Repetitive firing triggers clustering of Kv2.1 potassium channels in Aplysia neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.     

Developmental uncoupling between blastoderm and yolk cell in the embryo of the teleost Fundulus

During cleavage and blastula stages of embryos of the teleost Fundulus heteroclitus all of the cells are both electotonically coupled and dye coupled to one another, as determined by microelectrode impalements and spread of Lucifer Yellow. At about the time that gastrulation begins we observed a specific loss of junctional coupling between the yolk cell and cells of the blastoderm. Passage of Lucifer Yellow between the yolk cell and blastoderm was reduced at stage 12 (late blastula), and not detected at stage 13 and thereafter, although cells of the blastoderm remain dye coupled to one another through gastrula stages. Also, junctional electrical coupling between the yolk cell and blastoderm became substantially reduced at stage 13 and thereafter. The loss of coupling at this specific cell apposition and time and the large size of the yolk cell may prove useful in analyzing the underlying cellular mechanisms.     

Similarities and differences in the structure of segmentally homologous neurons that control the hearts in the leech,Hirudo medicinalis

Summary The neural circuit that controls the hearts in the leech comprises an ensemble of synaptically interconnected cardiac motor neurons (HE cells) and cardiac interneurons (HN cells). Both the HE cells and the HN cells constitute segmentally homologous sets. We have investigated the structure of these neurons by iontophoretic injection of Lucifer Yellow dye.Bilateral pairs of HE cells have been identified in segmental ganglia 3–19 of the nerve cord. Their structure was found to be nearly identical from ganglion to ganglion and from animal to animal.Bilateral pairs of HN cells have been identified in segmental ganglia 1–7 of the nerve cord. Their dendritic structure was found to vary from ganglion to ganglion. These segmental differences among HN cells were observed consistently from animal to animal. Some of the segmental differences in HN cell structure correlate with previously described physiological differences.     

Functional and morphological evidence for the existence of neurites from abdominal ganglion bag cell neurons in the head-ring ganglia of Aplysia

Summary Three lines of evidence are presented indicating that axons of the Aplysia neuroendocrine bag cells extend into the head-ring ganglia of the CNS. When the abdominal ganglion was bisected longitudinally, separating the two bag cell clusters, an afterdischarge induced in one cluster generated an afterdischarge in the other via activity through the head-ring ganglia to which each half abdominal ganglion was attached by connective nerves. This suggests that some axons of bag cells in each cluster communicate through the head-ring ganglia. Retrograde labelling of bag cells occurred when rhodamine-onjugated latex microspheres were injected into the cerebral or either pleural ganglion, a direct demonstration that bag cell axons extend into these ganglia. Finally, cell LP1 in the left pleural ganglion was inhibited during a bag cell afterdischarge, an action mimicked by application of alpha-bag cell peptide (BCP). Since BCP can act only close to its site of release due to susceptibility to peptidase activity, it is likely that LP1 inhibition is dependent on the local release of BCP from bag cell neurites in the pleural ganglion. These results open new possibilities for how bag cell afterdischarges may be initiated and broaden the distribution of their effects.Abbreviations ASW artificial sea water; -BCP -bag cell peptide - ELH egg-laying-hormone - IR immunorective - PB phosphate buffer - PVC pleurovisceral connective     

The distribution of acetylcholine receptor clusters and sites of transmitter release along chick ciliary ganglion neurite-myotube contacts in culture

Acetylcholine receptors accumulate along the length of cholinergic neuron-skeletal muscle contacts in vitro. The main purpose of this study was to describe, in a quantitative way, the distribution of acetylcholine receptor clusters induced by ciliary ganglion neurons over a period of time extending from hours to weeks after contacts are established. Neurites were filled with Lucifer Yellow and receptor clusters were identified with rhodamine-bungarotoxin. A cluster located within 5 micron of a nerve process or 10 micron of the base of a growth cone was considered to be a neurite-associated receptor patch (NARP). The first synaptic potentials were evoked 20 min after growth cone-myotube contact, and, after 24 h of co-culture, greater than 60% of the nerve-muscle pairs tested were functionally connected. NARPs appear rapidly; the first clusters were detected approximately 6 h after the neurons were plated. They were composed of several small subclusters or speckles of rhodamine-bungarotoxin fluorescence. The initial accumulation of receptors may occur at the advancing tips of nerve processes because NARPs were found at greater than 80% of the growth cone-muscle contacts examined between 12 and 24 h of co-culture. Over the 3-wk period examined, the mean incidence of NARPs ranged between 1.0 and 2.6 per 100 micron of neurite-myotube contact, with the peak observed on the second day of co-culture. During the first 3 d in culture, when the neurons were multipolar, nearly all of the primary processes induced one or more clusters. With time, as the neurons become unipolar (Role and Fischbach, 1987) NARPs persisted along the remaining dominant process. Measurements made during the third day of co-culture suggest that NARPs disappear along shorter neurites before they retract. Synaptic currents were detected by focal extracellular recording at 55% of the NARPs. The fact that spontaneous or evoked responses were not recorded at 45% suggests that contacts with clusters exhibit two functional states. Two types of presynaptic specialization at identified NARPs observed by scanning electron microscopy appear to be correlated with the functional state.     

Variability and frequent failure of lucifer yellow to pass between two electrically coupled neurons in Lymnaea stagnalis

The electrically coupled giant neurosecretory neurons VD1 and RPD2 of Lymnaea stagnalis were found to have coupling coefficients ranging from ca. 0.1-0.6. When the fluoroescent dye Lucifer Yellow was injected intracellularly into one of the neurons, in most preparations no dye was observed to pass through into the coupled cell body or the process leading to it. There was no apparent correlation between the amount of dye coupling and the length of time allowed for diffusion of the dye in the cells. In eight preparations, the electrical coupling coefficient was measured before dye was injected. There was no correlation between dye coupling and the electrical coupling coefficient.     

Physiological and anatomical properties of Leydig cells in the segmental nervous system of the leech

Summary Each of the 21 segmental ganglia in the American leechMacrobdella decora contains a pair of Leydig cells (ca. 45 m) each of which is located in a posteriolateral glial packet. Leydig cells exhibit spontaneous action potentials (1–10/s) whose duration and undershoot depend upon membrane polarization. The two Leydig cells within each ganglion are bidirectionally-coupled (V ₂/V ₁0.3). Pairs of ipsilateral Leydig cells within adjacent ganglia are mutually excitatory such that an impulse in one generates an impulse in the other. The interganglionic latency for any cell pair is constant regardless of the direction of impulse conduction and is unchanged by 20 mM Mg²⁺ saline. These data indicate that the interactions are not mediated by chemical synapses. Additionally, the results of collision experiments lead us to infer that ipsilateral Leydig cell pairs utilize common axonal pathways for interganglionic interactions. If Leydig cells are driven by current injection to fire impulses at frequencies of six to ten per s, cells in adjacent ganglia exhibit impulse failures. The combination of spontaneous activity, intraganglionic coupling and interganglionic interactions results in the generation of constant, low frequency impulse activity and can cause impulse reverberations.The branching pattern of Leydig cells filled with HRP is consistent with their functional properties and connectivity. Each cell sends axons to both adjacent ganglia through the ipsilateral connectives and projects to the periphery only by the lateral roots of these adjacent ganglia. This unusual morphology was verified Lucifer Yellow CH.In addition to intraganglionic dye-coupling, dye coupling was occasionally evident between ipsilateral cells in adjacent ganglia. Electron microscopy of Leydig cells depicts abundant 100 nm granules in both their somata and neuropilar processes. Although this fine structure suggests a neurosecretory role, we were unable to discern a peripheral function for these neurons.Abbreviation H R P horseradish peroxidase     

Changing patterns of ganglion cell coupling and connexin expression during chick retinal development

We have used dye injection and immunolabeling to investigate the relationship between connexin (Cx) expression and dye coupling between ganglion cells (GCs) and other cells of the embryonic chick retina between embryonic days 5 and 14 (E5-14). At E5, GCs were usually coupled, via soma-somatic or dendro-somatic contacts, to only one or two other cells. Coupling increased with time until E11 when GCs were often coupled to more than a dozen other cells with somata in the ganglion cell layer (GCL) or inner nuclear layer (INL). These coupled clusters occupied large areas of the retina and coupling was via dendro-dendritic contacts. By E14, after the onset of synaptogenesis and at a time of marked cell death, dye coupling was markedly decreased with GCs coupled to three or four partners. At this time, coupling was usually to cells of the same morphology, whereas earlier coupling was heterogeneous. Between E5 and E11, GCs were sometimes coupled to cells of neuroepithelial morphology that spanned the thickness of the retina. The expression of Cx 26, 32, and 43 differed and their distribution changed during the period studied, showing correlation with events such as proliferation, migration, and synaptogenesis. These results suggest specific roles for gap junctions and Cx's during retinal development.     

Hyperosmotic media inhibit voltage-dependent calcium influx and peptide release in Aplysia neurons

Summary The bag cell neurons of Aplysia provide a model system in which to investigate the effects of hyperosmolality on the electrical and secretory properties of neurons. Brief stimulation of these neurons triggers an afterdischarge of action potentials that lasts approximately 20–30 min, during which time they release several neuroactive peptides. We have found that pre-incubation of intact clusters of bag cell neurons in hyperosmotic media prior to stimulation prevents the initiation of afterdischarges. Furthermore, an increase in osmolality of the external medium during an ongoing afterdischarge causes its premature termination. Hyperosmotic media attenuate the release of peptide evoked by both electrically stimulated afterdischarges and potassium-induced depolarization. The ability of high potassium to depolarize the bag cell neurons is, however, not impaired. Exposure of isolated bag cell neurons to hyperosmotic media also inhibits the amplitude of action potentials evoked by depolarizing current injection and attenuates the voltage-dependent calcium current. In isolated bag cell neurons loaded with the calcium indicator dye, fura-2, hyperosmotic media reduced the rise in intracellular calcium levels that normally occurs in response to depolarization. Our results suggest that the effects of hyperosmotic media on peptide secretion in bag cell neurons can largely be attributed to their effects on calcium entry.This work was supported by NIH Grant NS-18492 to L.K. Kaczmarek.     

Morphology of identified neurosecretory and non-neurosecretory cells in the cockroach pars intercerebralis

Electrophysiologically identified cells of the cockroach pars intercerebralis (Periplaneta americana) were injected with the dye Lucifer Yellow for morphological examination and with horseradish peroxidase for ultrastructural marking. In addition to this, uninjected cells were also studied to elaborate the findings from the injected material. The two electrophysiologically distinct classes of cells (type I and type II) correspond to two distinct morphological and ultrastructural classes. Type I cells are the medial neurosecretory cells of the pars intercerebralis, which project their axons to the retrocerebral neuro-hemal complex. Their cell bodies have a mean diameter of 17 microns, and they contain neurosecretory granules 200 nm in diameter. Arborizations emanate from the axon in the anterior part of the protocerebral neuropil. The type II cell bodies are larger (38 microns in diameter). Their axons project into the contralateral circumesophageal connective. These cells were usually multipolar, having somatic arborizations in the anterior portocerebral neuropil. The cell bodies contain vesicles 40 nm in diameter, numerous trophospongia, and a multi-layered glial envelope.     

Dye coupling in the organ of Corti

Summary Dye-coupling in an in vitro preparation of the supporting cells of the guinea-pig organ of Corti was evaluated by use of the fluorescent dyes, Lucifer Yellow, fluorescein and 6 carboxyfluorescein. Despite the presence of good electrical coupling in Hensen cells (coupling ratios >0.6) the spread of Lucifer yellow was inconsistent. Hensen cells are very susceptible to photoinactivation, i.e., cell injury upon illumination of intracellular dye; and this in conjunction with Lucifer Yellow's charge and K+-induced precipitability may account for its variability of spread. Fluorescein and 6 carboxyfluorescein, on the other hand, spread more readily and to a greater extent than Lucifer Yellow, often spreading to cell types other than those of Hensen. Dye spread is rapid, occurring within a few minutes. These results suggest that molecules of metabolic importance also may be shared by the supporting cells of the organ of Corti.     

Sprouting and functional regeneration of an identified serotonergic neuron following axotomy

An identified serotonergic neuron (C1) in the cerebral ganglion of Helisoma trivolvis sprouts following axotomy and rapidly (seven to eight days) regenerates to recover its regulation of feeding motor output from neurons of the buccal ganglia. The morphologies of normal and regenerated neurons C1 were compared. Intracellular injection of the fluorescent dye, Lucifer Yellow, into neuron C1 was compared with serotonin immunofluorescent staining of the cerebral and buccal ganglia. The two techniques revealed different and complimentary representations of the morphology of neuron C1. Lucifer Yellow provided optimal staining of the soma, major axon branches, and dendritic arborization. Immunocytochemical staining revealed terminal axon branches on distant targets and showed an extensive plexus of fine fibers in the sheaths of ganglia and nerve trunks. In addition to C1, serotonin-like immunoreactivity was localized in approximately 30 other neurons in each of the paired cerebral ganglia. Only cerebral neurons C1 had axons projecting to the buccal ganglia. No neuronal somata in the buccal ganglia displayed serotonin-like immunoreactivity. Observations of regenerating neurons C1 demonstrated: Actively growing neurites, both in situ and in cell culture, displayed serotonin-like immunoreactivity; severed distal axons of C1 retained serotonin-like immunoreactivity for up to 28 days; axotomized neurons C1 regenerated to restore functional control over the feeding motor program.     

Physiological and morphological evidence for coupling in mouse salivary gland acinar cells

Three experimental techniques were employed to examine coupling between acinar cells of the mouse salivary gland. Passage of DC current pulses via intracellular microelectrodes between neighboring cells showed that small ions could be directly passed from one cell to another. Intracellular iontophoresis of the dye Lucifer Yellow CH into a single cell indicated that small molecules could spread by means of intercellular cytoplasmic bridges througout an acinus and, occasionally, into cells of adjacent acini. Freeze-fracture replicas of acinar cell membranes indicated the presence of gap junctions which were correlated with both electrical and dye coupling experiments. Suggestions are made for the function of direct intercellular exchange in salivary secretory cells. The role of electrical coupling in coordination of the activity of different secretory cell types is discussed as one possible function.     

Angiotensin (1–7) re-establishes heart cell communication previously impaired by cell swelling: Implications for myocardial ischemia

The influence of hypertonic solution on dye coupling was investigated in cell pairs isolated from the left ventricle of adult Sprague Dawley rats.The hypertonic solution together with Lucifer Yellow CH, were dialyzed into one cell of the pair using the whole cell clamp tecnique, and the diffusion of dye in the dialyzed as well as in non-dialyzed cell, was followed by measuring the intensity of fluorescence in both cells as a function of time.The results indicated that: (1) Lucifer Yellow CH dialyzed into one cell of the pair diffuses easily into the nondialyzed cell through gap junctions; (2) the intracellular dialysis of an hypertonic solution into one cell of the pair, increases the area of the dialyzed cell and reduced the area of the non-dialyzed cell suggesting intercellular movement of water; (3) the hypertonic solution dialyzed into one cell of the pair abolished the dye coupling; (4) the gap junction permeability (Pj) estimated before and after administration of hypertonic solution showed an appreciably decrease of Pj; (5) angiotensin (1–7) (Ang (1–7) (10–9 M) administered to the bath re-established the dye coupling abolished by hypertonic solution and reduced the cell area; (6) the effect of Ang (1–7) was related to the activation of Mas receptor and was dependent on the activation of PKA. Conclusions: the reestablishment of dye coupling elicited by Ang (1–7) seen in cell pairs dialyzed with hypertonic solution, might indicate that under similar conditions like that seen during myocardial ischemia, the peptide might be of benefit preventing the impairment of cell communication and impulse propagation associated with cardiac reentrant arrhytmias.     

Reduced junctional permeability at interrhombomeric boundaries.

Intercellular communication is considered to have a role during pattern specification processes in early embryonic development. This report analyzes the changing gap junctional communication properties of chick neuroepithelial cells depending on their position relative to the segmental partitions of the rhombencephalon. Intercellular electrical coupling and dye transfer were studied with microelectrode techniques. Neuroepithelial cells were electrically coupled irrespective of their location relative to interneuromeric boundaries. Iontophoretic injection of biocytin or Lucifer Yellow into single cells inside the rhombomeres was followed by transjunctional diffusion to the surrounding cells. In contrast, dye transfer was strictly limited when the diffusion zone contacted the cells forming the interneuromeric limits. Label injected into the boundary cells did not spread to other cells at all. Avian interrhombomeric boundaries are thus sites of reduced junctional permeability during early morphogenesis.     

Intercellular communication of notochord cells during their differentiation in Cynops orientalis

Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye.Lucifer Yellow,Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions.high incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement.With the subsidence of cell movements,both dye coupling and gap junctions were reduced to lower levels.It was,therefore,Suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap Junctions of altered configuration occurred in notochord cells in late taibud stage.The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.     

Effects of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B26-34), and the 8-amino acid alpha-bag cell peptide (alpha-BCP1-8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both alpha-BCP1-8 and B26-34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells is mimicked by alpha-BCP1-8. The excitatory effect of the atrial gland extract cannot be duplicated with B26-34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B26-34 seems to mimick alpha-BCP1-8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.     

Functional gap junctions in the early sea urchin embryo are localized to the vegetal pole.

Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance (G(j)) of 26 nS that decreased to 12 nS before the next cleavage division. G(j) between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction.     

Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer

This report describes a method of marking nerve cells which is approximately 100 times more sensitive than those previously available. The method depends upon intracellular injection of a new, highly fluorescent dye, Lucifer Yellow CH, which can be viewed both in living tissue and after fixation and embedding. The intense fluorescence of the dye makes injected neurons visible in cleared wholemounts, where the complex three-dimensional structure of neurons is readily apparent.Three new observations have been made with Lucifer Yellow. First, many of the invertebrate neurons studied possess an extensive and complex array of fine processes not visible with other techniques. Second, dye spreads rapidly within an injected cell. Third, dye frequently spreads from the injected cell directly to certain other cells. The movement of dye from cell to cell, termed “dye-coupling,” occurred primarily, but not exclusively, between cells known to be electrically coupled.Dye-coupling in the turtle retina revealed striking and distinctive patterns of connections. Type I horizontal cells appear to be multiply connected to each other in an extensive net. Type II horizontal cells are often connected to each other in a hexagonal array. Individual type I and type II cells, widely separated, are frequently dye-coupled; in one case, they were connected by a dyefilled axon.Dye-coupling, readily observed because of the low molecular weight and the intense fluorescence of the new dye, may serve as a general method of tracing certain functional connections by morphological means, and of studying the transfer of small molecules between cells. Preliminary results suggest that systems of dye-coupled cells are substantially more common than was previously believed.     

Communication compartments in the gastrulating mouse embryo

We characterized the pattern of gap junctional communication in the 7.5-d mouse embryo (at the primitive streak or gastrulation stage). First we examined the pattern of dye coupling by injecting the fluorescent tracers, Lucifer Yellow or carboxyfluorescein, and monitoring the extent of dye spread. These studies revealed that cells within all three germ layers are well coupled, as the injected dye usually spread rapidly from the site of impalement into the neighboring cells. The dye spread, however, appeared to be restricted at specific regions of the embryo. Further thick section histological analysis revealed little or no dye transfer between germ layers, indicating that each is a separate communication compartment. The pattern of dye movement within the embryonic ectoderm and mesoderm further suggested that cells in each of these germ layers may be subdivided into smaller communication compartments, the most striking of which are a number of "box-like" domains. Such compartments, unlike the restrictions observed between germ layers, are consistently only partially restrictive. In light of these results, we further monitored ionic coupling to determine if some coupling might nevertheless persist between germ layers. For these studies, Lucifer Yellow was coinjected while ionic coupling was monitored. The injected Lucifer Yellow facilitated the identification of the impalement sites, both in the live specimen and in thick sections in the subsequent histological analysis. By using this approach, all three germ layers were shown to be ionically coupled, indicating that gap junctional communication is maintained across the otherwise dye-uncoupled "germ layer compartments." Thus our results demonstrate that partially restrictive communication compartments are associated with the delamination of germ layers in the gastrulating mouse embryo. The spatial distribution of these compartments are consistent with a possible role in the underlying development.