Pre- and post-synaptic effects of manipulating surface charge with divalent cations at the photoreceptor synapse.

Persistence of horizontal cell (HC) light responses in extracellular solutions containing low Ca2+ plus divalent cations to block Ca2+ currents (ICa) has been attributed to Ca2+-independent neurotransmission. Using a retinal slice preparation to record both ICa and light responses, we demonstrate that persistence of HC responses in low [Ca2+]o can instead be explained by a paradoxical increase of Ca2+ influx into photoreceptor terminals arising from surface charge-mediated shifts in ICa activation. Consistent with this explanation, application of Zn2+ or Ni2+ caused a hyperpolarizing block of HC light responses that was relieved by lowering [Ca2+]o. The same concentrations of Zn2+ and Ni2+ reduced the amplitude of ICa at the rod dark potential and this reduction was relieved by a hyperpolarizing shift in voltage dependence induced by lowering [Ca2+]o. Block of ICa by Mg2+, which has weak surface charge effects, was not relieved by low [Ca2+]o. Recovery of HC responses in low [Ca2+]o was assisted by enhancement of rod light responses. To bypass light stimulation, OFF bipolar cells were stimulated by steps to -40 mV applied to presynaptic rods during simultaneous paired recordings. Consistent with surface charge theory, the post-synaptic current was inhibited by Zn2+ and this inhibition was relieved by lowering [Ca2+]o. Nominally divalent-free media produced inversion of HC light responses even though rod light responses remained hyperpolarizing; HC response inversion can be explained by surface charge-mediated shifts in ICa. In summary, HC light responses modifications induced by low divalent cation solutions can be explained by effects on photoreceptor light responses and membrane surface charge without necessitating Ca2+-independent neurotransmission. Furthermore, these results suggest that surface charge effects accompanying physiological changing divalent cation levels in the synaptic cleft may provide a means for modulating synaptic output from photoreceptors.

Autoradiographic distribution of peripheral benzodiazepine receptors in the retina of the albino rabbit, Lepus cunicula.

The distribution of peripheral benzodiazepine receptors (PBRs) in the retina of the albino rabbit, Lepus cunicula, was studied by autoradiography using [3H]-PK11195, a isoquinoline carboxamide, as a tracer. Autoradiograms obtained by directly placing the slides containing the retina sections on tritium-sensitive film provide evidence for the presence of PBRs in rabbit retina. Furthermore, the dark field examination of photomicrographs taken from autoradiograms showed two dense horizontal bands corresponding to the outer and inner photoreceptor segments, and to the inner plexiform layer. The retinal regions where [3H]-PK11195 binding was more dense are rich in mitochondria, suggesting that as in other neuronal tissues, retinal PBRs are involved in the mitochondrial activity.

The bicentennial of the Voltaic battery (1800-2000): the artificial electric organ.

Alessandro Volta invented the electric battery at the end of 1799 and communicated his invention to the Royal Society of London in 1800. The studies that led him to develop this revolutionary device began in 1792, after Volta read the work of Luigi Galvani on the existence of an intrinsic electricity in living organisms. During these studies, Volta obtained a series of results of great physiological relevance, which led him to anticipate some important ideas that marked the inception of modern neuroscience. These results have been obscured by a cultural tradition that has seen Volta exclusively as a physicist, lacking interest for biological problems and opposed in an irreversible way to the physiologist, Luigi Galvani.

Biological machines: from mills to molecules.

Although scientific progress is usually represented as being linear, it may, in fact, have a cyclical character--some discoveries may be forgotten or lost (at least temporarily), and themes may reappear through the centuries. Consider, for example, the concept of 'molecular machines', from the exciting phase of research that flourished in the seventeenth century, to the idea of machines that is at centre stage in modern cell biology.

Manipulation of synaptic sign and strength with divalent cations in the vertebrate retina: pushing the limits of tonic, chemical neurotransmission.

At the first synaptic level of the vertebrate retina, photoreceptor light responses are transmitted to second order neurones through a chemical synapse based on a tonic release of neurotransmitter modulated by graded changes of presynaptic potential. The possibility that such synapses could work through a Ca2+-independent process had been proposed by previous authors, based on the persistence of transmission process in low Ca2+ media containing Co2+ or Ni2+ ions. Recently, we were able to explain these results within the framework of the classical calcium-hypothesis of synaptic transmission by taking into account the modifications of presynaptic surface potential brought about by changes of divalent cation concentrations. Here we report data showing how a surface-charge hypothesis could account for several apparently paradoxical effects of divalent cation manipulations such as: the enhancement of neurotransmitter release induced by low Ca2+ media; the transmission "unblocking" effect of Zn2+, Co2+ and Ni2+; and the reversal of transmission polarity induced by application of low Ca2+ media containing Cd2+ or Mg2+ ions.

Resistance of retinal extracellular space to Ca2+ level decrease: implications for the synaptic effects of divalent cations.

Ion-sensitive microelectrodes were used to measure the variations of [Ca2+]o induced by application of low Ca2+ media in the superfused eyecup preparation of the Pseudemys turtle. The aim of the experiments was to evaluate the possibility, suggested by previous studies, that in the deep, sclerad, layers of the retina [Ca2+]o may remain high enough to sustain chemical synaptic transmission even after prolonged application of low-Ca2+ saline. It was found that, at depths of 100-200 micron from the vitreal surface, [Ca2+ ]o did not fall below 1 mM even after application for periods of 30-60 min of nominally Ca2+-free media, and it was >0.3 mM after 30-min application of media containing EGTA and with a Ca2+ concentration of 1 nM. Previous studies in isolated salamander photoreceptors have shown that a reduction of [Ca2+ ]o to 0.3-1.0 mM may result in a paradoxical increase of Ca2+ influx into synaptic terminals due to the reduced screening of negative charge on the external face of the plasma membrane. On the basis of these results, the persistence or enhancement of synaptic transmission from photoreceptors to horizontal cells observed in various retinas treated with low-Ca2+ media may be accounted for within the classical Ca2+-dependent theory of synaptic transmission without invoking a Ca2+-independent mechanism.

Calcium-independent release of neurotransmitter in the retina: a "copernican" viewpoint change.

The release of synaptic transmitter in chemical synapses is brought about by Ca2+ influx through voltage-dependent Ca2+ channels opened by depolarisation of presynaptic terminals. However, in some preparations transmitter release persists or increases in low-Ca2+ media, and it has therefore been proposed that transmitter release could also occur through a Ca2+-independent, carrier mediated process. In particular it has been suggested that this may be the case for synaptic transmission between photoreceptors and second order neurones of the vertebrate retina. From our recent experiments on synaptic transmission from photoreceptors to horizontal cells of turtle and salamander retinas, it appears that lowering extracellular Ca2+ can actually promote Ca2+ influx through voltage-activated Ca2+ channels via a modification of surface potential of plasma membranes. On the basis of this apparently paradoxical effect of low Ca2+ media, it is possible to reaccommodate the so-called Ca2+-independent release within the framework of Ca2+-dependent synaptic transmission without invoking unconventional mechanisms.

Marcello Malpighi and the difficult birth of modern life sciences.

All his life, Marcello Malpighi (1628-1694), the founder of modern microscopic anatomy, was unwillingly involved in difficult debates within a reactionary medical milieu that questioned the significance of modern science and its utility to medicine. Malpighi's responses to his detractors, included in posthumous works first published in 1697 by the Royal Society, offer an important insight into a critical phase of scientific progress in the 17th century and help to reveal the prevailing conception of science. In some ways, Malpighi's views predate important ideas in modern biology.

Does pattern electroretinogram spatial tuning alteration in Parkinson's disease depend on motor disturbances or retinal dopaminergic loss?

Systemic decrease of dopaminergic cells, such as in Parkinson's disease may produce visual alterations in humans. In order to show possible pattern electroretinogram (PERG) spatial tuning function (STF) alterations due to impaired dopaminergic transmission in humans, we studied a group of Parkinson's disease patients before and during treatment with the dopamine precursor, levodopa, and compared their performances with those of an age-matched control group. Moreover, in order to exclude the possible involvement of motor disabilities to produce PERG alterations, we also investigated PERG responses in post-traumatic parkinsonian patients who exhibited motor abnormalities as a consequence of focal lesions of basal ganglia, in the absence of systemic dopaminergic degeneration. Our results showed a clear decrease of PERG responses in Parkinson's disease patients particularly at medium spatial frequency range (2.7-4.0 cycles/degree) with a substantial preservation of responses at low frequencies. Levodopa therapy reversed these alterations in Parkinson's disease patients, resulting in the recovery of a normal tuning function shape. In contrast to Parkinson's disease, the tuning function appeared to be preserved in post-traumatic parkinsonian patients. Our results clearly establish a relationship between retinal alteration in PD patients and dopaminergic retinal function.

Animal electricity and the birth of electrophysiology: the legacy of Luigi Galvani.

Preceded by a companion paper on Galvani's life, this article is written on the occasion of the bicentenary of the death of Luigi Galvani. From his studies on the effects of electricity on frogs, the scientist of Bologna derived the hypothesis that animal tissues are endowed with an intrinsic electricity that is involved in fundamental physiological processes such as nerve conduction and muscle contraction. Galvani's work swept away from life sciences mysterious fluids and elusive entities like "animal spirits" and led to the foundation of a new science, electrophysiology. Two centuries of research work have demonstrated how insightful was Galvani's conception of animal electricity. Nevertheless, the scholar of Bologna is still largely misrepresented in the history of science, because the importance of his researches seems to be limited to the fact that they opened the paths to the studies of the physicist Alessandro Volta, which culminated in 1800 with the invention of the electric battery. Volta strongly opposed Galvani's theories on animal electricity. The matter of the scientific controversy between Galvani and Volta is examined here in the light of two centuries of electrophysiological studies leading to the modern understanding of electrical excitability in nerve and muscle. By surveying the work of scientists such as Nobili, Matteucci, du Bois-Reymond, von Helmholtz, Bernstein, Hermann, Lucas, Adrian, Hodgkin, Huxley, and Katz, the real matter of the debate raised by Galvani's discoveries is here reconsidered. In addition, a revolutionary phase of the 18th century science that opened the way for the development of modern neurosciences is reevaluated.

Luigi Galvani and animal electricity: two centuries after the foundation of electrophysiology.

Luigi Galvani and his famous experiments on frogs carried out in the second half of the 18th century belong more to legend than to the history of science. Galvani not only laid the foundations of a new science, electrophysiology, but also opened the way for the invention of the electric battery, and thus for the development of the physical investigations of electricity. However, in spite of the widespread celebration of his work, Galvani's scientific endeavours have been largely misrepresented in the history of science. The scholar of Bologna has a stereotyped image as an 'occasional' scientist, who started his studies by chance, largely ignored the scientific theories of his time and wandered aimlessly in mental elaborations until the physicist of Pavia, Alessandro Volta, entered the field, correctly interpreted Galvani's results and eventually developed the electric battery. With the present understanding of electrical phenomena in excitable membranes, it is now time to reconsider the real matter raised by Galvani's discoveries and by his hypothesis of an intrinsic 'animal electricity', and to make a clearer evaluation of a revolutionary phase of scientific progress.

Retinal horizontal cells: old cells, old experiments, new results.

The study of neural interactions in the vertebrate retina carried out after the pioneering studies of Svaetichin has provided important information on the functioning of nerve circuits in the central nervous system. Recently we have investigated the effects of changes of divalent cation concentration on the synaptic transmission between cones and horizontal cells of the turtle retina. Our results seemed apparently in contrast with the classical Ca2(+)-hypothesis of chemical synaptic transmission. Application of low Ca2+ media resulted in a recovery of synaptic transmission after application of divalent cations such as Ca2+, Zn2+ and Ni2+ traditionally considered as Ca2+ channel antagonists. Moreover, in the absence of exogenous divalent cations, low Ca2+ could result in an increase of transmitter release particularly if Mg2+ was omitted from the perfusing medium. These apparently paradoxical results can be reconciled with the postulates of the Ca2(+)-hypothesis of synaptic transmission by taking into account the effects of divalent cations on the fixed charges present at the external surface of cell membrane. It is possible that a similar interpretation could also account for the so-called "Ca2(+)-independent" transmission in other structures of the nervous system.

Calcium-independent synaptic transmission: artifact or fact?

The release of neurotransmitters at classical chemical synapses occurs via Ca2+ influx through voltage-dependent Ca2+ channels, which are opened following depolarization of presynaptic terminals. However, owing to a persistence or increase in the amount of transmitter released in preparations containing low concentrations of Ca2+, it has been proposed that transmitter release could also occur through a Ca(2+)-independent, carrier-mediated process. On the other hand, lowering extracellular [Ca2+] can actually promote Ca2+ influx through voltage-activated Ca2+ channels via a modification of the surface potential of plasma membranes. Therefore, the proposed Ca(2+)-independent transmitter release could be re-accommodated within the framework of the Ca2+ hypothesis of synaptic transmission by taking into account the surface-charge effects.

Low-calcium-induced enhancement of chemical synaptic transmission from photoreceptors to horizontal cells in the vertebrate retina.

According to the classical calcium hypothesis of synaptic transmission, the release of neurotransmitter from presynaptic terminals occurs through an exocytotic process triggered by depolarization-induced presynaptic calcium influx. However, evidence has been accumulating in the last two decades indicating that, in many preparations, synaptic transmitter release can persist or even increase when calcium is omitted from the perfusing saline, leading to the notion of a "calcium-independent release" mechanism. Our study shows that the enhancement of synaptic transmission between photoreceptors and horizontal cells of the vertebrate retina induced by low-calcium media is caused by an increase of calcium influx into presynaptic terminals. This paradoxical effect is accounted for by modifications of surface potential on the photoreceptor membrane. Since lowering extracellular calcium concentration may likewise enhance calcium influx into other nerve cells, other experimental observations of "calcium-independent" release may be reaccommodated within the framework of the classical calcium hypothesis without invoking unconventional processes.

The peripheral-type benzodiazepine receptor ligands [3H]Ro 5-4864 and [3H]PK 11195 bind to the retina of rabbit, but not of turtle.

The binding of [3H]flunitrazepam, [3H]Ro 5-4864, and [3H]PK 11195 to membrane preparations of the retina was studied in the turtle and rabbit. Only a single population of [3H]flunitrazepam binding sites was detected in the turtle, whereas two populations appeared to be present in the rabbit. No specific binding for [3H]Ro 5-4864 and [3H]PK 11195 could be detected in the turtle. In rabbit, both ligands bound with high affinity, revealing a significant population of binding sites (KD values of 24 +/- 2.3 and 2.2 +/- 0.8 nM, and Bmax values of 440 +/- 35 and 1,482 +/- 110 fmol/mg of protein, respectively). The binding was temperature- and protein-dependent. Displacement studies showed a similar rank order of potency of various unlabeled ligands against both [3H]Ro 5-4864 and [3H]PK 11195 (PK 11195 > Ro 5-4864 > flunitrazepam > flumazenil). These results suggest that peripheral-type benzodiazepine receptors are present in the retina of the rabbit, but not of the turtle.

Dihydropyridine-sensitive calcium current mediates neurotransmitter release from bipolar cells of the goldfish retina.

The release of neurotransmitter is evoked by activation of the Ca current (ICa) at presynaptic terminals. Though multiple types of ICa have been reported in various cells, little is known about the properties of presynaptic ICa in the vertebrate CNS. The aim of this article is to identify the type of ICa involved in the release of neurotransmitter from retinal bipolar cells. Bipolar cells with a large axon terminal were isolated enzymatically from the goldfish retina, and studied by the following techniques: (1) recordings of ICa in the whole-cell recording configuration, (2) visualization of intracellular free Ca2+ concentration ([Ca2+]i) with the Fura-2 imaging system, and (3) real-time electrophysiological bioassay of released excitatory amino acid transmitter by a voltage-clamped horizontal cell isolated from the catfish retina. The only ICa found in bipolar cells was the high-voltage-activated, dihydropyridine-sensitive type. This result supports the recent study by Heidelberger and Matthews (1992). When ICa was activated by a short depolarizing pulse, a rapid increase of [Ca2+]i was restricted to the axon terminal. A much slower and smaller increase of [Ca2+]i was sometimes observed at the cell body, probably due to the diffusion of intracellular free Ca2+ from the axon terminal. The increase of [Ca2+]i was completely suppressed by nicardipine, suggesting that Ca2+ entered through dihydropyridine-sensitive Ca channels located mainly at the axon terminal. Activating ICa of the bipolar cell evoked a transmitter-induced current in the excitatory amino acid probe (i.e., the catfish horizontal cell). Both currents were suppressed concomitantly by nifedipine but not by omega-conotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of the benzodiazepine ligand [3H]-RO 15-1788 to membrane preparations of the rabbit and turtle retina.

1. We have studied the binding of [3H]-RO 15-1788 to membrane preparations of the retina of rabbit (Lepus cunicula) and turtle (Pseudemys scripta elegans). 2. In both species, [3H]-RO 15-1788 binding was maximal at 0 degrees C and decreased with increasing temperature. It was saturable, protein concentration-dependent and specific. Flunitrazepam, unlabelled RO 15-1788 and ethyl-beta-carboline were the most effective displacers, whereas RO 5,4864 was ineffective. 3. In both turtle and rabbit retina, Scatchard analysis indicated the presence of a single binding site for [3H]-RO 15-1788. The KD was 0.75 nM in both turtle and rabbit, while the Bmax were 520 and 250 fmol/mg protein in turtle and rabbit respectively. A study of the association rate of [3H]-RO 15-1788 binding revealed faster kinetics in turtle, as compared to rabbit.

Involvement of D1 and D2 Dopamine Receptors in the Control of Horizontal Cell Electrical Coupling in the Turtle Retina.

We studied the actions of D1 and D2 dopamine agonists and antagonists on the coupling of horizontal cell axons in the turtle retina by a combination of pharmacological and electrophysiological methods. Both D1 and D2 receptors were identified in membrane fractions by radioligand binding using [3H]-SCH 23390 and [3H]-spiperone, respectively. The KD of both receptor classes were identical (0.21 nM) but D1 receptor density exceeded that of D2 receptors by more than four-fold. D1 agonists increased the activity of adenylate cyclase in a dose-dependent manner, whereas D2 agonists were without significant effect by themselves, nor did D2 antagonists block the D1-mediated increase in adenylate cyclase activity. Intracellular recordings and Lucifer Yellow dye injections were used to characterize the modifications of the receptive field profile of horizontal cell axons (H1AT) exposed to different pharmacological agents. Dopamine or D1 agonists (0.05 - 10 microM) induced a marked constriction of the H1AT receptive field, whereas D2 agonists elicited a small expansion of the receptive field. However, in the presence of a D1 antagonist, as well as IBMX to inhibit phosphodiesterase, D2 agonists (10 - 70 microM) induced a marked increase in the receptive field profile. These results indicate that both D1 and D2 dopamine receptors play a role in shaping the receptive field profile of the horizontal cell axon terminal in the turtle retina.

Binding of [3H] muscimol to the retina of rabbit and turtle.

1. We studied the binding of [3H]muscimol to membrane preparations of the retina of rabbit (Lepus cuniculus) and turtle (Pseudemys scripta elegans). 2. In both species, [3H]muscimol binding was maximal at 0 degrees C and decreased with increasing temperature, it was saturable, protein concentration dependent and specific. Muscimol, GABA and bicuculline were the most effective displacers, whereas baclofen and diaminobutyric acid were ineffective. 3. In the turtle retina, Scatchard analysis indicated the presence of a single site with a KD of 20.81 nM, and a Bmax of 3.620 pmol/mg prot. 4. In the rabbit, a single site could be identified in the nanomolar concentration range (KD of 12.8 nM, Bmax of 1.327). A study of the association rate of [3H]muscimol binding revealed a faster kinetics in turtle, as compared to rabbit.