Flash X-Ray (Fxr) Linear Induction Accelerator (Lia) Optimization Sensor Delay Correction

The radiographic goal of the FXR Optimization Project is to generate an x-ray pulse with peak energy of 19 MeV, spot-size of 1.5 mm, a dose of 500 rad, and duration of 60 ns. The electrical objectives are to generate a 3 kA electron-beam and refine our 16 MV accelerator so that the voltage does not vary more than 1%-rms. In a multi-cell linear induction accelerator, like FXR, the timing of the acceleration pulses relative to the beam is critical. The pulses must be timed optimally so that a cell is at full voltage before the beam arrives and does not drop until the beam passes. In order to stay within the energy-variation budget, the synchronization between the cells and beam arrival must be controlled to a couple of nanoseconds. Therefore, temporal measurements must be accurate to a fraction of a nanosecond. FXR Optimization Project developed a one-giga-sample per second (gs/s) data acquisition system to record beam sensor data. Signal processing algorithms were written to determine cell timing with an uncertainty of a fraction of a nanosecond. However, the uncertainty in the sensor delay was still a few nanoseconds. This error had to be reduced if we are to improve the quality of the electron beam. Two types of sensors are used to align the cell voltage pulse against the beam current. The beam current is measured with resistive-wall sensors. The cell voltages are read with capacitive voltage monitors. Sensor delays can be traced to two mechanisms: (1) the sensors are not co-located at the beam and cell interaction points, and (2) the sensors have different length jumper cables and other components that connect them to the standard-length coaxial cables of the data acquisition system. Using the physical locations and dimensions of the sensor components, and the dielectric constant of the materials, delay times were computed. Relative to the cell voltage, the beam current was theoretically reporting late by 7.7 ns. Two experiments were performed to verify and refine the sensor delay correction. In the first experiment, the beam was allowed to drift through a cell that was not pulsed. The beam induces a potential into the cell that is read by the voltage monitor. Analysis of the data indicated that the beam sensor signal was likely 7.1 ns late. In the second experiment, the beam current is calculated from the injector diode voltage that is the sum of the cell voltages. A 7 ns correction produced a very good match between the signals from the two types of sensors. For simplicity, we selected a correction factor that advanced the current signals by 7 ns. This should reduce the uncertainty in the temporal measurements to less than 1 ns

Cholinergic Interneurons Mediate Fast VGluT3-Dependent Glutamatergic Transmission in the Striatum

The neurotransmitter glutamate is released by excitatory projection neurons throughout the brain. However, non-glutamatergic cells, including cholinergic and monoaminergic neurons, express markers that suggest that they are also capable of vesicular glutamate release. Striatal cholinergic interneurons (CINs) express the Type-3 vesicular glutamate transporter (VGluT3), although whether they form functional glutamatergic synapses is unclear. To examine this possibility, we utilized mice expressing Cre-recombinase under control of the endogenous choline acetyltransferase locus and conditionally expressed light-activated Channelrhodopsin2 in CINs. Optical stimulation evoked action potentials in CINs and produced postsynaptic responses in medium spiny neurons that were blocked by glutamate receptor antagonists. CIN-mediated glutamatergic responses exhibited a large contribution of NMDA-type glutamate receptors, distinguishing them from corticostriatal inputs. CIN-mediated glutamatergic responses were insensitive to antagonists of acetylcholine receptors and were not seen in mice lacking VGluT3. Our results indicate that CINs are capable of mediating fast glutamatergic transmission, suggesting a new role for these cells in regulating striatal activity

Cerebral activations related to ballistic, stepwise interrupted and gradually modulated movements in parkinson patients

Patients with Parkinson's disease (PD) experience impaired initiation and inhibition of movements such as difficulty to start/stop walking. At single-joint level this is accompanied by reduced inhibition of antagonist muscle activity. While normal basal ganglia (BG) contributions to motor control include selecting appropriate muscles by inhibiting others, it is unclear how PD-related changes in BG function cause impaired movement initiation and inhibition at single-joint level. To further elucidate these changes we studied 4 right-hand movement tasks with fMRI, by dissociating activations related to abrupt movement initiation, inhibition and gradual movement modulation. Initiation and inhibition were inferred from ballistic and stepwise interrupted movement, respectively, while smooth wrist circumduction enabled the assessment of gradually modulated movement. Task-related activations were compared between PD patients (N = 12) and healthy subjects (N = 18). In healthy subjects, movement initiation was characterized by antero-ventral striatum, substantia nigra (SN) and premotor activations while inhibition was dominated by subthalamic nucleus (STN) and pallidal activations, in line with the known role of these areas in simple movement. Gradual movement mainly involved antero-dorsal putamen and pallidum. Compared to healthy subjects, patients showed reduced striatal/SN and increased pallidal activation for initiation, whereas for inhibition STN activation was reduced and striatal-thalamo-cortical activation increased. For gradual movement patients showed reduced pallidal and increased thalamo-cortical activation. We conclude that PD-related changes during movement initiation fit the (rather static) model of alterations in direct and indirect BG pathways. Reduced STN activation and regional cortical increased activation in PD during inhibition and gradual movement modulation are better explained by a dynamic model that also takes into account enhanced responsiveness to external stimuli in this disease and the effects of hyper-fluctuating cortical inputs to the striatum and STN in particular

Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically-modified animal models, together with biochemical and pharmacological approaches, we provide a high resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington’s disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases

Developmental regulation of CB1-mediated spike-time dependent depression at immature mossy fiber-CA3 synapses

Early in postnatal life, mossy fibres (MF), the axons of granule cells in the dentate gyrus, release GABA which is depolarizing and excitatory. Synaptic currents undergo spike-time dependent long-term depression (STD-LTD) regardless of the temporal order of stimulation (pre versus post and viceversa). Here we show that at P3 but not at P21, STD-LTD, induced by negative pairing, is mediated by endocannabinoids mobilized from the postsynaptic cell during spiking-induced membrane depolarization. By diffusing backward, endocannabinoids activate cannabinoid type-1 (CB1) receptors probably expressed on MF. Thus, STD-LTD was prevented by CB1 receptor antagonists and was absent in CB1-KO mice. Consistent with these data, in situ hybridization experiments revealed detectable level of CB1 mRNA in the granule cell layer at P3 but not at P21. These results indicate that CB1 receptors are transiently expressed on immature MF terminals where they counteract the enhanced neuronal excitability induced by the excitatory action of GABA

Endocannabinoids Generated by Ca2+ or by Metabotropic Glutamate Receptors Appear to Arise from Different Pools of Diacylglycerol Lipase

The identity and subcellular sources of endocannabinoids (eCBs) will shape their ability to affect synaptic transmission and, ultimately, behavior. Recent discoveries support the conclusion that 2-arachidonoyl glycerol, 2-AG, is the major signaling eCB, however, some important issues remain open. 2-AG can be synthesized by a mechanism that is strictly Ca2+-dependent, and another that is initiated by G-protein coupled receptors (GPCRs) and facilitated by Ca2+. An important question is whether or not the 2-AG in these cases is synthesized by the same pool of diacylglycerol lipase alpha (DAGLα). Using whole-cell voltage-clamp techniques in CA1 pyramidal cells in acute in vitro rat hippocampal slices, we investigated two mechanistically distinct eCB-mediated responses to address this issue. We now report that pharmacological inhibitors of DGLα have quantitatively different effects on eCB-mediated responses triggered by different stimuli, suggesting that functional, and perhaps physical, distinctions among pools of DAGLα exist

Striatal neuropeptides enhance selection and rejection of sequential actions

The striatum is the primary input nucleus for the basal ganglia, and receives glutamatergic afferents from the cortex. Under the hypothesis that basal ganglia perform action selection, these cortical afferents encode potential “action requests.” Previous studies have suggested the striatum may utilize a mutually inhibitory network of medium spiny neurons (MSNs) to filter these requests so that only those of high salience are selected. However, the mechanisms enabling the striatum to perform clean, rapid switching between distinct actions that form part of a learned action sequence are still poorly understood. Substance P (SP) and enkephalin are neuropeptides co-released with GABA in MSNs preferentially expressing D1 or D2 dopamine receptors respectively. SP has a facilitatory effect on subsequent glutamatergic inputs to target MSNs, while enkephalin has an inhibitory effect. Blocking the action of SP in the striatum is also known to affect behavioral transitions. We constructed phenomenological models of the effects of SP and enkephalin, and integrated these into a hybrid model of basal ganglia comprising a spiking striatal microcircuit and rate–coded populations representing other major structures. We demonstrated that diffuse neuropeptide connectivity enhanced the selection of unordered action requests, and that for true action sequences, where action semantics define a fixed structure, a patterning of the SP connectivity reflecting this ordering enhanced selection of actions presented in the correct sequential order and suppressed incorrect ordering. We also showed that selective pruning of SP connections allowed context–sensitive inhibition of specific undesirable requests that otherwise interfered with selection of an action group. Our model suggests that the interaction of SP and enkephalin enhances the contrast between selection and rejection of action requests, and that patterned SP connectivity in the striatum allows the “chunking” of actions and improves selection of sequences. Efficient execution of action sequences may therefore result from a combination of ordered cortical inputs and patterned neuropeptide connectivity within striatum

INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells

T cell antigen receptor-proximal signaling components, Rho-family GTPases, and formin proteins DIA1 and FMNL1 have been implicated in centrosome reorientation to the immunological synapse of T lymphocytes. However, the role of these molecules in the reorientation process is not yet defined. Here we find that a subset of microtubules became rapidly stabilized and that their α-tubulin subunit posttranslationally detyrosinated after engagement of the T cell receptor. Formation of stabilized, detyrosinated microtubules required the formin INF2, which was also found to be essential for centrosome reorientation, but it occurred independently of T cell receptor-induced massive tyrosine phosphorylation. The FH2 domain, which was mapped as the INF2 region involved in centrosome repositioning, was able to mediate the formation of stable, detyrosinated microtubules and to restore centrosome translocation in DIA1-, FMNL1-, Rac1-, and Cdc42-deficient cells. Further experiments indicated that microtubule stabilization was required for centrosome polarization. Our work identifies INF2 and stable, detyrosinated microtubules as central players in centrosome reorientation in T cellsThis work was supported by grants BFU2009-07886 and CONSOLIDER COAT CSD2009-00016 to M.A. Alonso, and BFU2011-22859 to I. Correas (all of them from the Ministerio de Economía y Competitividad, Spain), and grant S2010/BMD-2305 from the Comunidad de Madrid to I. Correa

Bmcc1s, a Novel Brain-Isoform of Bmcc1, Affects Cell Morphology by Regulating MAP6/STOP Functions

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology