Presynaptic mGluRs Control the Duration of Endocannabinoid-Mediated DSI.

GABA synapses in the brain undergo depolarization-induced suppression of inhibition (DSI) that requires activation of presynaptic cannabinoid type 1 receptors (CB1Rs). The brevity of DSI, lasting ∼1 min in most brain regions, has been ascribed to the transient production of 2-arachidonoylglycerol (2-AG). Here, we propose that the duration of DSI is controlled by heterologous interactions between presynaptic mGluRs and CB1Rs. By examining GABA synapses on parvocellular corticotropin-releasing hormone-expressing neurons in the paraventricular nucleus of the hypothalamus (PVN) of male and female mice, we show that DSI decays quickly in experimental conditions in which both GABA and glutamate are released from adjacent nerve terminals. Pharmacological inhibition of group I mGluRs prolongs DSI, whereas prior activation of mGluRs inhibits DSI, collectively suggesting that group I mGluRs quench presynaptic CB1R signaling. When photostimulation of genetically identified terminals is used to release only GABA, CB1R-dependent DSI persists for many minutes. Under the same conditions, activation of group I mGluRs reestablishes classical, transient DSI. The long-lasting DSI observed when GABA synapses are independently recruited functionally uncouples inhibitory input to PVN neurons. These observations suggest that heterologous interactions between mGluRs and CB1Rs control the temporal window of DSI at GABA synapses, providing evidence for a powerful new way to affect functional circuit connectivity in the brain.SIGNIFICANCE STATEMENT Postsynaptic depolarization liberates endocannabinoids, resulting in a rapid and transient decrease in release probability at GABA synapses. We discovered that mGluRs control the duration of depolarization-induced suppression of inhibition (DSI), most likely through heterologous desensitization of cannabinoid type 1 receptors by presynaptic mGluR5 By shortening the duration of DSI, mGluRs control the temporal window for retrograde signaling at GABA synapses. Physiological or pathological changes that affect glutamate spillover may profoundly affect network excitability by shifting the duration of cannabinoid inhibition at GABA synapses.

[Quick and simple synthesis of (11)C-(+)-alpha-dihydrotetrabenazine to be used as a PET radioligand of vesicular monoamine transporters]. / Síntesis rápida y simplificada de C-(+)-alpha-dihidrotetrabenazina para su utilización como radioligando PET de los transportadores vesiculares de monoaminas.

UNLABELLED: Dihydrotetrabenazine (2-hydroxy-3-isobutyl-9,10-dimethoxy-1,3,4,6,7-hexahydro-11bH-benzo[a]-quinolizine, DTBZ) has become the ideal radioligand for the presynaptic vesicular monoamine transporter VMAT2 based on its high binding affinity and optimal lipophilicity. OBJECTIVE: To develop an automatic procedure for labelling DTBZ with carbon-11, which has been shown to be a highly effective marker for in vivo studies of neuronal losses in animal models with Parkinson's disease using positron emission tomography (PET). MATERIALS AND METHODS: We have developed a new fully automated synthesis procedure to obtain 11C-(+)DTBZ quickly and simply through labelling the precursor -(+)desmethyldihy-drotetrabenazine- at room temperature in the presence of dimethyl sulfoxide (DMSO) and potassium hydroxide (KOH), using 11CH3I as primary precursor. The final purification was carried out by solid phase extraction using commercially available cartridges and the residual solvents (DMSO and ethyl ether) were eliminated by evaporation. RESULTS: The whole procedure was automated, and after 54 syntheses, an average production of 1.94 GBq of sterile, pyrogen-free 11C-(+)DTBZ with a radiochemical purity > 99 % was obtained with 5 minutes irradiation and 6 minutes of synthesis after 11CH3I production. 11C-(+)DTBZ binding to presynaptic dopamine nerve terminals has been demonstrated by MicroPET studies in Wistar rats and M. Fascicularis monkeys. CONCLUSIONS: This new synthesis procedure is quick and simple, due to optimised techniques, which have allowed elimination of residual solvents based on their polarity for the final purification. It is also applicable to other automatic syntheses for obtaining compounds labelled by methylation reactions.

Presynaptic calcium channels: structure, regulators, and blockers.

The central and peripheral nervous systems express multiple types of ligand and voltage-gated calcium channels (VGCCs), each with specific physiological roles and pharmacological and electrophysiological properties. The members of the Ca(v)2 calcium channel family are located predominantly at presynaptic nerve terminals, where they are responsible for controlling evoked neurotransmitter release. The activity of these channels is subject to modulation by a number of different means, including alternate splicing, ancillary subunit associations, peptide and small organic blockers, G-protein-coupled receptors (GPCRs), protein kinases, synaptic proteins, and calcium-binding proteins. These multiple and complex modes of calcium channel regulation allow neurons to maintain the specific, physiological window of cytoplasmic calcium concentrations which is required for optimal neurotransmission and proper synaptic function. Moreover, these varying means of channel regulation provide insight into potential therapeutic targets for the treatment of pathological conditions that arise from disturbances in calcium channel signaling. Indeed, considerable efforts are presently underway to identify and develop specific presynaptic calcium channel blockers that can be used as analgesics.

Alpha2-adrenoceptor subtypes--unexpected functions for receptors and ligands derived from gene-targeted mouse models.

Alpha2-adrenoceptors belong to the group of nine adrenoceptors which mediate the biological actions of the endogenous catecholamines adrenaline and noradrenaline. Studies with gene-targeted mice carrying deletions in the genes encoding alpha2A-, alpha2B- or alpha2C-adrenoceptors have provided new insight into adrenergic receptor biology: (1) In principle, all three alpha2-receptor subtypes may operate as presynaptic inhibitory feedback receptors to control the release of noradrenaline and adrenaline or other transmitters from neurons. (2) Pharmacological effects of non-selective alpha2-ligands could be assigned to specific receptor subtypes, e.g. hypotension, sedation and analgesia are mediated via alpha2A-receptors. (3) Alpha2-adrenoceptor deficient mice have helped to uncover novel and unexpected functions of these receptor, e.g. feedback control of catecholamine release via alpha2C-receptors in adrenal chromaffin cells and control of angiogenesis during embryonic development. (4) Additional pharmacological targets for alpha2-adrenoceptor ligands were identified, e.g. inhibition of cardiac HCN2 and HCN4 pacemaker channels by clonidine.

Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2.

Biochemical and genetic studies place the amyloid precursor protein (APP) at the center stage of Alzheimer's disease (AD) pathogenesis. Although mutations in the APP gene lead to dominant inheritance of familial AD, the normal function of APP remains elusive. Here, we report that the APP family of proteins plays an essential role in the development of neuromuscular synapses. Mice deficient in APP and its homolog APP-like protein 2 (APLP2) exhibit aberrant apposition of presynaptic marker proteins with postsynaptic acetylcholine receptors and excessive nerve terminal sprouting. The number of synaptic vesicles at presynaptic terminals is dramatically reduced. These structural abnormalities are accompanied by defective neurotransmitter release and a high incidence of synaptic failure. Our results identify APP/APLP2 as key regulators of structure and function of developing neuromuscular synapses.

GABAB receptor protein and mRNA distribution in rat spinal cord and dorsal root ganglia.

The presence of metabotropic receptors for GABA, GABAB, on primary afferent terminals in mammalian spinal cord has been previously reported. In this study we provide further evidence to support this in the rat and show that the GABAB receptor subunits GABAB1 and GABAB2 mRNA and the corresponding subunit proteins are present in the spinal cord and dorsal root ganglion. We also show that the predominant GABAB1 receptor subunit mRNA present in the afferent fibre cell body appears to be the 1a form. In frozen sections of lumbar spinal cord and dorsal root ganglia (DRG) GABAB receptors were labelled with [3H]CGP 62349 or the sections postfixed with paraformaldehyde and subjected to in situ hybridization using oligonucleotides designed to selectively hybridize with the mRNA for GABAB(1a), GABAB(1b) or GABAB2. For immunocytochemistry (ICC), sections were obtained from rats anaesthetized and perfused-fixed with paraformaldehyde. The distribution of binding sites for [3H]CGP 62349 mirrored that previously observed with [3H]GABA at GABAB sites. The density of binding sites was high in the dorsal horn but much lower in the ventral regions. By contrast, the density of mRNA (pan) was more evenly distributed across the laminae of the spinal cord. The density of mRNA detected with the pan probe was high in the DRG and distributed over the neuron cell bodies. This would accord with GABAB receptor protein being formed in the sensory neurons and transported to the primary afferent terminals. Of the GABAB1 mRNA in the DRG, approximately 90% was of the GABAB(1a) form and approximately 10% in the GABAB(1b) form. This would suggest that GABAB(1a) mRNA may be responsible for encoding presynaptic GABAB receptors on primary afferent terminals in a manner similar to that we have previously observed in the cerebellar cortex. GABAB2 mRNA was also evenly distributed across the spinal cord laminae at densities equivalent to those of GABAB1 in the dorsal horn. GABAB2 mRNA was also detected to the same degree within the DRG. Immunocytochemical analysis revealed that GABAB(1a), GABAB(1b) and GABAB2 were all present in the spinal cord. GABAB(1a) labelling appeared to be more dense than GABAB(1b) and within the superficial dorsal horn GABAB(1a) was present in the neuropil whereas GABAB(1b) was associated with cell bodies in this region. Both 1a and 1b immunoreactivity was expressed in motor neurons in lamina IX. GABAB2 immunoreactivity was expressed throughout the spinal cord and was evident within the neuropil of the superficial laminae.

Age-related cognitive deficits, impaired long-term potentiation and reduction in synaptic marker density in mice lacking the beta-amyloid precursor protein.

Mutations in the beta-amyloid precursor protein are strongly associated with some cases of familial Alzheimer's disease. The normal physiological role of beta-amyloid precursor protein in the brain was evaluated in a cross-sectional analysis of mice deficient in beta-amyloid precursor protein. Compared with wild-type control mice the beta-amyloid precursor protein-null mice developed age-dependent deficits in cognitive function and also had impairments in long-term potentiation. In addition, the brains of the beta-amyloid precursor protein-null mice had marked reactive gliosis in many areas, especially in the cortex and hippocampus. A subpopulation of mice (n = 15) died prematurely (between three and 18 months of age). Analysis of another six mice from the same population that were showing weight loss and hypolocomotor activity exhibited a marked reactive gliosis as detected by immunoreactivity for glial fibrillary acidic protein and a profound loss of immunoreactivities for the presynaptic terminal vesicle marker proteins synaptophysin and synapsin and the dendritic marker microtubule-associated protein-2 in many brain areas, but most predominantly in the cortex and hippocampus. These results suggest that normal beta-amyloid precursor protein may serve an essential role in the maintenance of synaptic function during ageing. A compromise of this function of the beta-amyloid precursor protein may contribute to the progression of the memory decline and the neurodegenerative changes seen in Alzheimer's disease.

Distinct structures and functions of related pre- and postsynaptic carbohydrates at the mammalian neuromuscular junction.

Carbohydrates that terminate in beta-linked N-acetylgalactosamine (betaGalNAc) residues are concentrated in the postsynaptic apparatus of the skeletal neuromuscular junction and have been implicated in the differentiation of the postsynaptic membrane. We now report that distinct synapse-specific betaGalNAc-containing carbohydrates are associated with motor nerve terminals. Two monoclonal antibodies that recognize distinct betaGalNAc-containing epitopes, CT1 and CT2, both stain synaptic sites on skeletal muscle fibers. However, CT1 selectively stains nerve terminal, whereas CT2 selectively stains the postsynaptic apparatus. Likewise, CT1 and CT2 selectively stain motoneuron-like and muscle cell lines, respectively. Using the cell lines, we identify distinct CT1- and CT2-reactive glycolipids and glycoproteins. Finally, we show that GalNAc modulates the adhesion of motoneuron-like cells to recombinant fragments of a synaptic cleft component, laminin beta2. Together, these results show that pre- as well as postsynaptic membranes bear and are affected by distinct but related synapse-specific carbohydrates.

Structure of the presynaptic (Y2) receptor-specific neuropeptide Y analog ANA-NPY.

Neuropeptide Y analog ANA-NPY or [Leu-17, Gln-19, Ala-20, Ala-23, Leu-28, Leu-31]NPY(13-36)-amide binds to postjunctional or Y1 receptors to raise blood pressure and to prejunctional or Y2 receptors to inhibit neurotransmitter release. ANA-NPY affects Y2 receptors in the same way as intact NPY but exhibits far less potent effects on Y1 receptors. The structure of ANA-NPY was examined using two-dimensional proton nuclear magnetic resonance spectroscopy. Complete assignment of all backbone and side chain hydrogens was accomplished with totally correlated spectroscopy (TOCSY) experiments providing through-bond 1H-1H connectivities, and nuclear Overhauser effect spectroscopy (NOESY), providing the through-space and sequential backbone connectivities. The tertiary solution structure of the peptide was performed using distance geometry and dynamic simulated annealing. ANA-NPY exhibits a helical structure with strong amphipathic character with a bend around Glu-24 indicating that the C-terminal segment 25-35 forms a single alpha-helical motif.

Characterization of a presynaptic glutamate receptor.

Glutamate receptors mediate excitatory neurotransmission in the brain and are important in the formation of memory and in some neurodegenerative disorders. A complementary DNA clone that encoded a 33-kilodalton protein (GR33) was obtained by screening a library with an antibody generated against glutamate binding proteins. The sequence of GR33 is identical to that of the recently reported presynaptic protein syntaxin. When GR33 was expressed in Xenopus oocytes, it formed glutamate-activated ion channels that are pharmacologically similar to those of N-methyl-D-aspartate receptors but with different electrophysiological properties. Mutation of the leucine 278 residue in the single putative transmembrane segment of GR33 affects the properties of the channel. Thus, in vivo GR33 may be a presynaptic glutamate receptor.