Anterior Pagoda Neurons Research Articles

Growth-inhibiting extracellular matrix proteins also inhibit electrical activity by reducing calcium and increasing potassium conductances

Inhibitionof neurite sprouting and electrical activity by extracellular matrix (ECM) glycoproteins was studied during neurite regeneration by using anterior pagoda (AP) neurons of the leech. Adult isolated neurons were plated in culture inside ganglion capsules, which among many ECM proteins, contain a group of inhibitory peanut lectin- (PNA) binding glycoproteins. These proteins inhibit neurite production and contribute to the formation of a bipolar outgrowth pattern by AP neurons. Addition of PNA lectin to the culture medium to block the inhibitory effects of ECM glycoproteins induced an increase of neurite sprouting, the loss of the bipolar pattern, and also an increase in the amplitude and duration of action potentials evoked by intracellular current injection. PNA lectin had independent effects on neurite sprouting and electrical activity, since there was no correlation between the total neurite length and the amplitude of the action potentials. Moreover, action potentials were increased by the presence of PNA lectin even in neurons that did not grow. The changes induced by PNA lectin on the active conductances underlying the action potentials were estimated by quantitative model simulations. We predict that the increases in the amplitude and duration of the action potential induced by PNA lectin were due to an increase in a calcium conductance and a reduction in the delayed rectifier potassium conductance. Our results suggest that inhibitory ECM glycoproteins may use independent signaling pathways to inhibit neurite sprouting and electrical activity. These proteins affect the action potential by changing the proportion of inward and outward active conductances.

Extracellular matrix glycoproteins inhibit neurite outgrowth of different types of identified leech neurons in culture

We explored the contribution of inhibitory peanut-binding extracellular matrix glycoproteins to the regeneration of characteristic outgrowth patterns by different types of identified neurons. Adult leech neurons were isolated one by one and plated in culture on a substrate that consisted of the capsules that encase the CNS ganglia. On the inside surface of this substrate, a combination of growth-promoting and -inhibiting extracellular matrix glycoproteins regulates the regeneration of distinctive outgrowth patterns by different neuron types. The role of inhibitory glycoproteins that bind to peanut lectin was studied by perturbation experiments in which peanut lectin was added to the culture medium. The effects of peanut lectin on the outgrowth patterns depended on the specific cell type that was tested. Anterior pagoda neurons, which on capsules produce a bipolar outgrowth pattern, in the presence of the lectin multiplied the number of primary neurites and the total neurite length and also lost their bipolarity. Annulus erector motoneurons, which on capsules grow poorly, in the presence of peanut lectin sprouted 70% more neurites and duplicated their total neurite length. By contrast, Retzius neurons which grow profusely on ganglion capsules, in the presence of peanut lectin increased the number of primary neurites without increasing their total neurite length or branch points. When neurons were plated on plastic, peanut lectin added to the culture medium did not affect the growth of neurons, thus showing that the effects of peanut lectin were induced by blocking the binding of neurons to inhibitory glycoproteins on the capsules. These results show that regeneration of different neuron types has different regulation by inhibitory extracellular matrix molecules.

Calcium Dynamics and Compartmentalization in Leech Neurons

Calcium dynamics in leech neurons were studied using a fast CCD camera. Fluorescence changes (DeltaF/F) of the membrane impermeable calcium indicator Oregon Green were measured. The dye was pressure injected into the soma of neurons under investigation. DeltaF/F caused by a single action potential (AP) in mechanosensory neurons had approximately the same amplitude and time course in the soma and in distal processes. By contrast, in other neurons such as the Anterior Pagoda neuron, the Annulus Erector motoneuron, the L motoneuron, and other motoneurons, APs evoked by passing depolarizing current in the soma produced much larger fluorescence changes in distal processes than in the soma. When APs were evoked by stimulating one distal axon through the root, DeltaF/F was large in all distal processes but very small in the soma. Our results show a clear compartmentalization of calcium dynamics in most leech neurons in which the soma does not give propagating action potentials. In such cells, the soma, while not excitable, can affect information processing by modulating the sites of origin and conduction of AP propagation in distal excitable processes.

Ultrastructure of electrical synapses between nociceptive and anterior pagoda neurons in the CNS of the leech ( Whitmania pigra )

We have used electron microscopy to measure quantitatively the morphology of electrical synapses in a circuit that has been proposed to account for the positional discrimination of the leech. Injection of a presynaptic nociceptive sensory neuron and the postsynaptic anterior pagoda neuron with HRP showed gap junctions in the neuropil. After double labeling, La(3+)-treated ganglia revealed labeled gap junctions from 2.0 to 3.5 nm wide. Between the labeled axon terminals, there were innexons with diameters of 8 to 10 nm. The innexon's central pore diameter was 2 nm, and the mean of the center-to-center distance between two innexons was 30 nm. Except for the gap junction areas of nociceptive sensory neuron axon terminals, the other ultrastructural parameters measured by freeze fracture were similar to those of samples labeled with HRP and filled with La(3+). These data suggested that the gap width, innexon diameter, and its central pore do not on their own account for the mechanism of positional discrimination, which may depend rather on the differences in distribution and number of gap junctions.

Native extracellular matrix induces a well-organized bipolar outgrowth pattern with neurite extension and retraction in cultured neurons

Cultured anterior pagoda (AP) neurons from the leech develop characteristic outgrowth patterns that depend on the molecular composition of the substrate. This article analyzes how native substrates from the central nervous system (CNS), such as the extracellular matrix (ECM) inside the capsules that enwrap the ganglia, determine the outgrowth patterns of AP neurons. When plated on the internal side of ganglion capsules, the remaining primary portion (stump) of AP neurons sprouted two main branches in opposite directions with bifurcations. This T-shaped pattern was distinctive for AP neurons and was different from the patterns of the same cell type plated on the external side of the capsule or on leech laminin extracts, in which they generated multiple neurites and branching points. AP neurons plated on tritonized CNS homogenates reproduced the outgrowth pattern displayed on ganglion capsules, in terms of the number of primary neurites, their length, their orientation, and the number of branch points. The development of the T-shaped outgrowth pattern of AP neurons on ganglion capsules and CNS homogenates started by the sprouting of one branch that later bifurcated, followed by a second branch in the opposite direction after a lag of several hours. Extension of the second branch and retraction of secondary neurites of the first were synchronous and contributed to refine the T-shaped pattern. These results suggest that during development or regeneration of the CNS, particular sets of ECM proteins have multiple effects regulating the number, direction, extension, and retraction of neurites.

The A-like potassium current of a leech neuron increases with age in cell culture.

Single leech neurons isolated and maintained in culture sprout and form electrical and chemical synapses, as they do in vivo, retaining most of the electrical properties of the intact membrane. However, some cells, such as Retzius, Anterior Pagoda (AP) cells and motoneurons, exhibit consistent changes of biophysical characteristics, which mimic those induced by axotomy in vivo and are reversed after reconnection. To improve our understanding of the mechanisms involved in these alterations and of their physiological significance, we investigated the early changes in outward currents developed by cultured AP neurons, using the patch-clamp technique in the whole-cell recording configuration. Different currents were isolated and a differential sensitivity to the time spent in culture and to internal calcium was observed. Three potassium currents were dissected: an A-like current, a delayed rectifier and a third unidentified component. The A-like potassium current was significantly increased with neuronal age in cell culture and was a function of the internal Ca(2+) concentration, whereas the two other potassium currents remained unchanged. Intracellular recordings performed from axotomized neurons of cultured ganglia revealed clear-cut alterations in spike adaptation, which might be due to changes of the A-like current.

New electrical properties of neurons induced by a homeoprotein.

To explore possible neurogenic functions of the genes of the Hox/HOM complexes, we injected the mRNA from the leech homeobox genes Lox1 and Lox4 into adult neurons that normally do not express them. The ectopic expression of Lox1 induced a specific transformation in the electrical properties of certain identified neurons: action potential amplitude increased about threefold after the injections. This effect of Lox1 expression was restricted, among cell types examined, to the anterior pagoda neurons (APs) and the nut neurons. This effect was also restricted to Lox1 ectopic expression; the action potentials of APs and nut neurons were not enlarged when the mRNAs of either Lox4, another leech Hox/HOM gene, or beta-galactosidase were injected. Lox1 mRNA injection did not affect the resting potential, input resistance, or axonal morphology of the transformed APs, raising the possibility that it acts via the modification of voltage-dependent ion channels. Thus, a specific homeobox gene can transform key neuronal characteristics in a cell-specific manner. We may thus add electrophysiologic properties to other aspects of neuronal identity determined by homeobox gene expression.

Outgrowth patterns and directed growth of identified neurons induced by native substrates in culture.

In this study, we have tested how various identified leech neurons in culture grow on surfaces that they normally contact in situ. Neurons were cultured either on ganglion capsules from which neurons had been removed or on skin. On these substrates, outgrowth patterns were characteristic for each cell type. Retzius cells plated on capsules extended bundles of thick, fasciculated processes with few branching points and in the opposite direction a tangle of fine neurites. Anterior pagoda (AP) neurons plated on capsules extended two single processes in opposite directions but failed to grow on skin. Sensory P and N neurons on capsules extended multiple processes. On skin, P neurons extended only two long branches in opposite directions over the superficial body wall. N neurons on skin extended multiple processes. Varicosities were common in the processes of P and N neurons on capsules or skin. The branching patterns described here bore closer resemblance to those in the developing or adult nervous system than to those on Concanavalin A or laminin-enriched extract. Pairs of Retzius or AP neurons plated at a distance on the same capsule extended neurites from one neuron toward the other and formed contacts. Such directed growth failed in hybrid pairs of Retzius and AP neurons or in pairs plated on laminin-enriched extract or Concanavalin A. Our results suggest that multiple growth-promoting molecules anchored to the extracellular matrix may cooperate in regulating the branching pattern of neurons, fasciculation, and direction of growth.

Photoinactivation of the giant neuropil glial cells in the leech Hirudo medicinalis: effects on neuronal activity and synaptic transmission.

1. We studied the effects of photoinactivation of neuropil glial (NG) cells of the leech Hirudo medicinalis on neuronal activity and synaptic transmission. Each segmental ganglion contains two of these giant glial cells, which are electrically and dye coupled. 2. One of the two NG cells in an isolated segmental ganglion was filled with the dye Lucifer yellow (LY). Subsequent irradiation of the ganglion with laser light (440 nm) to photolyze LY caused irreversible depolarization of both NG cells. The NG cells that were filled with LY depolarized from -73 +/- 1.1 (SE) mV to -22 +/- 2.4 mV within 25 +/- 2.8 min of continuous irradiation (n = 22). The other NG cell, which was not directly filled with LY, depolarized with some delay. 3. Photoinactivation of the NG cells caused an irreversible depolarization of Retzius neurons and noxious (N) sensory cells by a mean of 14 mV (n = 36) and 9 mV (n = 24), respectively. In addition, the input resistance was reduced by 54% in Retzius cells and by 34% in N cells. Spikes could not be evoked in Retzius cells after the inactivation of the NG cells, either by intracellular current injection or by electrical nerve stimulation. Similarly, anterior pagoda neurons, annulus erector neurons, and the excitor neurons of the ventrolateral circular muscles became inexcitable. However, N cells, heart interneurons, and most of the heart motor neurons, touch cells, and pressure cells could still generate spontaneous or evoked action potentials. 4. Photoinactivation of the NG cells impaired the electrical connection between the two Retzius neurons. The electrical coupling was completely eliminated in six of eight cell pairs and reduced by 66% in two others. 5. Photoinactivation of the NG cells in the 3rd and 4th segmental ganglion caused a complete block of the chemical synapse between reciprocal inhibitory heart interneurons in these ganglia; the bursting rhythm either stopped or changed to a tonic activity, whereas inhibitory postsynaptic potentials could not be recorded in either heart interneuron anymore. 6. Laser irradiation alone had no effect on neuronal activity and synaptic transmission. Addition of glutathione (10 mM) and ascorbic acid (10 mM) to the saline to bind extracellular radicals that might be produced by the irradiation did not suppress the effects caused by photoinactivation of NG cells. 7. Elevation of bath K+ concentration to 12 mM, acidification of the saline to pH 5.5, and alkalinization to pH 8.5 for 6 min each did not mimick the effects on membrane properties of Retzius cells as produced by inactivation of NG cells. The results suggest some role of glial cells in the maintenance of neuronal activity and electrical and chemical synaptic transmission.

Action potential initiation site depends on neuronal excitation

The initiation site in neurons is where the excitatory and inhibitory inputs sum to generate action potentials. It is generally considered to be at a fixed location, typically at the axon hillock or initial segment, although action potentials, or impulses, could in theory arise at a site that shifts dynamically. The data reported here show that the initiation site can shift in a graded manner, by as much as 175 microm, depending on the level of neuronal excitation. Laser axotomy reveals that the Anterior Pagoda (AP) neuron of the leech is excitable within the synaptic neuropil before its axon bifurcates. Using an electrophysiological technique to measure relative delays in impulses arriving at different sites, we have found that depolarization, either by applied current or by synaptic input, can shift the site of impulse initiation in the cell proximally toward the soma and neurites receiving synaptic input. Impulse initiation in this region should enhance the efficacy of inputs synapsing there. Conversely, hyperpolarization can shift the initiation site distally. A shifting initiation site, therefore, may be a mechanism by which synaptic inputs can rapidly enhance or suppress the active response of the AP neuron to other synaptic inputs.

Correlation of receptive field position of mechanosensory neurons and the strength of their connections to AP neurons in the CNS of the leech (Whitmania pigra)

An identified neuron of unknown function in the CNS of the leech, the anterior pagoda (AP) cell, receives multiple synaptic inputs from mechanosensory neurons that innervate the skin. Impulses in touch (T), pressure (P) and nociceptive (N) sensory cells on both sides of the ganglion produced electrical coupling potentials on both AP cells. Sensory cells with receptive fields contralateral to the cell body of the AP neuron always gave rise to larger synaptic potentials. In addition sensory cells supplying dorsal skin gave rise to larger synaptic potentials than those with lateral or ventral fields. It is suggested that integration by the AP cell can provide information about the position of mechanical stimuli impinging on the body wall of the animal.

Interactions during a critical period inhibit bilateral projections in embryonic neurons

The anterior pagoda (AP) neurons in the CNS of the medicinal leech are found as homologous pairs in 20 of the 21 midbody ganglia. Each AP is the mirror image of its mate, extending its main axon across the midline of the CNS and eventually into the contralateral body wall, thereby attaining a unilateral pattern of innervation. Certain features of the adult AP morphology are known to arise through interactions among homologs early in development (Gao and Macagno, 1987b), but it is not known whether the contralateral nature of the projection pattern is due to intrinsic "one-sidedness" or rather to cell-cell interactions that inhibit the formation of a second, ipsilateral projection. In the experiments described in this report, we tested the possibility that an AP's contralateral homolog itself inhibits the formation of bilateral projections. One AP was photoablated in the intact embryo early in development and then the response of the remaining AP was examined. We found that an AP can extend bilaterally symmetrical projections when its homolog is missing, but only during a critical period that, interestingly, begins when an AP's interactions with other specific neurons come to an end. To determine whether synaptic communication between AP homologs could be responsible for the timing of this critical period, we recorded electrophysiologically from pairs of embryonic AP neurons. Although no detectable chemical signaling was observed, AP cells were electrically coupled throughout the entire critical period. Further, the junctions between these neurons were permeated by 5-HT, whereas larger molecules such as carboxyfluorescein were impermeant. This dye coupling decreased with age even while electrical coupling persisted, suggesting but not proving that the properties of the gap junctions between AP neurons may also change with time. We conclude that unilateral AP cells possess the intrinsic ability to project bilaterally, but are inhibited from doing so by age-dependent interactions with homologous neurons, possibly mediated by gap-junctional communication.

Calcium wave fronts that cross gap junctions may signal neuronal death during development

Embryonic anterior pagoda (AP) neurons in the leech interact with their segmental homologs in adjacent ganglia through transient axons that overlap during a critical period of development and then retract. However, when an AP neuron is ablated mechanically or by irradiation during this period, an adjacent homolog responds by reinitiating growth of its overlapped axon and thereby taking over vacated territory (Gao and Macagno, 1987b; Gao, 1989). The death of an AP cell is therefore communicated to its homolog, but the mechanism underlying this signaling is presently unknown. Since it was recently found that AP homologs are electrically and dye coupled through their transient axons (Wolszon et al., 1994), we investigated the possibility that gap junctions may mediate the cell death signal that could occur between developing neurons. Among several candidate intercellular signals, we began by studying calcium dynamics in embryonic AP cells, in situ, since calcium is known to cross gap junctions and is implicated in cell death in many systems. We found that elements that usually increase [Ca2+]i in adult neurons, such as releasable internal stores or voltage-dependent calcium channels, were not present at the critical period. Instead, mechanisms that reduce free calcium, such as buffering and pumping, were the most robust. When a large, focal calcium rise was produced in an AP axon by making a lesion with a UV microbeam (leading to eventual death of these neurons), calcium did not rise quickly throughout the cell, but rather moved in a slow (0.05-0.25 micron/sec) wave front away from the lesion site, into other processes of the damaged cell. Furthermore, when a calcium wave front reached the growth cone of a transient axon, it crossed at the gap junctions into the coupled axon of the neighboring AP neuron, but went no further. Since it is known that an AP responds to a neighbor's death by reinitiating growth only in that axon that contacts the dying cell (Gao and Macagno, 1987b; Gao, 1989), these observations are consistent with calcium playing a role in the signaling of cell death to homologs that are coupled to a dying cell.

Growth cone "collapse" in vivo: are inhibitory interactions mediated by gap junctions?

In the leech embryo, oppositely directed axons of homologous anterior pagoda (AP) neurons overlap with each other extensively within the nerves that link adjacent ganglia, and inhibit each other's further growth (Gao and Macagno, 1987b). During this 5-8 d period of inhibition, the axons begin to grow thin, and eventually they retract completely. However, deletion of one overlapping AP cell results in the renewed growth of the remaining AP cell's axon, which then innervates territory vacated by the killed cell. Thus, each neuron can detect the presence of the other, and adjust its branching pattern accordingly. To begin to explore how these neurons detect and inhibit each other, we tested for direct communication between them. Dye fills with fluorescent chromophores suggested direct contact between their axons at the light level, and this was confirmed by serial-section electron microscopic analysis. Morphological features resembling aspects of gap junctions were observed where the projections were closely apposed, and subsequent electrophysiological recordings demonstrated electrical coupling between the mutually inhibited axons. Confirmation that these projections communicate via gap junctions was obtained using intracellular injection of 5-HT as a tracer, followed by anti-5-HT immunohistochemistry. The tracer passed selectively between AP neurons. We propose that the gap junctions formed between the transient projections of the developing AP neurons may mediate the exchange of the signals that permit homologs to recognize each other and to inhibit the further forward progress of these projections.