strychnine and lucifer-yellow

The effects of Ca2+, Sr2+, and Ba2+ on spontaneous and evoked glycinergic inhibitory postsynaptic currents (mIPSCs and eIPSCs) were studied using the "synaptic bouton" preparation of rat spinal neurons and conventional whole cell recording under voltage-clamp conditions. In response to application of Ca2+-free solution, the frequency of mIPSC initially rapidly decreased to 40 approximately 50% of control followed by a gradual further decline in mIPSC frequency to approximately 30% of control. Once mIPSC frequency had significantly decreased in Ca2+-free solution, application of Ca2+, Sr2+, or Ba2+ increased mIPSC frequency. The rank order of effect in restoring mIPSCs was Ba2+>>Ca2+>Sr2+. Moreover, the application of excess external [K+]o solution (30 mM) containing Sr2+ or Ba2+ after 2 h in Ca2+-free solution also increased mIPSC frequency in the order Sr2+>or==Ba2+>Ca2+. The mean mIPSC amplitude was not affected at all. In contrast, eIPSCs produced by focal stimulation of single boutons were completely abolished in Ca2+-free solution or when Ca2+ was replaced by Sr2+ or Ba2+ (2 mM each). However, eIPSCs were restored in increased concentrations of Sr2+ or Ba2+ (5 mM each). The results show that these divalent cations affect mIPSC and eIPSCs differently and indicate that the mechanisms underlying transmitter release that generates eIPSCs and mIPSC in presynaptic nerve terminals are different. The different mechanisms might be explained by the different sensitivity of synaptotagmin isoforms to Ca2+, Sr2+, and Ba2+.

Topics: Animals; Animals, Newborn; Cations, Divalent; Electric Stimulation; Glycine; Glycine Agents; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Interneurons; Isoquinolines; Neural Inhibition; Patch-Clamp Techniques; Rats; Rats, Wistar; Spinal Cord; Strychnine; Time Factors

1. Whole-cell patch recording and puff pipette techniques were used to identify glutamate receptor mechanisms on bipolar cell (BC) dendrites in the zebrafish retinal slice. Recorded neurons were stained with Lucifer Yellow, to correlate glutamate responses with BC morphology. 2. BC axon terminals (ATs) consisted of swellings or varicosities along the axon, as well as at its end. AT stratification patterns identified three regions in the inner plexiform layer (IPL): a thick sublamina a, with three bands of ATs, a narrow terminal-free zone in the mid-IPL, and a thin sublamina b, with two bands of ATs. BCs occurred with ATs restricted to sublamina a(Group a), sublamina b(Group b) or with ATs in both sublaminae (Group a/b). 3. OFF-BCs belonged to Group a or Group a/b. These cells responded to glutamate or kainate with a CNQX-sensitive conductance increase. Reversal potential (Erev) ranged from -0.6 to +18 mV. Bipolar cells stimulated sequentially with both kainate and glutamate revealed a population of glutamate-insensitive, kainate-sensitive cells in addition to cells sensitive to both agonists. 4. ON-BCs responded to glutamate via one of three mechanisms: (a) a conductance decrease with Erev approximately 0 mV, mimicked by L-(+)-2-amino-4-phosphonobutyric acid (APB) or trans-1-amino-1, 3-cyclopentanedicarboxylic acid (trans-ACPD), (b) a glutamate-gated chloride conductance increase (IGlu-like) characterized by Erev >= ECl (where ECl is the chloride equilibrium potential) and partial blockade by extracellular Li+/Na+ substitution or (c) the activation of both APB and chloride mechanisms simultaneously to produce a response with outward currents at all holding potentials. APB-like responses were found only among BCs in Group b, with a single AT ramifying deep within sublamina b; whereas, cells expressing IGlu-like currents had one or more ATs, and occurred within Groups b or a/b. 5. Multistratified cells (Group a/b) were common and occurred with either ON- or OFF-BC physiology. OFF-BCs typically had one or more ATs in sublamina a and only one AT in sublamina b. In contrast, multistratified ON-BCs had one or more ATs in sublamina b and a single AT ramifying deep in sublamina a. Multistratified ON-BCs expressed the IGlu-like mechanism only. 6. Visual processing in the zebrafish retina involves at least 13 BC types. Some of these BCs have ATs in both the ON- and OFF-sublaminae, suggesting a significant role for ON- and OFF-inputs throughout the IPL.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Aminobutyrates; Animals; Axons; Chlorides; Cycloleucine; Dendrites; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Glutamic Acid; In Vitro Techniques; Isoquinolines; Kainic Acid; Neurons; Picrotoxin; Retina; Strychnine; Zebrafish

1. By using the whole-cell recording configuration of the patch-clamp technique in a spinal cord slice preparation, we have made recordings from visually identified neurones in the lateral horn of the thoracic and lumbar spinal cord of neonatal rats (newborn to 14 days postnatal). 2. Some of the recorded neurones were labelled with the fluorescent dye Lucifer Yellow (n = 27). Their morphology was typical for sympathetic preganglionic neurones (SPNs). Based on the size of the cell soma and the electrophysiological properties, unlabelled neurones were also regarded as SPNs. 3. Spontaneous synaptic activity of different patterns could be observed in 73% of the recorded neurones (n = 106). It reversed at the chloride equilibrium potential (ECl) and could be reversibly blocked by strychnine (1-10 microM), but not by bicuculline (10 microM) or SR95531 (5-10 microM). 4. Synaptic activity could be elicited by focal electrical stimulation in the vicinity of the recorded neurone. These evoked synaptic events exhibited features similar to the spontaneous synaptic activity. 5. Application of glycine (100 microM-1 mM) by a fast microperfusion system induced a chloride current in twenty-seven out of thirty cells tested. The currents were reversibly blocked by strychnine (1-10 microM), but were only weakly sensitive to bicuculline (10 microM). Stability of current responses to glycine was increased by inclusion of ATP (4 mM) in the intracellular medium. 6. Application of gamma-aminobutyric acid (GABA; 100 microM-1 mM) by the fast microperfusion system induced a chloride current in all twenty neurones tested. These currents were reversibly blocked by bicuculline (10 microM). Strychnine (1-10 microM) blocked this current only weakly. Run-down of GABA-induced currents was prevented to a great extent by inclusion of ATP (4 mM) in the pipette. 7. These results suggest that the inhibitory synaptic activity recorded from SPNs in thin, transverse slices of neonatal rat spinal cord is mediated by glycine receptor-gated Cl- channels. GABAA receptor-gated Cl- channels might be activated by inputs from other spinal segments and/or descending pathways from higher brain regions.

Topics: Animals; Animals, Newborn; Chloride Channels; Chlorides; Electric Stimulation; Evoked Potentials; Fluorescent Dyes; gamma-Aminobutyric Acid; Ganglia, Sympathetic; Glycine; In Vitro Techniques; Isoquinolines; Membrane Potentials; Neurons; Rats; Rats, Wistar; Strychnine; Synapses