Functional requirement for class I MHC in CNS development and plasticity.

Class I major histocompatibility complex (class I MHC) molecules, known to be important for immune responses to antigen, are expressed also by neurons that undergo activity-dependent, long-term structural and synaptic modifications. Here, we show that in mice genetically deficient for cell surface class I MHC or for a class I MHC receptor component, CD3zeta, refinement of connections between retina and central targets during development is incomplete. In the hippocampus of adult mutants, N-methyl-D-aspartate receptor-dependent long-term potentiation (LTP) is enhanced, and long-term depression (LTD) is absent. Specific class I MHC messenger RNAs are expressed by distinct mosaics of neurons, reflecting a potential for diverse neuronal functions. These results demonstrate an important role for these molecules in the activity-dependent remodeling and plasticity of connections in the developing and mature mammalian central nervous system (CNS).

Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX.

Genetic disorders affecting cellular responses to DNA damage are characterized by high rates of translocations involving antigen receptor loci and increased susceptibility to lymphoid malignancies. We report that the Nijmegen breakage syndrome protein (NBS1) and histone gamma-H2AX, which associate with irradiation-induced DNA double-strand breaks (DSBs), are also found at sites of VDJ (variable, diversity, joining) recombination-induced DSBs. In developing thymocytes, NBS1 and gamma-H2AX form nuclear foci that colocalize with the T cell receptor alpha locus in response to recombination activating gene (RAG) protein-mediated VDJ cleavage. Our results suggest that surveillance of T cell receptor recombination intermediates by NBS1 and gamma-H2AX may be important for preventing oncogenic translocations.

gamma-Aminobutyric acid receptor distribution in the mushroom bodies of a fly (Calliphora erythrocephala): a functional subdivision of Kenyon cells?

Antibodies against the Drosophila gamma-aminobutyric acid (GABA) receptor subunit RDL were used to investigate the significance of inhibitory inputs to the mushroom bodies in the blowfly (Calliphora erythrocephala) brain. The pedunculus and the lobes of the mushroom body, which mainly consist of Kenyon cell fibers, revealed strong immunoreactivity against RDL. Pedunculi, alpha- and beta-lobe show characteristic unstained core structures with concentric labeling along the neuropile axis. The gamma-lobes in contrast exhibit a compartmentalized RDL-immunoreactive pattern. These data suggest an important role of GABAergic inhibition in the pedunculus and the lobes of insect mushroom bodies. It is most likely that the RDL-immunoreactivity in the mushroom bodies is closely related to Kenyon cell fibers suggesting that Kenyon cells are an inhomogeneous class of neurons, only part of which receive inhibitory GABAergic input from extrinsic elements. GABAergic inhibition, therefore, may play a substantial role in the process of learning and memory formation in the insect mushroom bodies.

Cholinergic and GABAergic pathways in fly motion vision.

BACKGROUND: The fly visual system is a highly ordered brain structure with well-established physiological and behavioral functions. A large number of interneurons in the posterior part of the third visual neuropil, the lobula plate tangential cells (LPTCs), respond to visual motion stimuli. In these cells the mechanism of motion detection has been studied in great detail. Nevertheless, the cellular computations leading to their directionally selective responses are not yet fully understood. Earlier studies addressed the neuropharmacological basis of the motion response in lobula plate interneurons. In the present study we investigated the distribution of the respective neurotransmitter receptors in the fly visual system, namely nicotinic acetylcholine receptors (nAChRs) and GABA receptors (GABARs) demonstrated by antibody labeling. RESULTS: The medulla shows a laminar distribution of both nAChRs and GABARs. Both receptor types are present in layers that participate in motion processing. The lobula also shows a characteristic layering of immunoreactivity for either receptor in its posterior portion. Furthermore, immunostaining for nAChRs and GABARs can be observed in close vicinity of lobula plate tangential cells. Immunostaining of GABAergic fibers suggests that inhibitory inputs from the medulla are relayed through the lobula to the lobula plate rather than through direct connections between medulla and lobula plate. CONCLUSIONS: The interaction of excitatory and inhibitory pathways is essential for the computation of visual motion responses and discussed in the context of the Reichardt model for motion detection.

A preparation of the blowfly (Calliphora erythrocephala) brain for in vitro electrophysiological and pharmacological studies.

We describe a method for the preparation and maintenance of the blowfly (Calliphora erythrocephala) brain in a recording chamber under in vitro conditions in a semi-slice configuration. Large identification neurones in the posterior part of the 3rd optic lobe (lobula plate) can be penetrated easily with microelectrodes. The so-called vertical system (VS) cells which respond to vertical image motion in vivo could be encountered best because their axons are escorted individually by specific tracheae. Fluorescent stained cells show their natural shape as being in vivo. Electrophysiological properties of the cells investigated so far, i.e., resting potential (about -40 mV) and firing properties (single rebound spikes), are comparable to recordings in intact flies. Initial pharmacological experiments on VS cells in this preparation reveal that iontophoretical application of acetylcholine and carbamylcholine results in depolarization. VS cells also respond to bath-applied nicotine (1 microM) with a slow depolarization of their membrane potential in normal fly saline as well as in a Ca(2+)-free saline, suggesting direct cholinergic input via nicotinic receptors. The suitability of the preparation for a wide range of electrophysiological and pharmacological studies is discussed.

Cholinergic and GABAergic receptors on fly tangential cells and their role in visual motion detection.

1. To identify some of the neurotransmitters involved in the processing of visual motion information the pharmacology of transmitter receptors on motion-sensitive visual interneurons (VS and HS cells) was investigated in an in vitro preparation of the blowfly (Calliphora erythrocephala) brain. Cholinergic and GABAergic drugs were applied in the bath and iontophoretically while recording intracellularly from HS and VS cells. 2. Bath-applied carbachol (10 and 100 microM) leads to a depolarization in HS and VS cells. One micromolar nicotine also has a depolarizing effect. Both agonists are effective in 0 Ca2+/high Mg(2+)-saline, too, which isolates the cells synaptically. The muscarinic agonists pilocarpine and oxotremorine have no effects on the membrane potential. 3. Iontophoretic application of acetylcholine, carbachol, and nicotine depolarizes VS and HS cells. The iontophoretic carbachol response is antagonized by alpha-bungarotoxin (EC50 = 0.19 microM), mecamylamine (EC50 = 0.32 microM), d-tubocurarine (EC50 = 9.5 microM), and bicuculline but not by decamethonium and scopolamine. 4. Bath application of muscimol strongly hyperpolarizes VS cells in normal fly saline. The gamma-aminobutyric acid-C (GABAC)-receptor agonist cis-4-aminocrotonic acid (CACA) has no effects. The hyperpolarizing response to iontophoretic applied muscimol is present in 0 Ca2+/high Mg2+ saline as well as in Co(2+)-containing saline. The muscimol response is reduced in low chloride saline and thus chloride sensitive. The muscimol response is blocked by picrotoxinin (EC50 = 3.4 microM) but not by the GABAA receptor antagonist bicuculline. 5. Taken together the primary responses of the lobula plate tangential cells appear to be nicotinic cholinergic and GABAergic. 6. The pharmacology of natural synaptic input to VS cells was investigated by extracellular electrical stimulation of the medulla. Such evoked excitatory postsynaptic potentials (EPSPs) are blocked reversibly in 0 Ca2+/high Mg2+ saline. The nicotinic antagonists mecamylamine (1 microM) and d-tubocurarine (50-100 microM) abolish or diminish the EPSPs, respectively. 7. The pharmacological data are incorporated into a semicellular model of a visual motion detector favoring a role of lobula plate tangential cells in certain steps of visual motion processing. Cholinergic and GABAergic inputs are an ideal cellular implementation of a linear subtraction of the signals arising from local motion-sensitive elements with opposite preferred direction. Such a mechanism enhances direction-selectivity and, together with dendritic integration, increases the sensitivity of the tangential cells for wide-field motion.

Mechanisms of dendritic calcium signaling in fly neurons.

We examined the mechanisms underlying dendritic calcium accumulation in lobula plate tangential cells of the fly visual system using an in vitro preparation of the fly brain. Local visual stimulation evokes a localized calcium signal in the dendrites of these cells in vivo. Here we show that a similar localized calcium accumulation can be elicited in vitro by focal iontophoretic application of the cholinergic agonist carbachol. The calcium signal had at least two sources: first, voltage-dependent calcium channels contributed to the carbachol-induced signal and were concentrated on the dendrite, the soma, and the terminal ramification of the axon. However, the dendritic calcium signal induced by carbachol stimulation was only weakly dependent on membrane depolarization. The most likely explanation for the second, voltage-independent part of the dendritic calcium signal is calcium entry through nicotinic acetylcholine receptors. We found no indication of second-messenger or calcium-mediated calcium release from intracellular stores. In summary, the characteristic spatiotemporal calcium signals in the dendrites of lobula plate tangential cells can be reproduced in vitro, and result from a combination of voltage- and ligand-gated calcium influx.

Caspase 8 activity in membrane blebs after anti-Fas ligation.

Previous studies of thymocyte apoptosis using a series of cell-permeable fluorogenic peptide substrates showed that Fas cross-linking triggered a caspase cascade in which cleavage of the IETDase (caspase 8-selective) substrate was the earliest caspase activity measured by flow cytometry. This result was expected in light of the abundant evidence for caspase 8 activation as an initiating event in the Fas death pathway. However, when apoptosis was induced by anti-Fas in CTL and the caspase cascade examined by this approach, IETDase activation followed increases in LEHDase, YVHDase, and VEIDase activities (selective for caspases 9, 1, and 6, respectively). When examined by confocal microscopy, anti-Fas-treated CTL showed the early appearance of IETDase-containing plasma membrane vesicles and their release from the CTL surface, followed by activation of other caspase activities in the cell interior. Since these vesicles were not included in the flow cytometry analysis, the early IETDase activity had been underestimated. In contrast to anti-Fas, induction of apoptosis in these CTL by IL-2 withdrawal resulted in early IETDase activity in the cytoplasm, with no plasma membrane vesiculation. Thus, anti-Fas-induced initiation of caspase activity at the plasma membrane may in some cells result in local proteolysis of submembrane proteins, leading to generation of membrane vesicles that are highly enriched in active caspase 8.

The familial Mediterranean fever protein, pyrin, associates with microtubules and colocalizes with actin filaments.

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and intense inflammation. FMF attacks are unique in their sensitivity to the microtubule inhibitor colchicine, contrasted with their refractoriness to the anti-inflammatory effects of glucocorticoids. The FMF gene, MEFV, was recently identified by positional cloning; it is expressed at high levels in granulocytes and monocytes. The present study investigated the subcellular localization of the normal gene product, pyrin. These experiments did not support previously proposed nuclear or Golgi localizations. Instead fluorescence microscopy demonstrated colocalization of full-length GFP- and epitope-tagged pyrin with microtubules; this was markedly accentuated in paclitaxel-treated cells. Moreover, immunoblot analysis of precipitates of stabilized microtubules with recombinant pyrin demonstrated a direct interaction in vitro. Pyrin expression did not affect the stability of microtubules. Deletion constructs showed that the unique N-terminal domain of pyrin is necessary and sufficient for colocalization, whereas disease-associated mutations in the C-terminal B30.2 (rfp) domain did not disrupt this interaction. By phalloidin staining, a colocalization of pyrin with actin was also observed in perinuclear filaments and in peripheral lamellar ruffles. The proposal is made that pyrin regulates inflammatory responses at the level of leukocyte cytoskeletal organization and that the unique therapeutic effect of colchicine in FMF may be dependent on this interaction. (Blood. 2001;98:851-859)