Subtypes of GABAergic Neurons Project Axons in the Neocortex

γ-aminobutyric acid (GABA)ergic neurons in the neocortex have been regarded as interneurons and speculated to modulate the activity of neurons locally. Recently, however, several experiments revealed that neuronal nitric oxide synthase (nNOS)-positive GABAergic neurons project cortico-cortically with long axons. In this study, we illustrate Golgi-like images of the nNOS-positive GABAergic neurons using a nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) reaction and follow the emanating axon branches in cat brain sections. These axon branches projected cortico-cortically with other non-labeled arcuate fibers, contra-laterally via the corpus callosum and anterior commissure. The labeled fibers were not limited to the neocortex but found also in the fimbria of the hippocampus. In order to have additional information on these GABAergic neuron projections, we investigated green fluorescent protein (GFP)-labeled GABAergic neurons in GAD67-Cre knock-in/GFP Cre-reporter mice. GFP-labeled axons emanate densely, especially in the fimbria, a small number in the anterior commissure, and very sparsely in the corpus callosum. These two different approaches confirm that not only nNOS-positive GABAergic neurons but also other subtypes of GABAergic neurons project long axons in the cerebral cortex and are in a position to be involved in information processing

cDNA cloning and nucleotide sequence of rat muscle-specific enolase (ββ enolase)

AbstractThe nucleotide sequence of rat muscle-specific enolase cDNA was determined by sequencing three cDNA clones encoding this enolase isozyme. The nearly full-length cDNA consists of 13-bp 5′- and 84-bp 3′-noncoding regions and a poly(A) tail in addition to a 1302-bp coding region encoding a polypeptide composed of 434 amino acid residues. The deduced primary structure of this enolase isozyme is about 80% similar to those determined previously for rat neuron-specific and non-neuronal enolase isozymes. Southern blot analysis suggested strongly the existence of a single copy of the muscle-specific enolase gene per haploid genome. The mRNA for this enolase isozyme was detected in rat skeletal muscle on day 1 after birth and its level increased rapidly during 10–30 days after birth without any change in its size (1500 bases)

Neuronal protease-activated receptor 1 drives synaptic retrograde signaling mediated by the endocannabinoid 2-arachidonoylglycerol

金沢大学医薬保健研究域保健学系Protease-activated receptor 1 (PAR1) is a member of the G-protein coupled receptors that are proteolytically activated by serine proteases. Recent studies suggest a definite contribution of PAR1 to brain functions, including learning and memory. However, cellular mechanisms by which PAR1 activation influences neuronal activity are not well understood. Here we show that PAR1 activation drives retrograde endocannabinoid signaling and thereby regulates synaptic transmission. In cultured hippocampal neurons from rat, PAR1 activation by thrombin or PAR1-specific peptide agonists transiently suppressed inhibitory transmission at cannabinoid-sensitive, but not cannabinoid-insensitive, synapses. The PAR1-induced suppression of synaptic transmission was accompanied by an increase in paired-pulse ratio, and was blocked by a cannabinoid CB 1 receptor antagonist. The PAR1-induced suppression was blocked by pharmacological inhibition of postsynaptic diacylglycerol lipase (DGL), a key enzyme for biosynthesis of the major endocannabinoid 2-arachidonoylglycerol (2-AG), and was absent in knock-out mice lacking the α isoform of DGL. The PAR1-induced IPSC suppression remained intact under the blockade of metabotropic glutamate receptors and was largely resistant to the treatment that blocked Ca2+ elevation in glial cells following PAR1 activation, which excludes the major contribution of glial PAR1 in IPSC suppression. We conclude that activation of neuronal PAR1 triggers retrograde signaling mediated by 2-AG, which activates presynaptic CB1 receptors and suppresses transmitter release at hippocampal inhibitory synapses. Copyright © 2011 the authors

Heterogeneous presynaptic distribution of monoacylglycerol lipase, a multipotent regulator of nociceptive circuits in the mouse spinal cord.

Monoacylglycerol lipase (MGL) is a multifunctional serine hydrolase, which terminates anti-nociceptive endocannabinoid signaling and promotes pro-nociceptive prostaglandin signaling. Accordingly, both acute nociception and its sensitization in chronic pain models are prevented by systemic or focal spinal inhibition of MGL activity. Despite its analgesic potential, the neurobiological substrates of beneficial MGL blockade have remained unexplored. Therefore, we examined the regional, cellular and subcellular distribution of MGL in spinal circuits involved in nociceptive processing. All immunohistochemical findings obtained with light, confocal or electron microscopy were validated in MGL-knockout mice. Immunoperoxidase staining revealed a highly concentrated accumulation of MGL in the dorsal horn, especially in superficial layers. Further electron microscopic analysis uncovered that the majority of MGL-immunolabeling is found in axon terminals forming either asymmetric glutamatergic or symmetric gamma-aminobutyric acid/glycinergic synapses in laminae I/IIo. In line with this presynaptic localization, analysis of double-immunofluorescence staining by confocal microscopy showed that MGL colocalizes with neurochemical markers of peptidergic and non-peptidergic nociceptive terminals, and also with markers of local excitatory or inhibitory interneurons. Interestingly, the ratio of MGL-immunolabeling was highest in calcitonin gene-related peptide-positive peptidergic primary afferents, and the staining intensity of nociceptive terminals was significantly reduced in MGL-knockout mice. These observations highlight the spinal nociceptor synapse as a potential anatomical site for the analgesic effects of MGL blockade. Moreover, the presence of MGL in additional terminal types raises the possibility that MGL may play distinct regulatory roles in synaptic endocannabinoid or prostaglandin signaling according to its different cellular locations in the dorsal horn pain circuitry

Ablation of Glutamate Receptor GluR delta 2 in Adult Purkinje Cells Causes Multiple Innervation of Climbing Fibers by Inducing Aberrant Invasion to Parallel Fiber Innervation Territory

Glutamate receptor GluRδ2 is exclusively expressed in Purkinje cells (PCs) from early development and plays key roles in parallel fiber (PF) synapse formation, elimination of surplus climbing fibers (CFs), long-term depression, motor coordination, and motor learning. To address its role in adulthood, we previously developed a mouse model of drug-induced GluRδ2 ablation in adult PCs (Takeuchi et al., 2005). In that study, we demonstrated an essential role to maintain the connectivity of PF-PC synapses, based on the observation that both mismatching of presynaptic and postsynaptic specializations and disconnection of PF-PC synapses are progressively increased after GluRδ2 ablation. Here, we pursued its role for CF wiring in adult cerebellum. In parallel with the disconnection of PF-PC synapses, ascending CF branches exhibited distal extension to innervate distal dendrites of the target and neighboring PCs. Furthermore, transverse CF branches, a short motile collateral rarely forming synapses in wild-type animals, displayed aberrant mediolateral extension to innervate distal dendrites of neighboring and remote PCs. Consequently, many PCs were wired by single main CF and other surplus CFs innervating a small part of distal dendrites. Electrophysiological recording further revealed that surplus CF-EPSCs characterized with slow rise time and small amplitude emerged after GluRδ2 ablation, and increased progressively both in number and amplitude. Therefore, GluRδ2 is essential for maintaining CF monoinnervation in adult cerebellum by suppressing aberrant invasion of CF branches to the territory of PF innervation. Thus, GluRδ2 fuels heterosynaptic competition and gives PFs the competitive advantages over CFs throughout the animal's life

Dissociating orexin-dependent and -independent functions of orexin neurons using novel Orexin-Flp knock-in mice

Uninterrupted arousal is important for survival during threatening situations. Activation of orexin/hypocretin neurons is implicated in sustained arousal. However, orexin neurons produce and release orexin as well as several co-transmitters including dynorphin and glutamate. To disambiguate orexin-dependent and -independent physiological functions of orexin neurons, we generated a novel Orexin-flippase (Flp) knock-in mouse line. Crossing with Flp-reporter or Cre-expressing mice showed gene expression exclusively in orexin neurons. Histological studies confirmed that orexin was knock-out in homozygous mice. Orexin neurons without orexin showed altered electrophysiological properties, as well as received decreased glutamatergic inputs. Selective chemogenetic activation revealed that both orexin and co-transmitters functioned to increase wakefulness, however, orexin was indispensable to promote sustained arousal. Surprisingly, such activation increased the total time spent in cataplexy. Taken together, orexin is essential to maintain basic membrane properties and input-output computation of orexin neurons, as well as to exert awake-sustaining aptitude of orexin neurons

Drebrin Isoforms Critically Regulate NMDAR- and mGluR-Dependent LTD Induction

Drebrin is an actin-binding protein that is preferentially expressed in the brain. It is highly localized in dendritic spines and regulates spine shapes. The embryonic-type (drebrin E) is expressed in the embryonic and early postnatal brain and is replaced by the adult-type (drebrin A) during development. In parallel, NMDA receptor (NMDAR)-dependent long-term depression (LTD) of synaptic transmission, induced by low-frequency stimulation (LFS), is dominant in the immature brain and decreases during development. Here, we report that drebrin regulates NMDAR-dependent and group 1 metabotropic glutamate receptor (mGluR)-dependent LTD induction in the hippocampus. While LFS induced NMDAR-dependent LTD in the developing hippocampus in wild-type (WT) mice, it did not induce LTD in developing drebrin E and A double knockout (DXKO) mice, indicating that drebrin is required for NMDAR-dependent LTD. On the other hand, LFS induced robust LTD dependent on mGluR5, one of group 1 mGluRs, in both developing and adult brains of drebrin A knockout (DAKO) mice, in which drebrin E is expressed throughout development and adulthood. Agonist-induced mGluR-dependent LTD was normal in WT and DXKO mice; however, it was enhanced in DAKO mice. Also, mGluR1, another group 1 mGluR, was involved in agonist-induced mGluR-dependent LTD in DAKO mice. These data suggest that abnormal drebrin E expression in adults promotes group 1 mGluR-dependent LTD induction. Therefore, while drebrin expression is critical for NMDAR-dependent LTD induction, developmental conversion from drebrin E to drebrin A prevents robust group 1 mGluR-dependent LTD

The Endocannabinoid 2-Arachidonoylglycerol Produced by Diacylglycerol Lipase α Mediates Retrograde Suppression of Synaptic Transmission

SummaryEndocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase α (DGLα) and β (DGLβ). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGLα knockout mice, whereas the retrograde suppression was intact in DGLβ knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGLα knockout brains, whereas the 2-AG content was normal in DGLβ knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGLα, but not by DGLβ, mediates retrograde suppression at central synapses

Differential CRH expression level determines efficiency of Cre- and Flp-dependent recombination

Corticotropin-releasing hormone expressing (CRH+) neurons are distributed throughout the brain and play a crucial role in shaping the stress responses. Mouse models expressing site-specific recombinases (SSRs) or reporter genes are important tools providing genetic access to defined cell types and have been widely used to address CRH+ neurons and connected brain circuits. Here, we investigated a recently generated CRH-FlpO driver line expanding the CRH system-related tool box. We directly compared it to a previously established and widely used CRH-Cre line with respect to the FlpO expression pattern and recombination efficiency. In the brain, FlpO mRNA distribution fully recapitulates the expression pattern of endogenous Crh. Combining both Crh locus driven SSRs driver lines with appropriate reporters revealed an overall coherence of respective spatial patterns of reporter gene activation validating CRH-FlpO mice as a valuable tool complementing existing CRH-Cre and reporter lines. However, a substantially lower number of reporter-expressing neurons was discerned in CRH-FlpO mice. Using an additional CRH reporter mouse line (CRH-Venus) and a mouse line allowing for conversion of Cre into FlpO activity (CAG-LSL-FlpO) in combination with intersectional and subtractive mouse genetic approaches, we were able to demonstrate that the reduced number of tdTomato reporter expressing CRH+ neurons can be ascribed to the lower recombination efficiency of FlpO compared to Cre recombinase. This discrepancy particularly manifests under conditions of low CRH expression and can be overcome by utilizing homozygous CRH-FlpO mice. These findings have direct experimental implications which have to be carefully considered when targeting CRH+ neurons using CRH-FlpO mice. However, the lower FlpO-dependent recombination efficiency also entails advantages as it provides a broader dynamic range of expression allowing for the visualization of cells showing stress-induced CRH expression which is not detectable in highly sensitive CRH-Cre mice as Cre-mediated recombination has largely been completed in all cells generally possessing the capacity to express CRH. These findings underscore the importance of a comprehensive evaluation of novel SSR driver lines prior to their application