Cindy Pfeiffer-Linn

GABA-mediated inhibition of visual interneurons in the crayfish medulla

Summary1.The actions of GABA on three classes of visual interneurons in crayfish, Procambarus clarkii, medulla externa are examined. The effect of GABA on the visual response is compared to GABAs action on agonist-elicited responses purported to mediate the visual response.2.GABA produces a shunting type of inhibition in medullary amacrine cells which is associated with a small depolarization (Figs. 2, 3), a large increase in input conductance (Gn) and a reversal potential close to rest (Fig. 4). GABA is a potent antagonist to the depolarizing action of acetylcholine (ACh) (Fig. 5).3.GABA depolarizes dimming fibers (Fig. 2), and the response is mediated by an increase in Gn (Fig. 6). GABA antagonizes the light-elicited IPSP and the hyperpolarizing action of ACh (Fig. 7).4.Sustaining fibers (SF) do not appear to have GABA receptors but GABA inhibits the excitatory visual input pathway to the SFs (Fig. 8). Conversely, the GABA antagonist, bicuculline, potentiates the SF light response (Fig. 9).5.GABA has at least three different modes of antagonist action in the medulla: i) Increased conductance and depolarization in dimming fibers and medullary amacrine neurons; ii) Decreased chloride conductance in tangential cells; and iii) An inhibitory action on the visual pathway which drives SFs.

NMDA receptors in invertebrates

The concept of an NMDA receptor has evolved over some thirty years. The initial impetus was the need to better distinguish “glutamate preferring” from “aspartate preferring” receptors among neurons of the mammalian spinal cord (Curtis and Watkins, 1963; McCulloch et al., 1974). This search gave rise to substantial evidence for a diversity of ionotropic excitatory amino acid (EAA) receptors with quantitatively different sensitivities for various EAA analogues. The properties of these receptors are reviewed in Mayer and Westbrook (1987), Shinozaki (1988), Cotman et al. (1988), Sansom and Usher-wood (1990) and Watkins (1989). The vertebrate receptors are named after the preferred selective agonist. The most important distinction is between the NMDA and non-NMDA (quisqualate and kinate) receptors. Although both receptor types are highly sensitive to glutamate and mediate nonspecific cation conductances, the NMDA receptor is particularly significant because the ion channel is voltage dependent and highly permeable to Ca*+ (Mayer and Westbrook, 1987). To our knowledge there has never been a major effort to determine whether any invertebrate species possesses an NMDA receptor. Rather, the modest data available have been produced in the context of structure-activity studies to better characterize particular glutamate and/or aspartate receptors. Although there are some gross similarities between some vertebrate and invertebrate EAA receptors, significant differences are invariably found in the details. Altogether we have identified 21 studies in which NMDA action was tested on an invertebrate glutamate/aspartate receptor. Although the overwhelming majority of results are negative, a few positive indications emerge. These are summarized in Table 1. In the foregoing these results are considered relative to the vertebrate NMDA receptor and the EAA receptors of the invertebrate species tested. Operationally, the NMDA receptor is characterized by the effects of selective agonists and antagonists, the action of modulators (Mg’+, glycine), voltage dependence in the presence of Mg*+, a large single channel conductance and ionic permeability. When agonist affinities are measured by the capacity to displace selective antagonists from the NMDA receptor, L-glutamate is the most potent (Watkins, 1989; Oliverman and Watkins, 1989). NMDA, ibotenate and L-homocysteate are strong competitors for the NMDA binding site while L-quisqualate, L-kainate and AMPA are not. D-a-aminoadipate (Davies and Watkins, 1979) was the first competitive antagonist discovered, to exhibit a preference for the NMDA receptor. Subsequently, more potent blockers were developed in which a phosphonate group is substituted for the w-carboxyl group and additional methylene groups extend the molecule length (Evans et al., 1982). The most effective of these are 2-amino-5phosphonopentanoate (APV or D-AP5) and 2-amino-7-phosphonoheptanoate (D-AP7). Recently these have been superseded by higher affinity antagonists derived from piperdine (Olverman and Watkins, 1989). Activation of the NMDA receptor requires glytine (K, N 200 nM), a requirement normally satisfied in vivo by the interstitial fluid (Johnson and Ascher, 1987; Thomson et al., 1989). The receptor’s glycine binding site is insensitive to strychnine but is competitively blocked by kynurenate, 7-Cl-kynurenate and HA-966 (Ascher and Johnson, 1989). M

Dopamine modulates unitary conductance of single PL-type calcium channels in Roccus chrysops retinal horizontal cells.

+(Kd = 72 nM) blocks current flow through the open NMDA receptor channel and imparts a voltage-dependence to channel activation (Davies and Watkins, 1977; Ault et al., 1980; Ascher and Nowak, 1988). At 1 .O mM Mg*+, membrane depolarizations to above 30 mV are sufficient to overcome the Mg*+ block. Co*+ and Mn*+ can mimic the action of Mg*+ but Ba*+, Ca*+ and Cd*+ do not. In physiological salines the single channel conductance is about 50 pS which is substantially larger than that of the non-NMDA receptor channels. Elevated external Ca*+ reduces the single channel conductance at negative membrane potentials from 56 pS (in 0 Caz+) to 15 pS at 100 mM Ca*+ (Ascher and Nowak, 1988). The NMDA channel is permeable to Na + , K + and Ca*+ and the Ca*+ permeability is unusually high; Pc,Z+/PN,+ * 1.0 (Ascher and Johnson, 1989). In normal physiological salines the reversal potential of the NMDA-elicited current is -0 mV. In many neurons, NMDA receptors are interspersed among non-NMDA receptors (Jahr and Stevens, 1987; Cull-Candy and Usowicz, 1987) and the voltage-sensitivity and slower kinetics of the NMDA activated channels are thought to have a

A NEURONAL NICOTINIC ACETYLCHOLINE RECEPTOR IN CRAYFISH NEURONS

1. Dopamine modulation of the PL‐type calcium channel of white bass retinal horizontal cells was studied in isolated, cultured neurons. Single‐channel recordings were made of calcium channels in outside‐out patches, under conditions which favoured the expression of calcium channel activity. 2. Analysis of single‐channel properties revealed that dopamine potentiated the activity of the sustained calcium channel in three ways. First, it increased unitary conductance through individual channels. Under the influence of dopamine, single‐channel conductance doubled. 3. Dopamine also increased the probability of channel opening and increased channel mean open time. The probability of opening increased 4‐fold while mean open time doubled. 4. The mean closed time was also affected. The time between individual openings was not affected but the closed time between bursts of openings was shortened by over 50%. 5. The effects of dopamine were mediated via the activation of a D1‐type receptor and the resulting activation of a cAMP‐mediated second messenger system. 6. The combination of the effects of dopamine significantly increased the net calcium influx into the cell.

Sodium dependency of the inward potassium rectifier in horizontal cells isolated from the white bass retina

In warm-blooded vertebrates, neuronal nicotinic acetylcholine receptors (nAChRs) are distinguished from muscle endplate receptors by their ligand affinities and sensitivity to several toxins. In the crayfish optic lobe, synaptic and acetylcholine (ACh)-elicited responses are blocked by toxins (F-toxin and neosurugatoxin) selective for neuronal nAChRs and are insensitive to the alpha-neurotoxins selective for endplate nAChRs.

Synaptic Mechanisms of a Dual Channel Contrast Detection System in the Crayfish Optic Lobe

The ionic properties underlying the inwardly rectifying potassium current in cultured voltage-clamped white bass horizontal cells were studied. Anomalous rectification was apparent upon membrane hyperpolarization with a reversal potential depolarized from the predicted value of EK. In raised extracellular potassium, the current increased and the reversal potential shifted toward a more depolarized membrane potential. Solutions containing decreased sodium caused a rapid decrease in the inward rectifier current but only slightly affected the reversal potential. Extracellular cesium or barium caused a reversible voltage-dependent reduction of the inward current. We interpret these results to mean that the inward rectifying channel in white bass horizontal cells is mainly permeable to potassium ions, but is sodium dependent. It may shape the photoresponses of the horizontal cells and may contribute to a hyperpolarization activated conductance increase measured in situ.

Dopamine modulates in a differential fashion T- and L-type calcium currents in bass retinal horizontal cells.

The sustaining and dimming fibers (SF, DF) of the crayfish optic lobe constitute a dual channel contrast detection system and mediate the dorsal light reflex. The receptive fields and dynamic properties of SFs and DFs are shaped by dendritic geometry and interactions among presynaptic columnar and multicolumnar neurons (amacrine and tangential cells) of the laminaand medulla externa. The visual response of each multicolumnar neuron reflects the antagonistic interaction of two postsynaptically acting neurotransmitters. These transmitters (Ach, GABA, Glutamate) are contained in columnar interneurons.