Protein kinase Cγ negatively regulates the intrinsic excitability in zebrin-negative cerebellar Purkinje cells.

Protein kinase C γ (PKCγ), a neuronal isoform present exclusively in the central nervous system, is most abundantly expressed in cerebellar Purkinje cells (PCs). Targeted deletion of PKCγ causes a climbing fiber synapse elimination in developing PCs and motor deficit. However, physiological roles of PKCγ in adult mouse PCs are little understood. In this study, we aimed to unravel the roles of PKCγ in mature mouse PCs by deleting PKCγ from adult mouse PCs of PKCγfl/fl mice via cerebellar injection of adeno-associated virus (AAV) vectors expressing Cre recombinase under the control of the PC-specific L7-6 promoter. Whole cell patch-clamp recording of PCs showed higher intrinsic excitability in PCs virally lacking PKCγ [PKCγ-conditional knockout (PKCγ-cKO) PCs] than in wild-type (WT) mouse PCs in the zebrin-negative module, but not in the zebrin-positive module. AAV-mediated PKCγ re-expression in PKCγ-deficient mouse PCs in the zebrin-negative module restored the enhanced intrinsic excitability to a level comparable to that of wild-type mouse PCs. In parallel with higher intrinsic excitability, we found larger hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents in PKCγ-cKO PCs located in the zebrin-negative module, compared with those in WT mouse PCs in the same region. However, pharmacological inhibition of the HCN currents did not restore the enhanced intrinsic excitability in PKCγ-cKO PCs in the zebrin-negative module. These results suggested that PKCγ suppresses the intrinsic excitability in zebrin-negative PCs, which is likely independent of the HCN current inhibition.

Anxiety control by astrocytes in the lateral habenula.

The potential role of astrocytes in lateral habenula (LHb) in modulating anxiety was explored in this study. The habenula are a pair of small nuclei located above the thalamus, known for their involvement in punishment avoidance and anxiety. Herein, we observed an increase in theta-band oscillations of local field potentials (LFPs) in the LHb when mice were exposed to anxiety-inducing environments. Electrical stimulation of LHb at theta-band frequency promoted anxiety-like behavior. Calcium (Ca2+) levels and pH in the cytosol of astrocytes and local brain blood volume changes were studied in mice expressing either a Ca2+ or a pH sensor protein specifically in astrocytes and mScarlet fluorescent protein in the blood plasma using fiber photometry. An acidification response to anxiety was observed. Photoactivation of archaerhopsin-T (ArchT), an optogenetic tool that acts as an outward proton pump, results in intracellular alkalinization. Photostimulation of LHb in astrocyte-specific ArchT-expressing mice resulted in dissipation of theta-band LFP oscillation in an anxiogenic environment and suppression of anxiety-like behavior. These findings provide evidence that LHb astrocytes modulate anxiety and may offer a new target for treatment of anxiety disorders.

Improving cell-specific recombination using AAV vectors in the murine CNS by capsid and expression cassette optimization.

The production of cell-type- and age-specific genetically modified mice is a powerful approach for unraveling unknown gene functions. Here, we present a simple and timesaving method that enables adeno-associated virus (AAV)-mediated cell-type- and age-specific recombination in floxed mice. To achieve astrocyte-specific recombination in floxed Ai14 reporter mice, we intravenously injected blood-brain barrier-penetrating AAV-PHP.eB vectors expressing Cre recombinase (Cre) using the astrocyte-specific mouse glial fibrillary acidic protein (mGfaABC1D) promoter. However, we observed nonspecific neuron-predominant transduction despite the use of an astrocyte-specific promoter. We speculated that subtle but continuous Cre expression in nonastrocytic cells triggers recombination, and that excess production of Cre in astrocytes inhibits recombination by forming Cre-DNA aggregates. Here, we resolved this paradoxical event by dividing a single AAV into two mGfaABC1D-promoter-driven AAV vectors, one expressing codon-optimized flippase (FlpO) and another expressing flippase recognition target-flanked rapidly degrading Cre (dCre), together with switching the neuron-tropic PHP.eB capsid to astrocyte-tropic AAV-F. Moreover, we found that the FlpO-dCre system with a target cell-tropic capsid can also function in neuron-targeting recombination in floxed mice.

State-dependent modulation of positive and negative affective valences by a parabrachial nucleus-to-ventral tegmental area pathway in mice.

Appropriately responding to various sensory signals in the environment is essential for animal survival. Accordingly, animal behaviors are closely related to external and internal states, which include the positive and negative emotional values of sensory signals triggered by environmental factors. While the lateral parabrachial nucleus (LPB) plays a key role in nociception and supports negative valences, it also transmits signals including positive valences. However, the downstream neuronal mechanisms of positive and negative valences have not been fully explored. In the present study, we investigated the ventral tegmental area (VTA) as a projection target for LPB neurons. Optogenetic activation of LPB-VTA terminals in male mice elicits positive reinforcement in an operant task and induces both avoidance and attraction in a place-conditioning task. Inhibition of glutamic acid decarboxylase (GAD) 65-expressing cells in the VTA promotes avoidance behavior induced by photoactivation of the LPB-VTA pathway. These findings indicate that the LPB-VTA pathway is one of the LPB outputs for the transmission of positive and negative valence signals, at least in part, with GABAergic modification in VTA.

Astrocytic GPCR-Induced Ca2+ Signaling Is Not Causally Related to Local Cerebral Blood Flow Changes.

Activation of Gq-type G protein-coupled receptors (GPCRs) gives rise to large cytosolic Ca2+ elevations in astrocytes. Previous in vitro and in vivo studies have indicated that astrocytic Ca2+ elevations are closely associated with diameter changes in the nearby blood vessels, which astrocytes enwrap with their endfeet. However, the causal relationship between astrocytic Ca2+ elevations and blood vessel diameter changes has been questioned, as mice with diminished astrocytic Ca2+ signaling show normal sensory hyperemia. We addressed this controversy by imaging cortical vasculature while optogenetically elevating astrocyte Ca2+ in a novel transgenic mouse line, expressing Opto-Gq-type GPCR Optoα1AR (Astro-Optoα1AR) in astrocytes. Blue light illumination on the surface of the somatosensory cortex induced Ca2+ elevations in cortical astrocytes and their endfeet in mice under anesthesia. Blood vessel diameter did not change significantly with Optoα1AR-induced Ca2+ elevations in astrocytes, while it was increased by forelimb stimulation. Next, we labeled blood plasma with red fluorescence using AAV8-P3-Alb-mScarlet in Astro-Optoα1AR mice. We were able to identify arterioles that display diameter changes in superficial areas of the somatosensory cortex through the thinned skull. Photo-stimulation of astrocytes in the cortical area did not result in noticeable changes in the arteriole diameters compared with their background strain C57BL/6. Together, compelling evidence for astrocytic Gq pathway-induced vasodiameter changes was not observed. Our results support the notion that short-term (<10 s) hyperemia is not mediated by GPCR-induced astrocytic Ca2+ signaling.

Interstitial-fluid shear stresses induced by vertically oscillating head motion lower blood pressure in hypertensive rats and humans.

The mechanisms by which physical exercise benefits brain functions are not fully understood. Here, we show that vertically oscillating head motions mimicking mechanical accelerations experienced during fast walking, light jogging or treadmill running at a moderate velocity reduce the blood pressure of rats and human adults with hypertension. In hypertensive rats, shear stresses of less than 1 Pa resulting from interstitial-fluid flow induced by such passive head motions reduced the expression of the angiotensin II type-1 receptor in astrocytes in the rostral ventrolateral medulla, and the resulting antihypertensive effects were abrogated by hydrogel introduction that inhibited interstitial-fluid movement in the medulla. Our findings suggest that oscillatory mechanical interventions could be used to elicit antihypertensive effects.

Virally induced CRISPR/Cas9-based knock-in of fluorescent albumin allows long-term visualization of cerebral circulation in infant and adult mice.

Albumin, a protein produced by liver hepatocytes, represents the most abundant protein in blood plasma. We have previously engineered a liver-targeting adeno-associated viral vector (AAV) that expresses fluorescent protein-tagged albumin to visualize blood plasma in mice. While this approach is versatile for imaging in adult mice, transgene expression vanishes when AAV is administered in neonates due to dilution of the episomal AAV genome in the rapidly growing liver. Here, we use CRISPR/Cas9 genome editing to insert the fluorescent protein mNeonGreen (mNG) gene into the albumin (Alb) locus of hepatocytes to produce fluorescently labeled albumin (Alb-mNG). We constructed a CRISPR AAV that includes ∼1 kb homologous arms around Alb exon 14 to express Alb-mNG. Subcutaneous injection of this AAV with AAV-CMV-Cas9 in postnatal day 3 mice resulted in two-photon visualization of the cerebral cortex vasculature within ten days. The expression levels of Alb-mNG were persistent for at least three months and were so robust that vasomotion and capillary blood flow could be assessed transcranially in early postnatal mice. This knock-in approach provides powerful means for micro- and macroscopic imaging of cerebral vascular dynamics in postnatal and adult mice.

Dynamic modulation of pulsatile activities of oxytocin neurons in lactating wild-type mice.

Breastfeeding, which is essential for the survival of mammalian infants, is critically mediated by pulsatile secretion of the pituitary hormone oxytocin from the central oxytocin neurons located in the paraventricular and supraoptic hypothalamic nuclei of mothers. Despite its importance, the molecular and neural circuit mechanisms of the milk ejection reflex remain poorly understood, in part because a mouse model to study lactation was only recently established. In our previous study, we successfully introduced fiber photometry-based chronic imaging of the pulsatile activities of oxytocin neurons during lactation. However, the necessity of Cre recombinase-based double knock-in mice substantially compromised the use of various Cre-dependent neuroscience toolkits. To overcome this obstacle, we developed a simple Cre-free method for monitoring oxytocin neurons by an adeno-associated virus vector driving GCaMP6s under a 2.6 kb mouse oxytocin mini-promoter. Using this method, we monitored calcium ion transients of oxytocin neurons in the paraventricular nucleus in wild-type C57BL/6N and ICR mothers without genetic crossing. By combining this method with video recordings of mothers and pups, we found that the pulsatile activities of oxytocin neurons require physical mother-pup contact for the milk ejection reflex. Notably, the frequencies of photometric signals were dynamically modulated by mother-pup reunions after isolation and during natural weaning stages. Collectively, the present study illuminates the temporal dynamics of pulsatile activities of oxytocin neurons in wild-type mice and provides a tool to characterize maternal oxytocin functions.

A blood-brain barrier-penetrating AAV2 mutant created by a brain microvasculature endothelial cell-targeted AAV2 variant.

Upon systemic administration, adeno-associated virus serotype 9 (AAV9) and the capsid variant PHP.eB show distinct tropism for the central nervous system (CNS), whereas AAV2 and the capsid variant BR1 transduce brain microvascular endothelial cells (BMVECs) with little transcytosis. Here, we show that a single amino acid substitution (from Q to N) in the BR1 capsid at position 587 (designated BR1N) confers a significantly higher blood-brain barrier (BBB) penetration capacity to BR1. Intravenously infused BR1N showed significantly higher CNS tropism than BR1 and AAV9. BR1 and BR1N likely use the same receptor for entry into BMVECs; however, the single amino acid substitution has profound consequences on tropism. This suggests that receptor binding alone does not determine the final outcome in vivo and that further improvements of capsids within predetermined receptor usage are feasible.

Excitatory subtypes of the lateral amygdala neurons are differentially involved in regulation of synaptic plasticity and excitation/inhibition balance in aversive learning in mice.

The amygdala plays a crucial role in aversive learning. In Pavlovian fear conditioning, sensory information about an emotionally neutral conditioned stimulus (CS) and an innately aversive unconditioned stimulus is associated with the lateral amygdala (LA), and the CS acquires the ability to elicit conditioned responses. Aversive learning induces synaptic plasticity in LA excitatory neurons from CS pathways, such as the medial geniculate nucleus (MGN) of the thalamus. Although LA excitatory cells have traditionally been classified based on their firing patterns, the relationship between the subtypes and functional properties remains largely unknown. In this study, we classified excitatory cells into two subtypes based on whether the after-depolarized potential (ADP) amplitude is expressed in non-ADP cells and ADP cells. Their electrophysiological properties were significantly different. We examined subtype-specific synaptic plasticity in the MGN-LA pathway following aversive learning using optogenetics and found significant experience-dependent plasticity in feed-forward inhibitory responses in fear-conditioned mice compared with control mice. Following aversive learning, the inhibition/excitation (I/E) balance in ADP cells drastically changed, whereas that in non-ADP cells tended to change in the reverse direction. These results suggest that the two LA subtypes are differentially regulated in relation to synaptic plasticity and I/E balance during aversive learning.

Development of microglia-targeting adeno-associated viral vectors as tools to study microglial behavior in vivo.

Here we describe the microglia-targeting adeno-associated viral (AAV) vectors containing a 1.7-kb putative promoter region of microglia/macrophage-specific ionized calcium-binding adaptor molecule 1 (Iba1), along with repeated miRNA target sites for microRNA (miR)-9 and miR-129-2-3p. The 1.7-kb genomic sequence upstream of the start codon in exon 1 of the Iba1 (Aif1) gene, functions as microglia preferential promoter in the striatum and cerebellum. Furthermore, ectopic transgene expression in non-microglial cells is markedly suppressed upon adding two sets of 4-repeated miRNA target sites for miR-9 and miR-129-2-3p, which are expressed exclusively in non-microglial cells and sponged AAV-derived mRNAs. Our vectors transduced ramified microglia in healthy tissues and reactive microglia in lipopolysaccharide-treated mice and a mouse model of neurodegenerative disease. Moreover, live fluorescent imaging allowed the monitoring of microglial motility and intracellular Ca2+ mobilization. Thus, microglia-targeting AAV vectors are valuable for studying microglial pathophysiology and therapies, particularly in the striatum and cerebellum.

DOPAnization of tyrosine in α-synuclein by tyrosine hydroxylase leads to the formation of oligomers.

Parkinson's disease is a progressive neurodegenerative disorder characterized by the preferential loss of tyrosine hydroxylase (TH)-expressing dopaminergic neurons in the substantia nigra. Although the abnormal accumulation and aggregation of α-synuclein have been implicated in the pathogenesis of Parkinson's disease, the underlying mechanisms remain largely elusive. Here, we found that TH converts Tyr136 in α-synuclein into dihydroxyphenylalanine (DOPA; Y136DOPA) through mass spectrometric analysis. Y136DOPA modification was clearly detected by a specific antibody in the dopaminergic neurons of α-synuclein-overexpressing mice as well as human α-synucleinopathies. Furthermore, dopanized α-synuclein tended to form oligomers rather than large fibril aggregates and significantly enhanced neurotoxicity. Our findings suggest that the dopanization of α-synuclein by TH may contribute to oligomer and/or seed formation causing neurodegeneration with the potential to shed light on the pathogenesis of Parkinson's disease.

Liver-secreted fluorescent blood plasma markers enable chronic imaging of the microcirculation.

Studying blood microcirculation is vital for gaining insights into vascular diseases. Blood flow imaging in deep tissue is currently achieved by acute administration of fluorescent dyes in the blood plasma. This is an invasive process, and the plasma fluorescence decreases within an hour of administration. Here, we report an approach for the longitudinal study of vasculature. Using a single intraperitoneal or intravenous administration of viral vectors, we express fluorescent secretory albumin-fusion proteins in the liver to chronically label the blood circulation in mice. This approach allows for longitudinal observation of circulation from 2 weeks to over 4 months after vector administration. We demonstrate the chronic assessment of vascular functions including functional hyperemia and vascular plasticity in micro- and mesoscopic scales. This genetic plasma labeling approach represents a versatile and cost-effective method for the chronic investigation of vasculature functions across the body in health and disease animal models.

Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation.

Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.

D-Cysteine Activates Chaperone-Mediated Autophagy in Cerebellar Purkinje Cells via the Generation of Hydrogen Sulfide and Nrf2 Activation.

Chaperone-mediated autophagy (CMA) is a pathway in the autophagy-lysosome protein degradation system. CMA impairment has been implicated to play a role in spinocerebellar ataxia (SCA) pathogenesis. D-cysteine is metabolized by D-amino acid oxidase (DAO), leading to hydrogen sulfide generation in the cerebellum. Although D-cysteine alleviates the disease phenotypes in SCA-model mice, it remains unknown how hydrogen sulfide derived from D-cysteine exerts this effect. In the present study, we investigated the effects of D-cysteine and hydrogen sulfide on CMA activity using a CMA activity marker that we have established. D-cysteine activated CMA in Purkinje cells (PCs) of primary cerebellar cultures where DAO was expressed, while it failed to activate CMA in DAO-deficient AD293 cells. In contrast, Na2S, a hydrogen sulfide donor, activated CMA in both PCs and AD293 cells. Nuclear factor erythroid 2-related factor 2 (Nrf2) is known to be activated by hydrogen sulfide and regulate CMA activity. An Nrf2 inhibitor, ML385, prevented CMA activation triggered by D-cysteine and Na2S. Additionally, long-term treatment with D-cysteine increased the amounts of Nrf2 and LAMP2A, a CMA-related protein, in the mouse cerebellum. These findings suggest that hydrogen sulfide derived from D-cysteine enhances CMA activity via Nrf2 activation.

Plasticity of neural connections underlying oxytocin-mediated parental behaviors of male mice.

The adult brain can flexibly adapt behaviors to specific life-stage demands. For example, while sexually naive male mice are aggressive to the conspecific young, they start to provide caregiving to infants around the time when their own young are expected. How such behavioral plasticity is implemented at the level of neural connections remains poorly understood. Here, using viral-genetic approaches, we establish hypothalamic oxytocin neurons as the key regulators of the parental caregiving behaviors of male mice. We then use rabies-virus-mediated unbiased screening to identify excitatory neural connections originating from the lateral hypothalamus to the oxytocin neurons to be drastically strengthened when male mice become fathers. These connections are functionally relevant, as their activation suppresses pup-directed aggression in virgin males. These results demonstrate the life-stage associated, long-distance, and cell-type-specific plasticity of neural connections in the hypothalamus, the brain region that is classically assumed to be hard-wired.

Protein kinase Cγ in cerebellar Purkinje cells regulates Ca2+-activated large-conductance K+ channels and motor coordination.

The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ-knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcgfl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcgfl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca2+-activated large-conductance K+ (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination.

Electrophysiological and Imaging Analysis of GFP-Tagged Protein Kinase C γ Translocation in Cerebellar Purkinje Cells.

The cerebellum contains the highest density of protein kinase C (PKC) in the central nervous system. PKCγ, the major isotype accounting for over half of the PKCs in the cerebellum, is expressed exclusively in Purkinje cells (PCs). Inactivated PKCγ, which is localized in the cytoplasm of PC dendrites and soma, begins to translocate to the cell membrane upon activation. However, the physiological conditions that induce PKCγ translocation in PC remain largely unknown. Here, we virally expressed PKCγ-GFP in PCs and examined the conditions that induced its translocation to PC dendrites by whole-cell patch clamp analysis combined with confocal GFP fluorescence imaging. A single or repetitive (150 pulses at 5 Hz for 30 s) electrical stimulation to a climbing fiber (CF), which produced a complex spike(s) in PC, failed to induce translocation of PKCγ-GFP to the dendritic shaft of PCs. Direct current injection (+ 2 nA for 3 s) to PC also did not induce the translocation, although PCs generated simple spikes continuously at high rates. In contrast, high-frequency parallel fiber (PF) stimulation (50 pulses at 50 Hz for 1 s), which triggered action potentials followed by sustained depolarization (known as mGluR1-mediated slow depolarization), caused translocation of cytoplasmic PKCγ-GFP to the membrane. Low-frequency PF stimulation (150 pulses at 5 Hz for 30 s) induced continuous simple spike firing but did not induce translocation. These results suggest that CF-triggered depolarization, which causes Ca2+ influx through voltage-gated Ca2+ channels throughout PC dendrites and somas, is insufficient to induce the translocation of PKCγ, instead requiring high-frequency PF stimulation that activates mGluR1.

A cortical cell ensemble in the posterior parietal cortex controls past experience-dependent memory updating.

When processing current sensory inputs, animals refer to related past experiences. Current information is then incorporated into the related neural network to update previously stored memories. However, the neuronal mechanism underlying the impact of memories of prior experiences on current learning is not well understood. Here, we found that a cellular ensemble in the posterior parietal cortex (PPC) that is activated during past experience mediates an interaction between past and current information to update memory through a PPC-anterior cingulate cortex circuit in mice. Moreover, optogenetic silencing of the PPC ensemble immediately after retrieval dissociated the interaction without affecting individual memories stored in the hippocampus and amygdala. Thus, a specific subpopulation of PPC cells represents past information and instructs downstream brain regions to update previous memories.