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H Korn

Publications and source records attributed to H Korn.

At least 73 records · Page 4Linked to original sources
PubMed1992

Mechanism of 4-aminopyridine action on voltage-gated potassium channels in lymphocytes.

The mechanism by which 4-aminopyridine (4-AP) blocks the delayed rectifier type potassium (K+) channels present on lipopolysaccharide-activated murine B lymphocytes was investigated using whole-cell and single channel patch-clamp recordings. 4-AP (1 microM-5 mM) was superfused for 3-4 min before applying depolarizing pulses to activate the channel. During the first pulse after application of 4-AP above 50 microM, the current inactivated faster, as compared with the control, but its peak was only reduced at high concentrations of 4-AP (Kd = 3.1 mM). During subsequent pulses, the peak current was decreased (Kd = 120 microM), but the inactivation rate was slower than in the control, a feature that could be explained by a slow unblocking process. After washing out the drug, the current elicited by the first voltage step was still markedly reduced, as compared with the control one, and displayed very slow activation and inactivation kinetics; this suggests that the K+ channels move from a blocked to an unblocked state slowly during the depolarizing pulse. These results show that 4-AP blocks K+ channels in their open state and that the drug remains trapped in the channel once it is closed. On the basis of the analysis of the current kinetics during unblocking, we suggest that two pathways lead from the blocked to the unblocked states. Computer simulations were used to investigate the mechanism of action of 4-AP. The simulations suggest that 4-AP must bind to both an open and a nonconducting state of the channel. It is postulated that the latter is either the inactivated channel or a site on closed channels only accessible to the drug once the cell has been depolarized. Using inside- and outside-out patch recordings, we found that 4-AP only blocks channels from the intracellular side of the membrane and acts by reducing the mean burst time. 4-AP is a weak base (pK = 9), and thus exists in ionized or nonionized form. Since the Kd of channel block depends on both internal and external pH, we suggest that 4-AP crosses the membrane in its nonionized form and acts from inside the cell in its ionized form.

4-Aminopyridine↗
PubMed1992

Partial glycinergic denervation induces transient changes in the distribution of a glycine receptor-associated protein in a central neuron.

The effect of partial glycinergic denervation on the cellular distribution of the 93 kDa peripheral polypeptide associated with the glycine receptor was studied at the level of the teleost Mauthner cell, an identified neuron of the goldfish brain (Carassius auratus). Previous studies using monoclonal antibodies raised against purified glycine receptors and immunoperoxidase staining have shown that these proteins are localized in clusters on the entire surface of this neuron. Specifically, the 93 kDa polypeptide was situated only on the cytoplasmic side of the postsynaptic membrane facing active zones. Unilateral electrolytic lesions of the vestibular complex caused the degeneration of some glycinergic afferents to this neuron. When the first signs of this response appeared, 3 d after the surgery, there was also a change in the ultrastructural distribution of the 93 kDa polypeptide in the deafferented cell. The synaptic protein apposed to degenerating axons did not spread onto adjacent extrasynaptic membranes, and it disappeared a few hours after the disruption of its presynaptic element. At the same time, a cytoplasmic immunoreactivity appeared as randomly distributed clusters in the deafferented Mauthner cell; these aggregates, not seen in control preparations, were never found inside membrane-bound organelles. In some preparations these clusters were localized along arrays at a relatively constant distance from the plasma membrane. The intracellular immunoreaction product was found in the soma and the initial part of the dendrites, gradually decreasing in number and intensity toward the extremities of these processes. At later postoperative stages, 10-15 d after surgery, the 93 kDa immunoreactivity remained only at postsynaptic membranes facing intact terminals. Similar alterations following denervation were observed in reticular neurons, at the level at which degenerating presynaptic terminals were also detected. In contrast, continuous 3-d blockade of synaptic transmission by strychnine, an antagonist of the glycine receptor, had no effect on either the distribution of the surface receptor clusters, or the 93 kDa peripheral protein linked to these receptors. Taken together, our results suggest that the ultrastructural distribution of the glycine receptor complex is regulated by "trophic" factors rather than by transmitter-evoked synaptic activity.

Afferent Pathways↗
PubMed1991

Differential distribution of GABA- and serotonin-containing afferents on an identified central neuron.

The distributions of gamma-aminobutyric acid (GABA)- and serotonin (5-HT)-containing terminals impinging on the surface of the Mauthner (M-) cell were studied at the light microscopic level using double immunofluorescent labeling and were compared with that of the glycine receptor. The latter was visualized indirectly, using a monoclonal mouse antibody which recognizes its 93-kDa associated protein. This neuron has two large principal dendrites: one extending ventrorostrally (ventral dendrite) and the other dorsolaterally (lateral dendrite). There are also two other classes of smaller processes: one that projects ventrally (small ventral dendrites) and one penetrating in the axon cap (cap dendrites), a peculiar neuropil surrounding the initial segment of the M-cell axon. A cellular regionalization of these afferent systems was found: GABA boutons, labeled for glutamic acid decarboxylase (GAD), were localized preferentially on the lateral dendrite while 5-HT-filled endings predominated on the ventral one. The density of these two classes of inputs was comparable in the other areas of the M-cell: less of their terminals were in contact with the soma outside the axon cap, and more numerous boutons, which presented either GABA or 5-HT immunoreactivities, were apposed to the small ventral dendrites. This preferential pattern of innervation differed with the ubiquitous presence of glycine receptor clusters on the M-cell membrane. Finally no evidence of a colocalization of GABA and 5-HT in afferent endings was detected at any portion of the M-cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Quantal analysis and synaptic efficacy in the CNS.

Quantal analysis of synaptic transmission at connections between neurons in the CNS has provided insights concerning the structural constraints on transmitter release and postsynaptic responsiveness. However, it has proven difficult in many cases to resolve the size and variability of a single quantum or to distinguish clear peaks in amplitude histograms of evoked responses, due in part to the superposition of background instrumental and biological noise. These limitations raise questions about recent attempts to use direct or indirect methods of quantal analysis in order to distinguish between pre- and postsynaptic loci of the modifications underlying long-term potentiation, particularly since the interpretations are model-dependent and the statistical treatments and experimental techniques employed incorporate simplifying assumptions not yet proven.

Animals↗
PubMed1991

A monoclonal antibody raised against the Mauthner cell also recognizes some reticular neurons.

A monoclonal antibody was raised against dissected Mauthner cells of goldfish, Carassius auratus. The immunoglobulin (mAb 222C2) recognized in this neuron a determinant that was localized on the soma of the Mauthner cell in front of the axon hillock and on the dorsomedial portion of the initial third of its ventral dendrite. When observed with electron microscopy, the staining was associated with polyribosomes and with the reticulum, close to the Golgi cisternae. The antibody also labelled other large neurons (10-40 microns) of the nuclei reticularis superior, medialis and inferior. In these cells, patchy immunolabelled elements could be detected, dispersed within cytoplasm. They did not exhibit the characteristic topological distribution observed in the Mauthner cell. On the basis of their size and location, this group of neurons may send axons to the spinal cord. No staining was observed in other areas of the brainstem, or in other structures such as the cerebellum or the optic tectum. The expression of this antigenic molecule in Mauthner and reticular cells suggests that these two sets of neurons are functionally and/or ontogenetically related. Although the molecular and functional characteristics of the antigenic molecule have not been determined, this antibody should be a useful marker for further developmental studies.

Animals↗
PubMed1991

Applicability of the coefficient of variation method for analyzing synaptic plasticity.

The classical coefficient of variation method for "quantal" analysis of synaptic responses allows unambiguous identification of pre- and postsynaptic loci underlying synaptic plasticity only when extensive simplifying restrictions are made. They include invariance of quantal parameters and the assumption that a single afferent produces the evoked potentials or currents. More general theoretical formulations and simulations demonstrate that the standard criteria do not always provide useful guidelines because when the other sources of physiological variance are included, putative pre- and postsynaptic domains may overlap. For example, data typically interpreted as indicating modifications at both sites can be due to a mechanism localized to only one of the two, if parameter variances are taken into consideration in the case of a single input cell, or if there are multiple inputs and the stimulus does not activate all of them reliably. With this perspective, other physiologically realistic hypotheses relevant to the expression of synaptic plasticity, such as that during long-term potentiation, can be envisioned.

Animals↗
PubMed1991

Silent synaptic connections and their modifiability.

Comparison of the two afferent systems illustrates certain features common to synaptic transmission as well as differences that might be important for synaptic plasticity. Transmission at both the inhibitory and excitatory connections is satisfactorily described by a simple binomial model that considers the average probability of release to be the same at each active site, although it should be stressed that the best evidence derives from the first set of afferents. Another similarity between the two systems is that short-term changes in synaptic efficacy, namely, facilitation and depression, appear to be due to changes in p. We previously suggested that both phenomena occur during repetitive stimulation, with the dominant effect depending upon the initial probability of release. It remains to be seen if depression dominates at other inhibitory connections, although it is already clear that one cannot generalize about excitation, because some excitatory junctions have an initial high p and exhibit a marked depression rather than the facilitation described here. We have found no evidence for the notion that some synapses within a connection may be silent. That idea has been proposed, but not proven, for other synaptic connections in the vertebrate central nervous system. Indeed, it will be difficult to assess as long as quantal release cannot be reliably detected at these junctions, and morphological confirmation at the ultrastructural level will also be required. On the other hand, evidence from a few peripheral junctions where one presynaptic afferent establishes hundreds of contacts with its target cell, does suggest the possibility of silent synapses, or at least an extremely low probability of release in those cases. These situations may correspond to extremes of our finding that as the number of release sites increases, p decreases. Regardless, the inverse relation between n and p suggests caution should be exercised in interpreting data indicating that synaptic plasticity is associated with increased numbers of synapses between two cells. Although we have not detected silent synapses within a transmitting connection, we have observed chemically silent connections between neurons, and the evidence reviewed here suggests transmission may be blocked postsynaptically, as with the inhibitory connections, or presynaptically, as with the excitatory ones. Although the underlying mechanisms are only partially elucidated, it is also clear that such connections can be switched into a transmitting mode. Consequently, they may provide a significant reserve that might well become functional in different behavioral states or in response to certain patterns of activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Role of medullary networks and postsynaptic membrane properties in regulating Mauthner cell responsiveness to sensory excitation.

A benefit of studying well-defined networks at a cellular level is that it might be possible both to place these details in the context of the specific function of the network and to extract general principles applicable to more complex systems. The Mauthner cell system in teleosts is one such vertebrate network where a single impulse can trigger a vital escape reaction, the C start, in response to auditory or visual stimuli. We review here experiments concerned with the organization, at the cellular level, of the afferent circuits impinging on the Mauthner cell and with certain intrinsic membrane properties of the Mauthner cell that contribute to shaping the threshold and expression of the C start. One concept that emerges is related to the interaction between excitatory and inhibitory drives to the Mauthner cell. It seems that every major afferent drive to this neuron also excites a feedforward inhibitory network which, in turn, exerts a major role in establishing and regulating the threshold of the escape response. This design feature is complemented by the Mauthner cell's membrane properties which contribute to the behavioral threshold but exhibit nonlinearities, as excitation begins to overcome inhibition. Finally, we have compared in detail the frequency-dependent characteristics of inhibition and excitation, as revealed by studies of individual identified synaptic connections. This comparison emphasizes the notion that although inhibition is maximized for weak transient stimuli, it becomes depressed at auditory stimulus frequencies that facilitate excitatory transmission and evoke the escape response.

Acoustic Stimulation↗
PubMed1991

Use of confocal microscope for the cellular analysis of the glycine synaptic receptor.

The presence of glycine receptors was examined with a monoclonal antibody and indirect immunofluorescence on reticular neurons of the goldfish (Carassius auratus) brainstem. Images of thin (0.6 microns) optical sections were recorded from 80 microns thick specimen with a confocal microscope thus obviating the need for mechanical slicing. Due to the reduced out-of-focus noise, high resolution was obtained. Lookthrough projections were computer generated. Compared with classical methods involving serial sectioning, this approach allowed the analysis of the subcellular distribution of this receptor with a considerable gain of time and increased resolution. On the Mauthner cell, an identified reticulo-spinal neuron, we found, that the size of glycine receptor microdomains varies depending on the cellular localization, i.e. somatic or dendritic. Furthermore the intensity of fluorescence was uneven within individual clusters, probably reflecting differences in receptor concentration. These heterogeneities may influence the variance of synaptic inhibitory noise in different regions of the Mauthner cell.

Animals↗
PubMed1991

Immunohistochemical localization of glycine receptors and a linked polypeptide in the goldfish brain.

Three monoclonal antibodies were used to examine with immunocytochemistry the distribution of the glycine receptors and the related 93kd polypeptide in the adult goldfish brain (Carassius auratus). One immunoglobulin recognizes the 48kd strychnine-binding subunit of the receptor and the two others bind to the peripheral 93kd polypeptide which is coupled to the receptor molecules. Immunofluorescent spots were visualised with all three antibodies on the somatic and dendritic membrane of the cells. A differential intensity of immunofluorescence was detected in the three different brain regions examined: the brainstem, the cerebellum and the telencephalon. For both proteins, the highest fluorescence was observed in the brainstem, particularly on reticular and vestibular neurons. In the cerebellum both the density and the intensity of labelling was low. At the level of the Mauthner cell, an identified bulbar command neuron, the distribution of glycinergic receptors was identical with that of the 93kd polypeptide. Both subunits were visualized on the somatic and dendritic membrane up to their extremities. These proteins appear to be co-localized in neurons other than the Mauthner cell, since they are always co-expressed in the same brain region. Their distributions are comparable with that observed in the mouse and rat nervous system as reported from autoradiographic localization of [3H]strychnine binding.

Animals↗
PubMed1991

Generation of synaptic noise: selective involvement of neuronal subsets.

All central neurons are subjected to continuous and random variations of their membrane potential because of "spontaneous" activity in their presynaptic afferents. This activity, which is called synaptic noise, is presumed to be responsible for the uncertainty of the input-output relation in these cells. In the Mauthner cell of teleosts, noise is mainly inhibitory, and is generated by the release of neurotransmitter in a probabilistic manner. This inhibitory activity has been studied in detail previously. Taking advantage of this understanding, we have constructed a model of the inhibitory networks and their target in order to determine the conditions required to reproduce the main stochastic aspects of synaptic noise. We have used a combination of computer simulations and simple semianalytical arguments. We conclude that, surprisingly, cells in the presynaptic networks do not contribute equally to these background fluctuations. Rather, noise is generated primarily by the operation of subsets of afferent cells: the spectrum is either dominated by signals originating from interneurons which make few terminals on the Mauthner cell, or by the output of "burster" cells firing spike trains rather than single spikes. Both possibilities lead to specific predictions, one of which has already been verified.

Analysis of Variance↗
PubMed1991

Serotonergic facilitation of quantal release at central inhibitory synapses.

The teleost Mauthner (M)-cell is subjected to a powerful glycinergic inhibition that regulates its threshold for initiation of a vital escape reflex. The effects of 5-HT on this inhibition were studied in current- and voltage-clamp experiments because the M-cell and its afferents have a profuse serotonergic innervation, including axoaxonal contacts with terminals of the inhibitory interneurons. Local applications of the amine and/or of its uptake blockers markedly enhanced inhibitory currents evoked by synchronous activation of two identified presynaptic networks. In order to determine the site of this 5-HT action, we exploited the fact that in the M-cell, synaptic noise is predominantly inhibitory. Furthermore, its quantal components can be resolved providing a means to distinguish pre- and post-synaptic loci underlying variations in synaptic efficacy. As in other cell types, it consists of spontaneous exocytotic events and multiquantal responses triggered by action potentials in afferent neurons. 5-HT or its uptake blockers produced a long-lasting enhancement of this noise, manifested as a shift to the right of amplitude distribution histograms of the individual inhibitory postsynaptic currents (IPSCs) and an increase in the mean quantal content of noise. In contrast, the size of the miniature IPSC (mIPSCs) remained constant in these experiments and in another series where 5-HT was given in presence of TTX. In this case, the amine also increased the rate of occurrence of single quanta. Taken together, these data indicate that 5-HT acts presynaptically to increase the probability of evoked and spontaneous release of glycine. This conclusion was reinforced by evidence that the overall frequency of the responses in noise could be unaltered by 5-HT, indicating that the afferent firing pattern had remained constant. Extracellular recordings of the presynaptic volleys showed that action potentials were not prolonged and suggested that 5-HT closes K+ channels in the terminal membrane, leading to an increase of resistance and a larger depolarization in that region as a possible mechanism for the facilitation of release. While the enhancement of transmitter release persisted for 20 min or more, there was also a transient induction of a K+ inward-rectifying current in the M-cell. These results indicate that 5-HT increases the efficacy of inhibitory synaptic transmission in the vertebrate CNS by a presynaptic action.

Animals↗
PubMed1990

Long-term potentiation of electrotonic coupling at mixed synapses.

Long-term potentiation of chemical synapses is closely related to memory and learning. Studies of this process have concentrated on chemically mediated excitatory synapses. By contrast, activity-dependent modification of gap junctions, which also widely exist in higher structures such as hippocampus and neocortex, has not been described. Here we report that at mixed synapses between sensory afferents and an identified reticulospinal neuron, the electrotonic coupling potential can be potentiated, as well as the chemically mediated excitatory postsynaptic potential, for a prolonged time period using a stimulation paradigm like that which produces long-term potentiation in hippocampus. The effect on coupling is due to an increase in gap-junctional conductance. Our data indicate that the potentiation of both synaptic components requires an increase in intracellular calcium, involves activation of NMDA (N-methyl-D-aspartate) receptors, and is specific to the tetanized pathway.

Afferent Pathways↗
PubMed1990

Ion channel blockers inhibit B cell activation at a precise stage of the G1 phase of the cell cycle. Possible involvement of K+ channels.

Lymphocytes express voltage-activated K+ channels in their membrane. Combining the patch-clamp techniques of recording with immunological methods, we have analyzed the expression and the involvement of these channels during defined steps of LPS-induced B cell activation. We show that the number of K+ channels increased strongly when B cells entered in the G1 phase of the cell cycle. The involvement of ion channels in B cell proliferation was assessed using channel blockers that inhibit the K+ current. It was first found that TEA, but not TMA, quinine and verapamil totally suppressed both K+ current and DNA synthesis by stimulated lymphocytes as measured by [3H]TdR uptake or propiedium iodide staining. The drugs affected neither the induction by LPS of activation markers such as Ag of the murine class II MHC and type II receptor for the Fc region of IgG nor the initial cell enlargement that occur early during activation. These data indicate that functional K+ channels are not essential for the transition from the G0 to the G1 phases. In contrast, the same channel antagonists blocked the induction of transferrin receptor expression, characteristic of the final stages of G1. These drugs acted on cells already in G1, because their addition 30 h after LPS still suppressed DNA synthesis, and because they inhibited the proliferation of purified B cell blasts. The effect of tetraethylammonium was reversible, a lag period of 12 h occurring before the cells start DNA synthesis after drug removal. Taken together, these data demonstrate that the proliferation of LPS-stimulated B cells requires functional ion channels at a critical period in the G1 phase, taking place before transferrin receptor expression and the entry into the S phase. The involvement of voltage dependent K+ channels at this particular point is suggested by the parallel effects of the drugs used on K+ currents and DNA synthesis.

4-Aminopyridine↗
PubMed1990

Differential distribution of serotoninergic inputs on the goldfish Mauthner cell.

The morphology and distribution of the serotoninergic (5-HT) input to the Mauthner cell (M cell) of a teleost, Carassius auratus, were analyzed at the light microscopic level. Immunohistochemical methods revealed that 1) most fibers innervating the M cell originate from the ventral and lateroventral regions of the rhombencephalon; 2) two groups of fibers contribute to this innervation, thick ones (type I, 0.4-0.7 microns in diameter) with terminal endings and thin ones (type II, less than 0.2 microns) that issue numerous beaded varicosities 4-10 microns from the target cell and only occasional side endings contacting it; 3) the density of immunoreactive profiles is uneven over the whole cell and predominates on the ventral dendrite; and 4) the two sets of axons, although overlapping, do not have the same distribution. Specifically, both classes are present on the ventral dendrite, whereas type II fibers are the only ones observed on the soma, in the region of the initial segment of the axon, and in the vicinity of the lateral dendrite. Functionally identified inputs on the M cell also have a regionalized distribution, depending, for example, on whether they belong to excitatory or inhibitory networks. Thus we propose that 5-HT inputs have specific influences that are a function of their respective localization.

Animals↗
PubMed1990

Ion channels and B cell mitogenesis.

Given the presence of ionic channels at the membrane of lymphocytes, we have analyzed the effect of various channels blockers on B lymphocytes activation. TEA and 4-AP, two K+ channels blockers, quinine, a blocker of Ca2(+)-activated K+ channels, nickel and verapamil, two Ca2+ channels blockers, all inhibited LPS-induced B cell proliferation. However, these drugs neither inhibited the induction of Ia and Fc gamma RII expression nor cell enlargement and early RNA synthesis, indicating that the entry of B lymphocytes into G1 phase was not affected. In contrast, both late RNA synthesis and the induction of the TfR, which occur while the cell progress through G1, were inhibited by these blockers. These data show that TEA, quinine and verapamil block B lymphocyte activation during the G1 phase, probably between G1A and G1B. To question whether these effects were due to the block of voltage-activated K+ channels, we compared the ability of TEA, quinine, verapamil, 4-AP and nickel to block proliferation and K+ channels. A striking correlation was found for all the drugs but less for 4-AP. Moreover, TMA, a TEA analog unable to block K+ currents, did not affect B cell proliferation. Taken together, our data suggests that functional voltage-gated K+ channels are required at a precise stage of the G1 phase of the B cell cycle.

Animals↗