We have examined synaptic transmission between isolated pairs of chick GABAergic amacrine cells, maintained in sparse culture and identified by their binding of an amacrine cell-selective antibody. Using the perforated-patch method to whole-cell clamp both cells of a pair, postsynaptic currents were examined for step depolarizations of the "presynaptic" cell. Synaptic transmission, frequently reciprocal, was calcium dependent and reversibly blocked by bicuculline. Post-synaptic currents, excluding those due to ohmic electrical coupling, were elicited only for presynaptic voltage steps positive to about -40 mV and were always very noisy, suggesting that they were summed from relatively small numbers of quanta. Postsynaptic currents continued well after the termination of the 100 msec presynaptic voltage step when the step was to -10 mV, or positive to this value. This result is interpreted to imply that presynaptic calcium concentration remains elevated after the membrane is returned to its holding potential. When presynaptic voltages were kept low or else presynaptic voltage was uncontrolled, spontaneous quantal events mediated by GABAA receptors could often be seen. Quanta rose quickly (less than 4 msec) and decayed with a mean time constant of 19.3 msec. The amplitude distributions of quantal currents were positively skewed, sometimes showing rare quanta of exceptionally large amplitude. Peak conductance per quantum was about 300 pS, corresponding to the simultaneous opening of only 17 GABAA channels and corresponding to a net flux of only 32 x 10(3) Cl- ions per millivolt of driving force. Estimates of the maximum sustained release rate at individual release sites suggest an upper bound of between 19 and 42 quanta per second.

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Proinflammatory cytokines impact islet ␤-cell mass and function by altering the transcriptional activity within pancreatic ␤-cells, producing increases in intracellular nitric oxide abundance and the synthesis and secretion of immunomodulatory proteins such as chemokines. Herein, we report that IL-1␤, a major mediator of inflammatory responses associated with diabetes development, coordinately and reciprocally regulates chemokine and insulin secretion. We discovered that NF-B controls the increase in chemokine transcription and secretion as well as the decrease in both insulin secretion and proliferation in response to IL-1␤. Nitric oxide production, which is markedly elevated in pancreatic ␤-cells exposed to IL-1␤, is a negative regulator of both glucose-stimulated insulin secretion and glucose-induced increases in intracellular calcium levels. By contrast, the IL-1␤-mediated production of the chemokines CCL2 and CCL20 was not influenced by either nitric oxide levels or glucose concentration. Instead, the synthesis and secretion of CCL2 and CCL20 in response to IL-1␤ were dependent on NF-B transcriptional activity. We conclude that IL-1␤-induced transcriptional reprogramming via NF-B reciprocally regulates chemokine and insulin secretion while also negatively regulating ␤-cell proliferation. These findings are consistent with NF-B as a major regulatory node controlling inflammation-associated alterations in islet ␤-cell function and mass. chemokine; inflammation; insulin secretion; interleukin-1; nitric oxide THE PROGRESSION TO BOTH TYPE 1 (T1DM) and type 2 diabetes mellitus (T2DM) proceeds via immune cell-associated alterations in islet ␤-cell mass and function. Alterations in islet ␤-cell mass and function are two major determinants controlling the total amount of insulin produced and secreted in response to physiological stimuli (e.g., glucose). Proinflammatory cytokines such as IL-1␤ and IFN␥ contribute significantly to losses in islet ␤-cell viability and insulin secretion. Islet

The diverse functions of retinal amacrine cells are reliant on the physiological properties of their synapses. Here we examine the role of mitochondria as Ca2+ buffering organelles in synaptic transmission between GABAergic amacrine cells. We used the protonophore p-trifluoromethoxy-phenylhydrazone (FCCP) to dissipate the membrane potential across the inner mitochondrial membrane that normally sustains the activity of the mitochondrial Ca2+ uniporter. Measurements of cytosolic Ca2+ levels reveal that prolonged depolarization-induced Ca2+ elevations measured at the cell body are altered by inhibition of mitochondrial Ca2+ uptake. Furthermore, an analysis of the ratio of Ca2+ efflux on the plasma membrane Na-Ca exchanger to influx through Ca2+ channels during voltage steps indicates that mitochondria can also buffer Ca2+ loads induced by relatively brief stimuli. Importantly, we also demonstrate that mitochondrial Ca2+ uptake operates at rest to help maintain low cytosolic Ca2+ levels. This aspect of mitochondrial Ca2+ buffering suggests that in amacrine cells, the normal function of Ca2+-dependent mechanisms would be contingent upon ongoing mitochondrial Ca2+ uptake. To test the role of mitochondrial Ca2+ buffering at amacrine cell synapses, we record from amacrine cells receiving GABAergic synaptic input. The Ca2+ elevations produced by inhibition of mitochondrial Ca2+uptake are localized and sufficient in magnitude to stimulate exocytosis, indicating that mitochondria help to maintain low levels of exocytosis at rest. However, we found that inhibition of mitochondrial Ca2+ uptake during evoked synaptic transmission results in a reduction in the charge transferred at the synapse. Recordings from isolated amacrine cells reveal that this is most likely due to the increase in the inactivation of presynaptic Ca2+ channels observed in the absence of mitochondrial Ca2+ buffering. These results demonstrate that mitochondrial Ca2+ buffering plays a critical role in the function of amacrine cell synapses.

Nitric oxide (NO) is generated by multiple cell types in the vertebrate retina, including amacrine cells. We investigate the role of NO in the modulation of synaptic function using a culture system containing identified retinal amacrine cells. We find that moderate concentrations of NO alter GABA(A) receptor function to produce an enhancement of the GABA-gated current. Higher concentrations of NO also enhance GABA-gated currents, but this enhancement is primarily due to a substantial positive shift in the reversal potential of the current. Several pieces of evidence, including a similar effect on glycine-gated currents, indicate that the positive shift is due to an increase in cytosolic Cl-. This change in the chloride distribution is especially significant because it can invert the sign of GABA- and glycine-gated voltage responses. Furthermore, current- and voltage-clamp recordings from synaptic pairs of GABAergic amacrine cells demonstrate that NO transiently converts signaling at GABAergic synapses from inhibition to excitation. Persistence of the NO-induced shift in E(Cl-) in the absence of extracellular Cl- indicates that the increase in cytosolic Cl- is due to release of Cl- from an internal store. An NO-dependent release of Cl- from an internal store is also demonstrated for rat hippocampal neurons indicating that this mechanism is not restricted to the avian retina. Thus signaling in the CNS can be fundamentally altered by an NO-dependent mobilization of an internal Cl- store.

Vertebrate photoreceptors are unusual neurons in that they are capable of continuous calcium-mediated release of neurotransmitter (Trifonov, 1968;Hagins et al., 1970). In this study, we have examined the development and characteristics of calcium currents in chick cone cells placed in culture on embryonic day 8. Cone cells were identified by their lectin-binding properties, rhodopsin-like immunoreactivity, and the presence of an oil droplet. Using the whole-cell patch-clamp method, we have seen calcium currents in these cells after three days in culture, slightly before the appearance of synapses (Gleason & Wilson, 1989). Because cone calcium currents are blocked by cadmium and nifedipine but are enhanced by Bay K 8644, they most closely resemble L-type current (Nowycky et al., 1985). An unexpected feature of these currents is that their gating ranges varied widely between cells so that some cells showed the foot of their activation range at -70 mV and others as positive as -25 mV. Calcium imaging of fura-2 loaded cells was used to confirm the time course of calcium current development and describe the distribution of cytosolic calcium. As expected, depolarization of young cells failed to increase cytosolic calcium but in older cells an increase of threefold to fourfold was usually observed. Both at rest and during depolarization, most cone cells showed regional differences in internal calcium concentration. In the most mature cones, depolarization strongly elevated cytosolic calcium at the terminal end of the cell while producing a lesser change around the oil droplet and the ellipsoid region, suggesting that calcium channels are localized to the terminal.

Amacrine cells in the vertebrate retina receive glutamatergic input from bipolar cells and make synapses onto bipolar cells, ganglion cells, and other amacrine cells. Recent studies indicate that amacrine cells express metabotropic glutamate receptors (mGluRs) and that signaling within the inner plexiform layer (IPL) of the retina might be modulated by mGluR activity. This study tests the hypothesis that activation of mGluR5 modulates GABA(A) receptor function in retinal amacrine cells. Whole cell voltage-clamp recordings were combined with pharmacology to establish the identity of the ionotropic GABA receptors expressed in primary cultures of chick amacrine cells and to determine how mGluR5 activity affected the behavior of those receptors. Application of GABA (20 microM) produced currents that reversed at -58.2 +/- 0.9 mV, near the predicted Cl(-) reversal potential of -59 mV. The GABA(A) receptor antagonist, bicuculline (50 microM), completely blocked the GABA-gated currents. cis-4-Aminocrotonic acid (CACA; 100 microM), a GABA(C) receptor agonist, produced small currents that were not blocked by the GABA(C) antagonist, (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic acid (TPMPA; 20 microM), but were completely blocked by bicuculline. These results indicate that cultured amacrine cells express GABA(A) receptors exclusively. Activating mGluR5 with (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 300 microM) enhanced GABA-gated currents by 10.0 +/- 1.5%. Buffering internal Ca(2+) with BAPTA (10 mM) blocked the CHPG-dependent enhancement. Activation of PKC with the cell-permeable PKC activators (-)-7-octylindolactam V, phorbol 12-myristate 13 acetate (PMA), or 1-oleoyl-2-acetyl-sn-glycerol (OAG) also enhanced GABA-gated currents in a dose-dependent manner. Preactivation of PKC occluded the mGluR5-dependent enhancement, and inhibition of Ca-dependent PKC isotypes with Gö6976 (35 nM) suppressed the effects of mGluR5 activation, suggesting that mGluR5 and PKC are part of the same pathway. To determine if mGluR5-dependent enhancement occurred at synaptic GABA(A) receptors, postsynaptic currents were recorded in the presence of CHPG. On average, the mean amplitudes of the quantal events were increased by about 18% when mGluR5 was activated. These results indicate that activation of mGluR5 enhances GABA-gated current in cultured amacrine cells in a manner that is both Ca(2+)- and PKC-dependent. These results support the possibility that glutamate released from bipolar cells can modulate the function of GABAergic amacrine cells and alter signaling in the inner plexiform layer.

To begin to understand the modulatory role of glutamate in the inner retina, we examined the mechanisms underlying metabotropic glutamate receptor 5 (mGluR5)-dependent Ca 2+ elevations in cultured GABAergic amacrine cells. A partial sequence of chicken retinal mGluR5 encompassing intracellular loops 2 and 3 suggests that it can couple to both G q and G s . Selective activation of mGluR5 stimulated Ca 2+ elevations that varied in waveform from cell to cell. Experiments using high external K + revealed that the mGluR5-dependent Ca 2+ elevations are distinctive in amplitude and time course from those engendered by depolarization. Experiments with a Ca 2+ -free external solution demonstrated that the variability in the time course of mGluR5-dependent Ca 2+ elevations is largely due to the influx of extracellular Ca 2+ . The sensitivity of the initial phase of the Ca 2+ elevation to thapsigargin indicates that this phase of the response is due to the release of Ca 2+ from the endoplasmic reticulum. Pharmacological evidence indicates that mGluR5-mediated Ca 2+ elevations are dependent upon the activation of phospholipase C. We rule out a role for L-type Ca 2+ channels and cAMP-gated channels as pathways for Ca 2+ entry, but provide evidence of transient receptor potential (TRP) channel-like immunoreactivity, suggesting that Ca 2+ influx may occur through TRP channels. These results indicate that GABAergic amacrine cells express an avian version of mGluR5 that is linked to phospholipase C-dependent Ca 2+ release and Ca 2+ influx, possibly through TRP channels.