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M G Honig

Publications and source records attributed to M G Honig.

At least 19 recordsLinked to original sources
PubMed2000

Pathway tracing using biotinylated dextran amines.

Biotinylated dextran amines (BDA) are highly sensitive tools for anterograde and retrograde pathway tracing studies of the nervous system. BDA can be reliably delivered into the nervous system by iontophoretic or pressure injection and visualized with an avidin-biotinylated HRP (ABC) procedure, followed by a standard or metal-enhanced diaminobenzidine (DAB) reaction. High molecular weight BDA (10 k) yields sensitive and exquisitely detailed labeling of axons and terminals, while low molecular weight BDA (3 k) yields sensitive and detailed retrograde labeling of neuronal cell bodies. The detail of neuronal cell body labeling can be Golgi-like. BDA tolerates EM fixation and processing well and can, therefore, be readily used in ultrastructural studies. Additionally, BDA can be combined with other anterograde or retrograde tracers (e.g. PHA-L or cholera toxin B fragment) and visualized either by multi-color DAB multiple-labeling - if permanent labels are desired, or by using multiple simultaneous immunofluorescence - if fluorescence viewing is desired. In the same manner, BDA pathway tracing and neurotransmitter immunolabeling can be combined. Note that BDA pathway tracing can also be combined with anterograde or retrograde labeling with fluorescent dextran amines, if one wishes to exclusively use tracers with the favorable transport properties and sensitivities of dextran amines. In this case, the BDA can be visualized together with the fluorescent dextran amines using fluorescence labeling for the BDA, or the fluorescent dextran amines can be visualized together with the BDA by multicolor DAB labeling via immunolabeling of the fluorescent dextran amines using anti-fluorophore antisera. BDA is, thus, a flexible and valuable pathway tracing tool that has gained widespread popularity in recent years.

Animals↗
PubMed1999

Ultrastructural observations on the expression of axonin-1: implications for the fasciculation of sensory axons during axonal outgrowth into the chick hindlimb.

To help understand how axons interact as they grow into the developing chick hindlimb, we used electron microscopy in conjunction with immunoperoxidase staining for the cell adhesion molecule axonin-1 to label sensory axons. The results showed that sensory axons travel together in bundles, tightly apposed to one another. In contrast, motoneuron axons are more widely spaced, although motoneuron axons situated at the perimeter of sensory axon bundles are found in close contact with neighboring sensory axons. Sensory growth cones and lamellipodia tend to be located centrally within the bundles, with several lamellipodia typically being found stacked together. Strikingly, regions of close axonal apposition are accompanied by axonin-1 expression, suggesting that such contacts are indeed adhesive. Taken together, these observations suggest that groups of sensory axons of a similar age grow together, with some of the older sensory axons fasciculating along motoneuron axons and younger sensory axons later fasciculating along older sensory axons. Axons situated at the periphery of sensory bundles are typically partly labelled, such that axonin-1 is expressed on membranes apposing other labelled axons but not on those facing unlabelled axons or unlabelled Schwann cells. Thus, axonin-1 appears to become redistributed within the membranes of axons growing into the limb, as it does on cultured neurons. In contrast, the neuron-glia cell adhesion molecule (NgCAM), which binds heterophilically to axonin-1, appears uniformly distributed on even those axons that would have an asymmetric distribution of axonin-1. Thus, the localization of axonin-1 strongly suggests that it plays an important role in sensory axon fasciculation, but the relative contributions of its interactions with various potential ligands are unclear. Finally, we found that some sensory growth cones have lamellipodia that are spread over considerable expanses. This suggests that although fasciculation is important in sensory axon guidance, sensory axons may also explore the local environment.

Animals↗
PubMed1999

Cellular localization of huntingtin in striatal and cortical neurons in rats: lack of correlation with neuronal vulnerability in Huntington's disease.

Immunohistochemistry and single-cell RT-PCR were used to characterize the localization of huntingtin and/or its mRNA in the major types of striatal neurons and in corticostriatal projection neurons in rats. Single-label immunohistochemical studies revealed that striatum contains scattered large neurons rich in huntingtin and more numerous medium-sized neurons moderate in huntingtin. Double-label immunohistochemical studies showed that the large huntingtin-rich striatal neurons include nearly all cholinergic interneurons and some parvalbuminergic interneurons. Somatostatinergic striatal interneurons, which are medium in size, rarely contained huntingtin. Calbindin immunolabeling showed that the vast majority of the medium-sized striatal neurons that contain huntingtin are projection neurons, but only approximately 65% of calbindin-labeled projection neurons (localized to the matrix compartment of striatum) were labeled for huntingtin. Calbindin-containing projection neurons of the matrix compartment and calbindin-negative projection neurons of the striatal patch compartment contained huntingtin with comparable frequency. Single-cell RT-PCR confirmed that striatal cholinergic interneurons contain huntingtin, but only approximately 65% of projection neurons contained detectable huntingtin message. The finding that huntingtin is not consistently found in striatal projection neurons [which die in Huntington's disease (HD)] but is abundant in striatal cholinergic interneurons (which survive in Huntington's disease) suggests that the mutation in huntingtin that causes HD may not directly kill neurons. In contrast to the heterogeneous expression of huntingtin in the different striatal neuron types, we found all corticostriatal neurons to be rich in huntingtin protein and mRNA. One possibility raised by our findings is that the HD mutation may render corticostriatal neurons destructive rather than render striatal neurons vulnerable.

Animals↗
PubMed1998

In vitro studies of growth cone behavior support a role for fasciculation mediated by cell adhesion molecules in sensory axon guidance during development.

Axonal interactions, which are mediated by cell adhesion molecules (CAMs) as well as other types of membrane proteins, are important for sensory axon pathfinding in the developing chick hindlimb. We have previously shown that injection of antibodies that block the function of either G4/L1 or N-cadherin into the limb, starting when the first sensory axons reach the plexus, alters the segmental pattern of projections along cutaneous nerves. Specific removal of polysialic acid from NCAM using the enzyme endoneuraminidase N (Endo N) also resulted in significant changes in cutaneous projection patterns, while injection of antibodies against NCAM itself had no obvious effect (M. G. Honig and U. S. Rutishauser, 1996, Dev. Biol. 175, 325-337). To help understand the cellular basis for these findings, we developed a tissue culture system in which the axons from dorsal root ganglion explants grow within defined laminin lanes and examined whether the same treatments increased or decreased a growth cone's tendency to be closely associated with neighboring axons. After 2 days in culture, images of the cultures were recorded, antibodies or Endo N was added, and images of the same fields were recaptured an hour later. To quantify the results, growth cones located in defined regions of the laminin lanes were classified, before and after the perturbation, as "free" (i.e., growing primarily on the laminin substratum), "fasciculated" (i.e., growing tightly along other neurites), or "intermediate" (i.e., growing both on the laminin substratum and in contact with other neurites). We found that anti-G4/L1 and anti-N-cadherin, but not anti-NCAM, caused an increase in defasciculated growth cones, whereas Endo N resulted in an increase in fasciculated growth cones. These changes in fasciculation are consistent with the changes in cutaneous projections seen in our previous in ovo perturbations. The results from these tissue culture experiments thus provide strong support for the idea that one mechanism by which CAMs affect sensory axon pathfinding in vivo is by regulating the affinity of sensory growth cones for neighboring axons, which in turn can modulate the growth cone's ability to navigate through the surrounding environment.

Animals↗
PubMed1998

The spatial relationships among cutaneous, muscle sensory and motoneuron axons during development of the chick hindlimb.

Previous studies have suggested that interactions with other axons are important in sensory axon pathfinding in the developing chick hindlimb. Yet the nature of these interactions remains unknown, in part because information about the spatial relationships among the different kinds of axons is lacking. To obtain this information, we combined retrograde axonal tracing with an immunofluorescent labelling approach that distinguishes between sensory and motoneuron axons. This allowed us to follow the trajectories of sensory axons having a known destination, while also identifying their neighbors. We found that as sensory and motoneuron axons meet in the spinal nerves and travel into the limb, sensory axons remain bundled together. The large bundles that are present proximally gradually split into smaller bundles as the axons course distally in the spinal nerves; more distally, some bundles join to again form large bundles. Younger, later-growing sensory axons appear to grow primarily along bundles of older sensory axons that grew out earlier. Starting from very proximal levels, axons projecting along an individual cutaneous nerve are found together in bundles that are situated in characteristic regions of each spinal nerve. Some of these bundles are initially interspersed with bundles of axons projecting along other nerves, thereby indicating that the initial position of a cutaneous axon in the spinal nerves does not strictly determine its subsequent trajectory. As they travel distally, bundles of axons projecting along one cutaneous nerve gradually join one another, becoming increasingly separated from axons having different destinations. In contrast, muscle sensory axons are situated adjacent to motoneuron axons innervating the same muscle for much of their course. This suggests that muscle sensory axons may be guided to the appropriate muscles by fasciculating along motoneuron axons. Taken together, the results show that sensory axons projecting along different nerves are different from one another and respond to cues in their environment to navigate through the spinal nerves and plexus. Thus, sensory neurons must be intrinsically specified with respect to their peripheral targets. Sensory axons appear to respond differentially to the axons they encounter, segregating from axons that project along different nerves and often growing with axons destined for the same nerve, suggesting that fasciculation may aid pathfinding.

Animals↗
PubMed1996

Changes in the segmental pattern of sensory neuron projections in the chick hindlimb under conditions of altered cell adhesion molecule function.

In the developing chick hindlimb, pathfinding by sensory axons is affected by their interactions with other axons entering the limb. Cell adhesion molecules (CAMs) expressed on the growing axons are likely to influence these interactions. Accordingly, to elucidate how CAMs affect sensory axon pathfinding, we injected antibodies that block the functions of NCAM, G4/L1, or N-cadherin into the hindlimb, starting at St. 25, when all motoneuron axons but only a few sensory axons had entered the plexus. In each case, the segmental pattern of projections was assessed 2-3 days later by retrogradely labeling individual peripheral nerves. With all perturbations, the gross anatomical nerve pattern developed normally; that is, some sensory axons formed cutaneous nerves while other sensory axons projected to muscles. However, the segmental pattern of sensory projections was changed when either G4/L1 or N-cadherin function was blocked in that fewer sensory axons crossed the anterior-posterior axis of the plexus. A likely reason for this effect is that anti-G4/L1 and anti-N-cadherin each decreased the amount of fasciculation and that sensory axons are less able to travel across the plexus when they are defasciculated. Anti-G4/L1 affected both cutaneous and muscle sensory projections while anti-N-cadherin affected cutaneous but not muscle sensory projections, in accord with known differences in the expression of these two CAMs on sensory and motoneuron axons. Although anti-NCAM did not appear to alter sensory projections, when polysialic acid (PSA) was enzymatically removed from NCAM, there was a marked increase in cutaneous projections from the most proximate DRG, although muscle sensory projections were unchanged. PSA removal may cause an increase in fasciculation that forces sensory axons to track along neighboring axons. Thus, without PSA, cutaneous axons project more in accord with the relative anterior-posterior positions they had as they entered the plexus. Taken together, these studies suggest that axonal fasciculation mediated by CAMs and regulated by PSA influences the ability of sensory growth cones to navigate through the plexus and project along the correct peripheral nerves.

Afferent Pathways↗
PubMed1995

The effects of target tissues on the outgrowth of chick cutaneous and muscle sensory neurons.

In some developing systems, growing axons are attracted to their target site by a diffusible molecule released by the target tissue. In the chick hindlimb, this mechanism could explain how axons, after having reached the plexus region, grow to muscle or to skin. To begin to test this possibility for limb-innervating sensory neurons, we cocultured dorsal root ganglion explants and potential target tissues in three-dimensional collagen gels. In particular, we wanted to know if target tissues, at early stages of development, specifically attract axons of only the appropriate type of sensory neuron. To identify each type of sensory neuron, we used DiI to retrogradely label either cutaneous or muscle sensory neurons in the embryo, prior to culturing. The results showed that dermal and muscle explants could each enhance the outgrowth of both cutaneous and muscle sensory axons. In contrast, the epidermis and the connective tissue associated with developing muscle appeared to inhibit the outgrowth of both cutaneous and muscle sensory axons. These results suggest that, in the embryo, the dermis and muscle cells both release diffusible factors that cause sensory axons to diverge from the plexus, extend toward the sources of these factors, and thereby form discrete peripheral nerves. The inhibitory effects of epidermis and muscle-associated connective tissue may serve to limit the growth of sensory axons to the structures, i.e., dermis and muscle cells, that ultimately receive sensory innervation. However, since for each of the difference types of limb tissue, the responses of cutaneous and muscle sensory neurons were always similar to one another, sensory axons must not be responding to target-derived factors when they decide whether to grow to skin or to muscle.

Animals↗
PubMed1995

The expression of cell adhesion molecules on the growth cones of chick cutaneous and muscle sensory neurons.

In the developing chick hindlimb, axons that will project along a given peripheral nerve sort out together, as they traverse the plexus region at the base of the limb, and become segregated from axons that will project along other peripheral nerves. This sorting out may involve, at least in part, the differential expression of various cell adhesion molecules (CAMs). To begin to explore this possibility, we have compared the relative levels of immunofluorescent labeling on the growth cones of two populations of sensory neurons whose axons become segregated from one another: cutaneous and muscle sensory neurons. We took a tissue culture approach, since this allowed us to readily visualize the immunofluorescent labeling of individual growth cones, and identified the two cell types by previous retrograde labeling with DiI. Two dorsal root ganglion explants, one containing DiI-labeled cutaneous neurons and the other containing DiI-labeled muscle sensory neurons, from opposite sides of the same embryo, were plated together in each culture dish. For all the CAMs were studied (NCAM, polysialylated NCAM, G4/L1, axonin-1, SC1/DM-GRASP/BEN, and N-cadherin), the intensity of immunofluorescent labeling typically was fairly uniform on the growth cone, it filopodia, and the portion of the neurite just proximal to the growth cone. Only one CAM, axonin-1, exhibited labeling that was especially intense at sites of interneuronal contact. Quantification of labeling intensities using image analysis showed that cutaneous and muscle sensory growth cones did not consistently differ from one another in their levels of expression of G4/L1 or of axonin-1. The latter finding stands in contrast to recent reports claiming that axonin-1 is not expressed on muscle sensory neurons. Each of the other CAMs (NCAM, polysialylated NCAM, SC1/DM-GRASP/BEN, and N-cadherin) also showed considerable overlap in the distribution of labeling intensities between the two populations, but overall, expression levels on muscle sensory growth cones were greater than on cutaneous growth cones. How the differential expression of some CAMs could potentially contribute to the way that cutaneous and muscle sensory growth cones become segregated from one another and the implications of these results for sensory neuron specification are discussed.

Animals↗
PubMed1993

Growth cones respond in diverse ways upon encountering neurites in cultures of chick dorsal root ganglia.

To help in understanding the role that interactions with other axons may have in helping to guide sensory neuron growth cones along the appropriate pathways in the chick hindlimb, we have examined how sensory growth cones respond when they contact the processes of other sensory neurons. Videotaping of neurons in dissociated cell cultures, as they participated in such encounters, showed that sensory growth cones can demonstrate any one of a number of responses: crossing, fasciculation, parallel growth, or retraction. We observed a variety of responses even among neurons from a single segment, suggesting the possibility that this is due to a heterogeneity of cell-surface labels among the neurons present within an individual dorsal root ganglion. If sensory neurons behave in vivo as we found they do in culture, the diverse behaviors of sensory growth cones may contribute to the process by which different subsets of axons sort out into separate, discrete bundles as they extend into the periphery during development. The diversity of responses we observed stands in contrast to the results of Kapfhammer, J.P., and Raper, J.A., [(1987) J. Neurosci. 7, 1595-1600] who found that sensory growth cones always cross sensory neurites. Possible explanations for the differences in the results were explored and are discussed.

Animals↗
PubMed1992

Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies.

Fluorescent dextran amines have recently been reported to be useful for anterograde pathway tracing. However, fluorescent markers are not always ideal for detailed mapping studies. We therefore evaluated the efficacy of a biotinylated dextran amine (BDA) for anterograde labeling in several different preparations. BDA was visualized with an avidin-biotinylated HRP (ABC) procedure followed by a standard or metal-enhanced diaminobenzidine (DAB) reaction. After iontophoretic injections of BDA into neocortex-like telencephalic regions in pigeons or into visual or somatosensory cortex in rats, there was excellent and abundant labeling of axons and terminals in forebrain, midbrain and hindbrain target areas with 1-week survival times. Large pressure injections of BDA into the avian telencephalon were also found to result in extensive anterograde labeling. We then carried out a series of studies using 2-color DAB double-labeling to determine effective approaches for combining BDA labeling with other labeling methods. Using an isolated embryonic chick spinal cord-hindlimb preparation, we combined BDA labeling with another anterograde labeling method to differentially label two sets of projections. In these studies, sensory neuron and motoneuron projections into the limb from the same segmental level, or motoneuron projections into the limb from two separate segments were differentially labeled by using HRP (visualized first with a blue/black metal-DAB reaction) and BDA (visualized second with a brown DAB reaction). In other double-labeling studies, we combined BDA labeling of axons and terminals with immunohistochemical labeling of neurons. In these experiments, telencephalic neurons in pigeons or rats were labeled immunohistochemically for parvalbumin or substance P (using a brown DAB reaction) and BDA-labeled axons were labeled blue/black (using a metal-intensified DAB reaction). Double-labeling was successful regardless of whether the entire immunohistochemical labeling procedure preceded or followed the BDA labeling procedure. Together, these studies show that BDA is effective for anterograde pathway tracing and can be used in double-label studies with other labeling methods.

3,3'-Diaminobenzidine↗
PubMed1991

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons.

We have examined the physiological properties of transmission at newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons in vitro. Chick neurons were labeled with fluorescent carbocyanine dyes before they were placed into culture (Honig and Hume, 1986), and were studied by making intracellular recordings during the first 2 weeks of coculture. Evoked monosynaptic excitatory postsynaptic potentials (EPSPs) were not observed until 48 h of coculture. Beyond this time, the frequency with which connected pairs could be found did not vary greatly with time. With repetitive stimulation, the evoked monosynaptic EPSPs fluctuated in amplitude from trial to trial and showed depression at frequencies as low as 1 Hz. To gain further information about the quantitative properties of transmission at newly formed synapses, we analyzed the pattern of fluctuations of delayed release EPSPs. In mature systems, delayed release EPSPs are known to represent responses to single quanta, or to the synchronous release of a small number of quanta. For more than half of the connections we studied, the histograms of delayed release EPSPs were extremely broad. This result suggested that either quantal responses are drawn from a continuous distribution that has a large coefficient of variation or that there are several distinct size classes of quantal responses. The pattern of fluctuations of monosynaptic EPSPs was consistent with both of these possibilities, and was inconsistent with the possibility that monosynaptic EPSPs are composed of quantal subunits with very little intrinsic variation. Although variation in the size of responses to single quanta might arise in a number of ways, one attractive explanation for our results is that the density and type of acetylcholine receptors varies among the different synaptic sites on the surface of developing sympathetic ganglion neurons.

Action Potentials↗
PubMed1990

Double-labeling of tissue containing the carbocyanine dye DiI for immunocytochemistry.

The fluorescent carbocyanine dye DiI can be used for retrograde and anterograde labeling of neuronal pathways. To investigate the possible neurochemical identity of DiI-labeled neuronal cell bodies and terminals, we used a procedure for double-labeling of the same tissue with antisera to specific neuroactive substances. This procedure involves visualizing the immunohistochemical label with an FITC-conjugated secondary antiserum. Both labels can be viewed in the same tissue by fluorescence microscopy, and individual cell bodies and processes double-labeled with DiI and antiserum can be identified by switching between filter sets appropriate for rhodamine (to see the DiI labeling) and for fluorescein (to see the immunhistochemical labeling). The method has been used with primary antisera to excitatory and inhibitory amino acid neurotransmitters, as well as to neuropeptides, and is likely to be useful with antibodies against a wide variety of substances. Several other immunocytochemical methods were found to be incompatible with DiI labeling.

Animals↗
PubMed1989

Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing.

The fluorescent carbocyanine dyes dil and diO have an extensive history of use in cell biology, but their use as neuronal tracers is relatively recent. We found in 1985 that these molecules were excellent retrograde and anterograde tracers in the developing nervous system. We went on to show that these dyes were retained in neurons placed in culture, that they initially labelled the processes as well as the cell bodies of cultured neurons, and that they were seemingly non-toxic. We suggested that the major mechanism of translocation for these molecules was lateral diffusion in the membrane, rather than fast axonal transport. This suggestion was recently confirmed in a striking manner by Godement et al., when they showed that these dyes can be used to label axonal projections in fixed tissues. Labelling with carbocyanine dyes has already allowed several exciting advances in developmental neurobiology. In this article we review the properties of carbocyanine dyes and point out some of their uses and advantages.

Animals↗
PubMed1986

Altered sensory projections in the chick hind limb following the early removal of motoneurons.

Chick sensory neurons grow to their correct targets in the hindlimb from the outset during normal development and following various experimental manipulations. This may result not because sensory neurons respond to specific limb-derived cues, but because they interact in some way with motoneurons which are responsive to such cues. To test this possibility, we removed the ventral part of the neural tube, which contains motoneurons and their precursors, at stages 16 1/2-20 1/2 and later examined the pathways sensory neurons had taken within the limb. Muscle nerves generally were missing or were reduced in diameter beyond the extent expected simply from the absence of motoneuron axons. In many cases, cutaneous nerves were enlarged, presumably due to the addition of other sensory axons. This result suggests that, in the absence of motoneurons, sensory neurons that normally project to muscles are unable to do so and may instead project along cutaneous pathways. Sensory axons from different segments also crossed less extensively in the plexus region than they did in control embryos, suggesting that alterations in their trajectories may normally be facilitated by similar changes in motoneuron pathways. Thus, motoneurons greatly enhance sensory neuron growth to muscles and contribute significantly toward the achievement of the normal sensory projection pattern. Sensory axons may fasciculate with motoneuron axons, or motoneuron axons may provide an aligned substrate for sensory neurons to grow along. Alternatively, motoneuron axons may alter the environment, thereby making certain pathways in the limb permissive for sensory neuron growth.

Afferent Pathways↗
PubMed1986

The development of sensory projection patterns in embryonic chick hindlimb under experimental conditions.

In the chick, sensory neurons grow to their segmentally appropriate target sites in the hindlimb from the outset during normal development. To elucidate the underlying mechanisms, we performed various manipulations of the neural tube, including the neural crest, or of the hindlimb, before axonal outgrowth and assessed the resulting sensory projections using retrograde and anterograde HRP labeling and electrophysiological techniques. Previous experiments had shown that motoneurons are specified to project to their appropriate target muscles prior to axon outgrowth and that they respond to cues in the limb in order to grow to those targets (C. Lance-Jones and L. Landmesser, 1980, J. Physiol. (London) 302, 559-602; C. Lance-Jones and L. Landmesser, 1981, Proc. R. Soc. London, B 214, 19-52). When several segments of neural tube and neural crest were deleted, sensory neurons in the remaining segments still projected along their correct pathways, as did motoneurons. In situations in which motoneurons grew to their correct targets from altered positions with respect to the limb (e.g., small neural tube reversals), sensory neurons also tended to project along the segmentally appropriate pathways both to skin and to muscle. In situations in which motoneurons were displaced greater distances from their normal point of entry into the limb and made wrong connections (e.g., large neural tube reversals, anterior-posterior limb reversals), sensory neurons also projected incorrectly. The patterns of sensory projections to muscles were, in each situation, generally similar to the motoneuron projections. These results are consistent with the possibility that sensory neurons, like motoneurons, are specified with respect to their peripheral connectivity. Alternatively, the results suggest that motoneurons may play a role in the process of pathway selection by sensory neurons.

Afferent Pathways↗
PubMed1986

Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures.

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.

Animals↗
PubMed1986

Excitatory action of ATP on embryonic chick muscle.

It has been suggested that ATP might play a role in synaptic transmission at developing vertebrate neuromuscular junctions. To increase our understanding of the events underlying synapse formation, we have used intracellular recording and patch clamp recording to examine the response of chick myoblasts and myotubes to to ATP and other nucleotides, ATP, applied at micromolar concentrations, has a potent depolarizing action on chick myoblasts and myotubes. The ATP depolarization declines during prolonged application of ATP and shows no recovery for at least 20 min after the removal of ATP. The physiological event that underlies the ATP response has a reversal potential near O mV and is due to a conductance increase. However, contrary to our expectation, in a series of nearly 200 cell-attached and outside-out patch recordings, we did not detect single-channel currents that were related to ATP. The myotube ATP receptor is pharmacologically distinct from putative ATP receptors in other systems. It is not activated by ADP, AMP, or adenosine. Furthermore, the nonhydrolyzable ATP analogs, AMP-PNP, alpha,beta-meATP, and beta,gamma-meATP (respectively, 5-adenylylimido diphosphate; alpha,beta-methylene adenosine 5'-triphosphate; and beta,gamma-methylene adenosine 5'-triphosphate), which are potent ATP agonists in other systems, have no depolarizing action on myotubes. The ATP receptor is also distinct from the nicotinic ACh receptor since responses to ATP are unaffected by the nicotinic antagonists d-tubocurarine and alpha-bungarotoxin. We therefore applied alpha-bungarotoxin to nerve-muscle co-cultures in the hope of uncovering an additional component of the postsynaptic potential, which might represent a synaptic action of ATP. Under these experimental conditions no evidence indicative of a postsynaptic action of ATP released from nerve terminals was observed.

Action Potentials↗