Presynaptic Calcium Channel Inhibition Underlies CB1 Cannabinoid Receptor-Mediated Suppression of GABA Release.

CB1 cannabinoid receptors (CB1) are located at axon terminals and effectively control synaptic communication and thereby circuit operation widespread in the CNS. Although it is partially uncovered how CB1 activation leads to the reduction of synaptic excitation, the mechanisms of the decrease of GABA release upon activation of these cannabinoid receptors remain elusive. To determine the mechanisms underlying the suppression of synaptic transmission by CB1 at GABAergic synapses, we recorded unitary IPSCs (uIPSCs) at cholecystokinin-expressing interneuron-pyramidal cell connections and imaged presynaptic [Ca(2+)] transients in mouse hippocampal slices. Our results reveal a power function with an exponent of 2.2 between the amplitude of uIPSCs and intrabouton [Ca(2+)]. Altering CB1 function by either increasing endocannabinoid production or removing its tonic activity allowed us to demonstrate that CB1 controls GABA release by inhibiting Ca(2+) entry into presynaptic axon terminals via N-type (Cav2.2) Ca(2+) channels. These results provide evidence for modulation of intrabouton Ca(2+) influx into GABAergic axon terminals by CB1, leading to the effective suppression of synaptic inhibition

Strategically Positioned Inhibitory Synapses of Axo-axonic Cells Potently Control Principal Neuron Spiking in the Basolateral Amygdala.

Axo-axonic cells (AACs) in cortical regions selectively innervate the axon initial segments (AISs) of principal cells (PCs), where the action potentials are generated. These GABAergic interneurons can alter the activity of PCs, but how the efficacy of spike control correlates with the number of output synapses remains unclear. Moreover, the relationship between the spatial distribution of GABAergic synapses and the action potential initiation site along the AISs is not well defined. Using paired recordings obtained in the mouse basolateral amygdala, we found that AACs powerfully inhibited or delayed the timing of PC spiking by 30 ms, if AAC output preceded PC spiking with no more than 80 ms. By correlating the number of synapses and the probability of spiking, we revealed that larger numbers of presynaptic AAC boutons giving rise to larger postsynaptic responses provided more effective inhibition of PC spiking. At least 10-12 AAC synapses, which could originate from 2-3 AACs on average, were necessary to veto the PC firing under our recording conditions. Furthermore, we determined that the threshold for the action potential generation along PC axons is the lowest between 20 and 40 mum from soma, which axonal segment received the highest density of GABAergic inputs. Single AACs preferentially innervated this narrow portion of the AIS where action potentials were generated with the highest likelihood, regardless of the number of synapses forming a given connection. Our results uncovered a fine organization of AAC innervation maximizing their inhibitory efficacy by strategically positioning synapses along the AISs

Different output properties of perisomatic region-targeting interneurons in the basal amygdala

Perisomatic region of principal neurons in the cortical regions is innervated by three types of GABAergic interneurons, including parvalbumi n - containing basket cells (PVBCs) and axo - axonic cells (AACs), as well as cholecystokinin and type 1 cannabinoid receptor - expressing basket cells (CCK/CB1BCs). These perisomatic inhibitory cell types can also be found in the basal nucleus of the amygdala, however, their output properties are largely unknown. Here, we performed whole - cell recordings in morphologically identified interneurons in slices prepared from transgenic mice, in which the GABAergic cells could be selectively targeted. By investigating the passive and active membrane properties of interneurons located within the bas al amygdala , w e observed that the three interneuron types had distinct single - cell properties . For instance, the input resistance, spike rate, accommodation in discharge rate , or after - hyperpolarization width at the half maximal amplitude separated the three interneuron types . Furthermore , we performed paired recordings from interneurons and principal neurons to uncover the basic features of unitary inhibitory postsynaptic curr ents (uIPSCs). We found that, although there was no difference in the magnitude of responses measured in the principal neurons , the uIPSCs originated from the distinct interneuron types differed in the rise time, failure rate, latency and short - term dynami cs. Moreover, the asynchronous transmitter release induced by a train of action potentials was typical for the output synapses of CCK/CB1BCs. Our results suggest that , although the three perisomatic inhibitory cell types give rise to uIPSCs with similar ma gnitude, their distinct spiking characteristics may help to accomplish specific function in amygdala operation

High Enzyme Activity of a Binuclear Nickel Complex Formed with the Binding Loops of the NiSOD Enzyme

Detailed equilibrium, spectroscopic and superoxide dismutase (SOD) activity studies are reported on a nickel complex formed with a new metallopeptide bearing two nickel binding loops of NiSOD. The metallopeptide exhibits unique nickel binding ability and the binuclear complex is a major species with 2x(NH2,N-amide,S-,S-) donor set even in an equimolar solution of the metal ion and the ligand. Nickel(III) species were generated by oxidizing the Ni-II complexes with KO2 and the coordination modes were identified by EPR spectroscopy. The binuclear complex formed with the binding motifs exhibits superior SOD activity, in this respect it is an excellent model of the native NiSOD enzyme. A detailed kinetic model is postulated that incorporates spontaneous decomposition of the superoxide ion, the dismutation cycle and fast redox degradation of the binuclear complex. The latter process leads to the elimination of the SOD activity. A unique feature of this system is that the Ni-III form of the catalyst rapidly accumulates in the dismutation cycle and simultaneously the Ni-II form becomes a minor species

CRK5 Protein Kinase Contributes to the Progression of Embryogenesis of Arabidopsis thaliana

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins

The AtCRK5 Protein Kinase Is Required to Maintain the ROS NO Balance Affecting the PIN2-Mediated Root Gravitropic Response in Arabidopsis

The Arabidopsis AtCRK5 protein kinase is involved in the establishment of the proper auxin gradient in many developmental processes. Among others, the Atcrk5-1 mutant was reported to exhibit a delayed gravitropic response via compromised PIN2-mediated auxin transport at the root tip. Here, we report that this phenotype correlates with lower superoxide anion (O-2(center dot-)) and hydrogen peroxide (H2O2) levels but a higher nitric oxide (NO) content in the mutant root tips in comparison to the wild type (AtCol-0). The oxidative stress inducer paraquat (PQ) triggering formation of O-2(center dot-) (and consequently, H2O2) was able to rescue the gravitropic response of Atcrk5-1 roots. The direct application of H2O2 had the same effect. Under gravistimulation, correct auxin distribution was restored (at least partially) by PQ or H2O2 treatment in the mutant root tips. In agreement, the redistribution of the PIN2 auxin efflux carrier was similar in the gravistimulated PQ-treated mutant and untreated wild type roots. It was also found that PQ-treatment decreased the endogenous NO level at the root tip to normal levels. Furthermore, the mutant phenotype could be reverted by direct manipulation of the endogenous NO level using an NO scavenger (cPTIO). The potential involvement of AtCRK5 protein kinase in the control of auxin-ROS-NO-PIN2-auxin regulatory loop is discussed

Excitation of Diverse Classes of Cholecystokinin Interneurons in the Basal Amygdala Facilitates Fear Extinction

There is growing evidence that interneurons (INs) orchestrate neural activity and plasticity in corticoamygdala circuits to regulate fear behaviors. However, defining the precise role of cholecystokinin-expressing INs (CCK INs) remains elusive due to the technical challenge of parsing this population from CCK-expressing principal neurons (CCK PNs). Here, we used an intersectional genetic strategy in CCK-Cre;Dlx5/6-Flpe double-transgenic mice to study the anatomical, molecular and electrophysiological properties of CCK INs in the basal amygdala (BA) and optogenetically manipulate these cells during fear extinction. Electrophysiological recordings confirmed that this strategy targeted GABAergic cells and that a significant proportion expressed functional cannabinoid CB1 receptors; a defining characteristic of CCK-expressing basket cells. However, immunostaining showed that subsets of the genetically-targeted cells expressed either neuropeptide Y (NPY; 29%) or parvalbumin (PV; 17%), but not somatostatin (SOM) or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-α. Further morphological and electrophysiological analyses showed that four IN types could be identified among the EYFP-expressing cells: CCK/cannabinoid receptor type 1 (CB1R)-expressing basket cells, neurogliaform cells, PV+ basket cells, and PV+ axo-axonic cells. At the behavioral level, in vivo optogenetic photostimulation of the targeted population during extinction acquisition led to reduced freezing on a light-free extinction retrieval test, indicating extinction memory facilitation; whereas photosilencing was without effect. Conversely, non-selective (i.e., inclusive of INs and PNs) photostimulation or photosilencing of CCK-targeted cells, using CCK-Cre single-transgenic mice, impaired extinction. These data reveal an unexpectedly high degree of phenotypic complexity in a unique population of extinction-modulating BA INs

The mitogen-activated protein kinase 4-phosphorylated heat shock factor A4A regulates responses to combined salt and heat stresses

Heat shock factors regulate responses to high temperatures, salinity, water deprivation or heavy metals. Their function in stress combinations is however not known. The Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature and combination of these conditions. Fast translocation of HSFA4A-YFP protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by MAP kinases MPK3/6 but also by MPK4 and Ser309 is the dominant MAPK phosphorylation site. In vivo data suggest that HSFA4A can be substrate of other kinases as well. Changing Ser309 to Asp or Ala has altered intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes

Mechanistic Explanation for Differences Between Catalytic Activities of Dissolved and Aerogel Immobilized Cu(II) Cyclen

The copper(II) complex of 1,4,7,10-tetraazacyclododecane [Cu(II)-cyclen] was covalently immobilized in mesoporous silica aerogel. This immobilization significantly alters the catalytic activity of Cu(II)-cyclen when referenced to the dissolved complex in the oxidation of phenol by H2O2 in aqueous solution. In order to understand this phenomenon, the functionalized aerogel was characterized by scanning electron microscopy (SEM), N2 porosimetry, small angle neutron scattering (SANS), infrared spectroscopy (IR) and electron paramagnetic resonance spectroscopy (EPR). Aerogel morphology is typical of mesoporous silica aerogels, and the coordination mode of Cu(II) in the immobilized complex is well-related but not identical to solution phase Cu(II)-cyclen. The mechanisms of the catalytic reactions involving dissolved and immobilized Cu(II)-cyclen were explored by fine kinetic experiments using capillary electrophoresis (CE) and on-line UV–vis spectrophotometry. Hydroquinone, pyrocatechol and the related benzoquinones were identified as the main intermediates in both reaction systems. A detailed kinetic model is postulated based on global data fitting, which clearly highlights the mechanistic differences in the two systems. Interestingly, the activation of the catalyst by H2O2 is more effective in the case of the aerogel, but the total conversion of phenol is slower due to hindered mass transport compared to using dissolved Cu(II)-cyclen