn neurons. Primary afferent central terminals express a wide variety of receptors that modulate glutamate and peptide release. Regulation of the amount and timing of neurotransmitter release critically affects the integration of postsynaptic responses and the coding of sensory information. The role of GABA (γ-aminobutyric acid) receptors expressed on afferent central terminals is particularly important in sensory processing, both in physiological conditions and in sensitized states induced by chronic pain. During the last decade, techniques of opto- and chemogenetic stimulation and neuronal selective labeling have provided interesting insights on this topic. This review focused on the recent advances about the modulatory effects of presynaptic GABAergic receptors in spinal cord dorsal horn and the neural circuits involved in these mechanisms.

Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues.

Transcript labeling in intact tissues using in situ hybridization chain reaction has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. However, large tissue labeling in non-perfused or fresh-frozen rodent and postmortem human samples, which provide more flexible utilization than perfused tissues, is largely unexplored. In the present study, we optimized the combination of in situ hybridization chain reaction in fresh-frozen rodent brains and then evaluated the uniformity of neuronal labeling between two clearing methods, CLARITY and iDISCO+. We found that CLARITY yielded higher signal-to-noise ratios but more limited imaging depth and required longer clearing times, whereas, iDISCO+ resulted in better tissue clearing, greater imaging depth and a more uniform labeling of larger samples. Based on these results, we used iDISCO+-cleared fresh-frozen rodent brains to further validate this combination and map the expression of a few genes of interest pertaining to mood disorders. We then examined the potential of in situ hybridization chain reaction to label transcripts in cleared postmortem human brain tissues. The combination failed to produce adequate mRNA labeling in postmortem human cortical slices but produced visually adequate labeling in the cerebellum tissues. We next, investigated the multiplexing ability of in situ hybridization chain reaction in cleared tissues which revealed inconsistent fluorescence output depending upon the fluorophore conjugated to the hairpins. Finally, we applied our optimized protocol to assess the effect of glucocorticoid receptor overexpression on basal somatostatin expression in the mouse cortex. The constitutive glucocorticoid receptor overexpression resulted in lower number density of somatostatin-expressing neurons compared to wild type. Overall, the combination of in situ hybridization chain reaction with clearing methods, especially iDISCO+, may find broad application in the transcript analysis in rodent studies, but its limited use in postmortem human tissues can be improved by further optimizations.

Rostro-Caudal Specificity of Corticospinal Tract Projections in Mice.

Rostro-caudal specificity of corticospinal tract (CST) projections from different areas of the cortex was assessed by retrograde labeling with fluorogold and retrograde transfection following retro-AAV/Cre injection into the spinal cord of tdT reporter mice. Injections at C5 led to retrograde labeling of neurons throughout forelimb area of the sensorimotor cortex and a region in the dorsolateral cortex near the barrel field (S2). Injections at L2 led to retrograde labeling of neurons in the posterior sensorimotor cortex (hindlimb area) but not the dorsolateral cortex. With injections of biotinylated dextran amine (BDA) into the main sensorimotor cortex (forelimb region), labeled axons terminated selectively at cervical levels. With BDA injections into caudal sensorimotor cortex (hindlimb region), labeled axons passed through cervical levels without sending collaterals into the gray matter and then elaborated terminal arbors at thoracic sacral levels. With BDA injections into the dorsolateral cortex near the barrel field, labeled axons terminated at high cervical levels. Axons from medial sensorimotor cortex terminated primarily in intermediate laminae and axons from lateral sensorimotor cortex terminated primarily in laminae III-V of the dorsal horn. One of the descending pathways seen in rats (the ventral CST) was not observed in most mice.

Efficient photoactivatable Dre recombinase for cell type-specific spatiotemporal control of genome engineering in the mouse

Precise genetic engineering in specific cell types within an intact organism is intriguing yet challenging, especially in a spatiotemporal manner without the interference caused by chemical inducers. Here we engineered a photoactivatable Dre recombinase based on the identification of an optimal split site and demonstrated that it efficiently regulated transgene expression in mouse tissues spatiotemporally upon blue light illumination. Moreover, through a double-floxed inverted open reading frame strategy, we developed a Cre-activated light-inducible Dre (CALID) system. Taking advantage of well-defined cell-type-specific promoters or a well-established Cre transgenic mouse strain, we demonstrated that the CALID system was able to activate endogenous reporter expression for either bulk or sparse labeling of CaMKIIα-positive excitatory neurons and parvalbumin interneurons in the brain. This flexible and tunable system could be a powerful tool for the dissection and modulation of developmental and genetic complexity in a wide range of biological systems.

Using anxiety as a sex‐specific neuropsychiatric biomarker of Alzheimer’s disease

AbstractBackgroundNeuropsychiatric disturbances, such as depression and anxiety, are observed in 90% of Alzheimer’s disease (AD) patients and are frequent in those at risk for AD. However, until recently, much of the research narrowly targeted co‐morbid depression. Clinical reports have provided evidence that anxiety symptoms predict the conversion to AD, over and beyond the effects of depression, memory loss, and even atrophy. Similarly, epidemiological studies show that neurodegeneration and clinical symptoms occur more rapidly in females once diagnosed. Although most AD studies have been performed using male mice, females represent two‐thirds of the AD population and are more susceptible to depression and anxiety.MethodTo study how anatomical sex and anxiety impact AD progression, I used our activity‐dependent tagging system mice. These mice allow for brain‐wide indelible labeling of neurons activated during learning, which then can be compared with secondary neuronal ensembles activated during memory retrieval. The neurons activated at both time points represent a memory trace or engram. Here, we aimed to identify the neural ensembles linking anxiety and memory loss following AD progression by utilizing behavioral studies, calcium imaging and whole‐brain microscopy, in female and male mice.ResultWe found: 1) Female AD mice exhibited anxiety‐like behavior at an earlier age compared to controls and male mice, 2) AD female mice displayed memory deficits as early as 2 months of age, 3) Anxiety‐like behavior correlated with memory impairment only in AD female mice, and 4) Unlike their male counterparts, female AD mice showed a decline in memory traces in the CA3 of the hippocampus.ConclusionWe now believe that anxiety could represent a neuropsychiatric predictor of AD and are currently working to translate these findings to humans using the Alzheimer’s disease neuroimaging initiative (ADNI) dataset. We have found that in humans, anxiety predicts transition to dementia and that anxiety has a sex‐specific effect on brain atrophy.