Chick Sensory Neurons Research Articles

Design of Potent Peptide Mimetics of Brain-derived Neurotrophic Factor

Brain-derived neurotrophic factor (BDNF) has potential for the treatment of human neurodegenerative diseases. However, the general lack of success of neurotrophic factors in clinical trials has led to the suggestion that low molecular weight neurotrophic drugs may be better agents for therapeutic use. Here we describe small, dimeric peptides designed to mimic a pair of solvent-exposed loops important for the binding and activation of the BDNF receptor, trkB. The monomer components that make up the dimers were based on a monocyclic monomeric peptide mimic of a single loop of BDNF (loop 2) that we had previously shown to be an inhibitor of BDNF-mediated neuronal survival (O'Leary, P. D., and Hughes, R. A. (1998) J. Neurochem. 70, 1712-1721). Bicyclic dimeric peptides behaved as partial agonists with respect to BDNF, promoting the survival of embryonic chick sensory neurons in culture. We reasoned that the potency and/or efficacy of these compounds might be improved by reducing the conformational flexibility about their dimerizing linker. Thus, we designed a highly conformationally constrained tricyclic dimeric peptide and synthesized it using an efficient, quasi-one-pot approach. Although still a partial BDNF-like agonist, the tricyclic dimer was particularly potent in promoting neuronal survival in vitro (EC50 11 pm). The peptides described here, which are greatly reduced in size compared with the parent protein, could serve as useful lead compounds for the development of true neurotrophic drugs and indicate that the structure-based design approach could be used to obtain potent mimetics of other growth factors that dimerize their receptors.

Cholinergic modulation of neurofilament expression and neurite outgrowth in chick sensory neurons

The morphogenetic role of the neurotransmitter acetylcholine was studied in cultures of dorsal root ganglia (DRG) neurons obtained from E12 and E18 chick embryos. With this model we have evaluated neurofilament expression and neurite outgrowth following cholinergic agonist and antagonist treatment. Morphometric analysis undertaken to evaluate fiber outgrowth has indicated that E12 DRG cultures treated with cholinergic agonists, such as muscarine and carbachol, when compared with untreated cultures, have longer fibers and a higher number of fibers per neuron. Concomitant treatment with agonists and the antagonists atropine or mecamylamine counteracts the increase in fiber outgrowth, suggesting that the cholinergic agonist effects were mediated by both muscarinic and nicotinic receptors. The expression of the three neurofilament proteins was also evaluated. Western blot analysis showed that, in E12 DRG cultures, both muscarine and carbachol induce a significant increase in neurofilament protein expression and that this effect is inhibited by cholinergic antagonist treatment. Moreover, Northern blot analysis has demonstrated that the increased expression of 68- and 145-kDa neurofilament proteins is dependent on cholinergic modulation of the neurofilament transcripts. Modulated expression of neurofilament proteins by cholinergic agonists was not evident in E18 DRG cultures, suggesting that, when sensory neurons have completed their differentiation, the cholinergic system might be involved in other functions. In conclusion, our data demonstrate that, during sensory neuron development, acetylcholine modulates neurite outgrowth controlling neurospecific marker expression.

Low-threshold heat receptor in chick sensory neurons is upregulated independently of nerve growth factor after nerve injury

In mammals, the cloned low-threshold heat receptor, vanilloid receptor subtype 1 (VR1), is involved in the genesis of thermal hyperalgesia after inflammation. However, there is evidence that VR1 is not involved in the thermal hyperalgesia that occurs after nerve injury. In search for other heat receptors which might be involved in this phenomenon, we previously demonstrated that chick dorsal root ganglion neurons, which are insensitive to capsaicin, respond to low-threshold heat. Here, we investigated whether expression of the low-threshold noxious heat receptor in chicks is regulated by nerve growth factor (NGF), as VR1 is in mammals. Heat (44 °C) responsiveness of isolated dorsal root ganglion neurons of chicks was investigated (i) under culture conditions for up to 4 days with and without NGF and (ii) after a tight ligation of the sciatic nerve for up to 6 days, using cobalt-uptake method. In every case, a significant upregulation in the proportion of heat-responsive neurons was observed. On the molecular level, there was an increase of chick VR1 mRNA level in dorsal root ganglion cells cultured for 3 days in medium lacking NGF. In rat dorsal root ganglion neurons cultured for 1–4 days without NGF, patch-clamp experiments revealed that after 1 day almost all neurons responding to heat also responded to capsaicin, whereas after 3–4 days, more than one-half of the heat-responsive neurons did not respond to capsaicin. These data suggest the existence of low-threshold heat receptors in chick dorsal root ganglion neurons, the expression of which is regulated independently of NGF.

Growing and Working with Peripheral Neurons

Cultures of vertebrate peripheral neurons have been used to address a variety of issues related to the cell biology of the neuron. They are particularly amenable to experimental manipulations, such as microinjection, and can be cultured under a variety of different conditions designed to meet the needs of the particular experiment. This chapter focuses on cultures of rat sympathetic neurons from the superior cervical ganglia and on cultures of chick sensory neurons from the lumbosacral dorsal root ganglia. Information is provided on methods for dissection, preparation of culture dishes and substrates, composition of media, relevant growth factors, reduction of nonneuronal contamination, and maintenance of the cultures.

Pp60 c-src Is a Negative Regulator of Laminin-1-Mediated Neurite Outgrowth in Chick Sensory Neurons

Multiple protein tyrosine kinases regulate neurite outgrowth in the developing nervous system. To begin to unravel the complexity of this regulation, we addressed the role of one specific kinase, pp60 c-src , in chick dorsal root ganglion (DRG) neurons grown on laminin-1, a well-characterized system to study neurite outgrowth. Pharmacological inhibition of all tyrosine kinases by genestein treatment of chick DRG neurons significantly increased neurite number and length by ∼50%. Similar increases in these parameters occurred when src-family kinases were inhibited using PP2. To implicate pp60 c-src directly in neurite outgrowth, we inactivated it in DRG neuronal growth cones using Chromophore-Assisted Laser Inactivation (CALI). CALI of pp60 c-src resulted in an 85% inactivation of its kinase activity and a 63% reduction in phosphotyrosine immunofluorescence in neurons. Microscale CALI of pp60 c-src in DRG growth cones caused a significant and acute two-fold increase in neurite extension rate during irradiation. These findings demonstrate that pp60 c-src is a negative regulator of laminin-1-mediated neurite outgrowth in chick sensory neurons.

Microtubule Reconfiguration during Axonal Retraction Induced by Nitric Oxide

Axonal retraction is induced by different types of physiological cues and is responsible for the elimination of mistargeted axons. There is broad agreement that alterations in the cytoskeleton underlie axonal retraction. The prevailing view is that axonal retraction involves a wholesale depolymerization of microtubules and microfilaments. However, axons retracting physiologically display a very different morphology than axons induced to retract by experimental depolymerization of microtubules. Experimental depolymerization of microfilaments actually prevents retraction rather than causing it. We have proposed an alternative hypothesis, namely that axonal retraction involves a backward retreat of cytoskeletal elements rather than their wholesale depolymerization. In the present study, we sought to test this hypothesis with regard to microtubules. When a donor of nitric oxide was applied to cultured chick sensory neurons, the majority of axons retracted dramatically within 30–60 min. Retracting axons were characterized by an enlarged distal region, a thin trailing remnant, and sinusoidal bends along the shaft. Quantitative immunofluorescence analyses showed no detectable loss of microtubule mass during retraction, even with regard to the most labile microtubules. Instead, microtubules were reconfigured into coiling and sinusoidal bundles to accommodate the shortening of the axon. Stabilization of microtubules by taxol did not prevent the retraction, even at concentrations of the drug that actually caused microtubule levels to increase. The retractions induced by nitric oxide were remarkably similar to those observed when motor proteins are manipulated, suggesting that these retractions may result from alterations in the activities of the motors that configure microtubules.

Microtubule reconfiguration during axonal retraction induced by nitric oxide.

Axonal retraction is induced by different types of physiological cues and is responsible for the elimination of mistargeted axons. There is broad agreement that alterations in the cytoskeleton underlie axonal retraction. The prevailing view is that axonal retraction involves a wholesale depolymerization of microtubules and microfilaments. However, axons retracting physiologically display a very different morphology than axons induced to retract by experimental depolymerization of microtubules. Experimental depolymerization of microfilaments actually prevents retraction rather than causing it. We have proposed an alternative hypothesis, namely that axonal retraction involves a backward retreat of cytoskeletal elements rather than their wholesale depolymerization. In the present study, we sought to test this hypothesis with regard to microtubules. When a donor of nitric oxide was applied to cultured chick sensory neurons, the majority of axons retracted dramatically within 30-60 min. Retracting axons were characterized by an enlarged distal region, a thin trailing remnant, and sinusoidal bends along the shaft. Quantitative immunofluorescence analyses showed no detectable loss of microtubule mass during retraction, even with regard to the most labile microtubules. Instead, microtubules were reconfigured into coiling and sinusoidal bundles to accommodate the shortening of the axon. Stabilization of microtubules by taxol did not prevent the retraction, even at concentrations of the drug that actually caused microtubule levels to increase. The retractions induced by nitric oxide were remarkably similar to those observed when motor proteins are manipulated, suggesting that these retractions may result from alterations in the activities of the motors that configure microtubules.

Temporal regulation of neuropilin-1 expression and sensitivity to semaphorin 3A in NGF- and NT3-responsive chick sensory neurons.

The extracellular molecule semaphorin 3A (Sema3A) is proposed to be a negative guidance cue that participates in patterning DRG sensory axons in the developing chick spinal cord. During development Sema3A is first expressed throughout the spinal cord gray matter, but Sema3A expression later disappears from the dorsal horn, where small-caliber cutaneous afferents terminate. Sema3A expression remains in the ventral horn, where large-muscle proprioceptive afferents terminate. It has been proposed that temporal changes in the sensitivity of different classes of sensory afferents to Sema3A contribute to the different pathfinding of these sensory afferents. This study compared the expression of the semaphorin 3A receptor subunit, neuropilin-1, and the collapse response of growth cones to semaphorin 3A for NGF (cutaneous)- and NT3 (proprioceptive)-dependent sensory axons extended from E6-E10 chick embryos. Growth cones extended from E6 DRGs in NT3-containing medium expressed neuropilin-1 and collapsed in response to Sema3A. From E7 until E10 NT3-responsive growth cones expressed progressively lower levels of neuropilin-1, and were less sensitive to Sema3A. On the other hand, growth cones extended from DRGs in NGF-containing medium expressed progressively higher levels of neuropilin-1 and higher levels of collapse response to Sema3A over the period from E6-E10. Thus, developmental patterning of sensory terminals in the chick spinal cord may arise from changes in both Sema3A expression in the developing spinal cord and accompanying changes in neuronal expression of the Sema3A receptor subunit, neuropilin-1.

The reversal potential of Ca 2+-activated Cl − currents indicates that chick sensory neurons accumulate intracellular Cl −

In order to establish the physiological role of the Ca 2+-activated Cl − current ( I Cl(Ca)) of chick primary afferent neurons, I measured the reversal potential of this current using either the amphotericin perforated patch technique (that alters intracellular Cl −) or the gramicidin perforated patch technique (that does not perturb intracellular Cl −). In the amphotericin experiments at 35°C, I Cl(Ca) reversed at the Cl − equilibrium potential ( E Cl=−24 mV) set by the superfusate (147 mM Cl −) and the pipette solution (60 mM Cl −). In contrast, in the gramicidin experiments at 35°C, I Cl(Ca) reversed at −42±2 mV, midway between E Cl of the solutions and E Cl expected if Cl − were passively distributed. Thus the gramicidin perforated patch technique monitors Cl − currents without perturbing intracellular Cl −. Further, the data imply that chick dorsal root ganglia (DRG) neurons actively accumulate Cl −. I Cl(Ca) reversed at the same potential (−46±3 mV) at 20°C indicating that the non-equilibrium distribution of Cl − is maintained at the lower temperature. Thus, I Cl(Ca) is a depolarizing current that can contribute to the after-depolarization in chick DRG neurons and thereby alter Ca 2+ influx.

Low-threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin-insensitive.

The heat-transducing receptor VR1 cloned from rat sensory neurons can be activated by both noxious heat and capsaicin. As the response of sensory neurons to capsaicin is species dependent, it is conceivable that the responses to noxious heat and to capsaicin are transduced by distinct receptors across different species. Therefore, we investigated responses to noxious heat from a capsaicin-insensitive (chick) and a capsaicin-sensitive (rat) species. In chick, whole-cell patch-clamp experiments in isolated dorsal root ganglion neurons revealed two populations of neurons with different thresholds to noxious heat, activated at approximately 43 degrees C and approximately 53 degrees C. In cobalt uptake experiments, the proportion of neurons showing a heat-induced response increased with increasing heat stimuli. Application of capsaicin (1-10 microM) did not result in inward currents or cobalt uptake. Rat neurons yielded comparable results in heat experiments, but were capsaicin-sensitive. Although chick neurons are insensitive to capsaicin, the competitive capsaicin antagonist capsazepine (1-10 microM) was effective in blocking heat-induced responses, verified by patch-clamp and cobalt uptake methods. The noncompetitive capsaicin antagonist ruthenium red (10 microM) reduced to almost nil the proportion of heat-responsive neurons identified with the cobalt uptake method. These findings suggest that chick DRG neurons express a low-threshold heat-transducing receptor with a pharmacological profile distinct from the low-threshold heat receptor VR1 cloned from rat DRG neurons. The data support the idea that there might be heat receptor subtypes with differences in the capsaicin binding site.

Modulation of cholinergic marker expression by nerve growth factor in dorsal root ganglia.

The presence of cholinergic markers in sensory ganglia has suggested a possible functional role of acetylcholine both as a cofactor of morphogenesis in embryonic life and in sensory transduction during adult life. Acetylcholine, in fact, is able to excite cutaneous nociceptors and to modulate noxious stimuli. Nerve growth factor (NGF) overexpression induces the survival of nociceptive neurons, the expression of their specific markers, and hyperalgesia. On the other hand, NGF modulate the levels of cholinergic markers in several area of nervous system. Considering these observations, the present work aims to investigate whether NGF is able also to control the expression of cholinergic markers in chick sensory neurons in culture. We selected three developmental stages (E8, E12, and E18) representative of different phases of chick embryo development and performed observations on culture in which NGF was omitted at the plating time, withdrawn after the initial 24 hr of culture or maintained for 48 hr. In the experimental protocol devised, NGF did not significantly affect cell survival. At E12 a 48 hr treatment with NGF causes a significant but limited increase in acetylcholinesterase activity; activity increase was not observed when NGF was removed after 24 hr. No changes in acetylcholinesterase activity were observed at E8 and E18 stages. NGF appears to be more effective in the modulation of choline acetyltransferase activity. At E12, in fact, about a doubling of enzyme activity was measured after 24 or 48 hr of treatment with NGF. A response was also found at E18, when a 50% increase in choline acetyltransferase activity was observed just after 24 hr treatment. The behavior of muscarinic receptors in response to NGF differs compared to the two cholinergic enzymes. At E8 and E12 a profound increase in muscarinic receptor expression was observed. Conversely, at E18 NGF produces a 50% reduction of receptors. Considering these observations and the demonstrated role of muscarinic receptors in the desensitization of nociceptors, the reduction of muscarinic receptors in DRG after NGF treatment is in agreement with the proposed algogenic action of NGF in the skin.

Neurite Outgrowth Inhibition by Chondroitin Sulfate Proteoglycan: Stalling/Stopping Exceeds Turning in Human Neuroblastoma Growth Cones

Chondroitin sulfate proteoglycan (CSPG) inhibits outgrowth from embryonic chick and rodent neurons in vivo and in vitro and is upregulated during development and following injury. The role of CSPG in outgrowth from human neurons has been largely untested, but is critical for our understanding of regeneration in humans following nervous system injury. Here we determined the effects of CSPG on platelet-derived growth factor (PDGF)-stimulated neurite outgrowth from SH-SY5Y human neuroblastoma cells, a well-accepted model of neuronal differentiation. Cells were plated on glass coverslips adsorbed with laminin (LN), CSPG, or a patterned substratum consisting of alternating stripes of the two molecules. Similar to other studies using chick or rodent neurons, SH-SY5Y cells extend neurites on LN, displaying a 15.2% increase in the total neurite length/cell as compared to cells plated on glass. Cells plated on CSPG alone exhibited reduced neurite outgrowth compared to cells plated on glass or LN. Interestingly, SH-SY5Y growth cones extending on LN and then encountering a CSPG border display more stopping/stalling (62.3%) than turning (27.9%) behaviors. Soluble CSPG inhibits neurite initiation from SH-SY5Y cells plated on glass, but not on LN. These data demonstrate that several CSPG-elicited responses of human neuron-like cells are similar to those from nonhuman neurons. However, approximately 70% of SH-SY5Y growth cones stop or stall at a CSPG border while over 80% of chick sensory neurons turn at a CSPG border. The experimental difference between these models may well indicate a functional difference between animal and human neuronal regeneration.

Modulation of N-type Ca2+ channels by intracellular pH in chick sensory neurons.

Both physiological and pathological neuronal events, many of which elevate intracellular [Ca2+], can produce changes in intracellular pH of between 0.15 and 0.5 U, between pH 7.4 and 6.8. N-type Ca2+ channels, which are intimately involved in exocytosis and other excitable cell processes, are sensitive to intracellular pH changes. However, the pH range over which N-type Ca2+ channels are sensitive, and the sensitivity of N-type Ca2+ channels to small changes in intracellular pH, are unknown. We studied the influence of intracellular pH changes on N-type calcium channel currents in dorsal root ganglion neurons, acutely isolated from 14-day-old chick embryos. Intracellular pH was monitored in patch-clamp recordings with the fluorescent dye, BCECF, and manipulated in both the acidic and basic direction by extracellular application of NH4+ in the presence and absence of intracellular NH4+. Changes in intracellular pH between 6.6 and 7.5 produced a graded change in Ca2+ current magnitude with no apparent shift in activation potential. Intracellular acidification from pH 7.3 to 7.0 reversibly inhibited Ca2+ currents by 40%. Acidification from pH 7.3 to pH 6.6 reversibly inhibited Ca2+ currents by 65%. Alkalinization from pH 7.3 to 7.5 potentiated Ca2+ currents by approximately 40%. Channels were sensitive to pHi changes with high intracellular concentrations of the Ca2+ chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, which indicates that the effects of pHi did not involve a Ca2+-dependent mechanism. These data indicate that N-type Ca2+ channel currents are extremely sensitive to small changes in pHi in the range produced by both physiological and pathological events. Furthermore, these data suggest that modulation of N-type Ca2+ channels by pHi may play an important role in physiological processes that produce small changes in pHi and a protective role in pathological mechanisms that produce larger changes in pHi.

Tau is required for neurite outgrowth and growth cone motility of chick sensory neurons.

The role of the microtubule-associated protein (MAP) tau in axon growth remains controversial. Antisense experiments have suggested that tau is required for axon outgrowth, whereas genetic knockout and immunodepletion studies have suggested that tau plays no role in this process. To investigate the role of tau in both neurite outgrowth and growth cone motility, we have used a different approach, the chromophore-assisted laser inactivation (CALI) of tau in chick dorsal root ganglion (DRG) neurons in culture. This approach generates an acute loss of tau function that is not subject to compensation by other MAPs. Inactivation of tau in whole DRG neurons (including cell body and neurites) reduced neurite number and length. Inactivation of tau within regions of growth cones using micro-scale CALI caused a decrease in neurite extension rate by approximately 2-fold. Surprisingly, it also caused a approximately 20% decrease in the lamellipodial size within the inactivation region, whereas the filopodial motility was not affected. These results suggest that tau is required in neurite outgrowth and that tau also functions in lamellipodial motility at the growth cone leading edge.

Structure-activity relationships of conformationally constrained peptide analogues of loop 2 of brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) promotes the survival of various neuronal populations and thus shows potential in the treatment of neurodegenerative disease. However, BDNF is not pharmacokinetically optimal for use as a therapeutic agent. As a step toward the development of low-molecular-weight BDNF-like drugs, we have designed a series of small, conformationally constrained peptides of various sizes using the three-dimensional structure of BDNF derived by homology modeling as a template. When tested in cultures of embryonic chick sensory neurons the peptides produced concentration-dependent inhibition of BDNF-mediated neuronal survival and caused both a rightward shift and depression of the maximum of the BDNF concentration-response curve. The compounds had no effect on the survival response to nerve growth factor and were without intrinsic trophic or toxic effects when added to cultures alone. With the aid of pharmacodynamic simulations we demonstrated that the inhibitory activity of the active peptides is consistent with them acting as competitive antagonists of BDNF for its high-affinity receptor, trkB. An alanine scan of the largest peptide identified several residues important in mediating the inhibitory action of the peptides. We intend to use the data from these studies to develop small peptidic BDNF-like agonists.

Comparison of Neurite Outgrowth Induced by Intact and Injured Sciatic Nerves: A Confocal and Functional Analysis

Mechanisms regulating axon growth in the peripheral nervous system have been studied by means of an in vitro bioassay, the tissue section culture, in which regenerating neurons are grown on substrata made up of tissue sections. Sections from intact and degenerated sciatic nerves proved to be different in their ability to support neurite outgrowth of embryonic chick sensory neurons from both qualitative and quantitative points of view. On denervated nerve sections, the total length of neurites elaborated per neuron was almost twice that found on intact nerve sections. In addition, confocal microscopy revealed a striking difference between intact and denervated nerve substrata: on denervated nerve sections, neurites grew inside the internal structures of endoneurial Schwann cell tubes, within the underlying tissue sections, whereas on intact nerve sections neurites extended along endoneurial basal laminae but never entered Schwann cell tubes. Perturbation experiments were used to analyze some of the molecular determinants that control neurite outgrowth in this system. Antibodies directed against the beta1-integrin subunit inhibited neurite extension on both normal and degenerated rat sciatic nerve tissue. Strikingly, however, differential inhibition was observed using antibodies directed against extracellular matrix molecules. Anti-laminin-2 (merosin) antibodies drastically reduced both the percentage of growing neurons and the total length of neurites on denervated nerve sections, but they did not modify these parameters on sections of normal nerve. Taken together, these results suggest that laminin-2/merosin promotes neurite outgrowth in peripheral nerve environments but only after Wallerian degeneration, which is when axons are allowed to extend within endoneurial tubes.

Receptive properties of embryonic chick sensory neurons innervating skin.

Receptive properties of embryonic chick sensory neurons innervating skin. J. Neurophysiol. 78: 2560-2568, 1997. We describe a new in vitro skin-nerve preparation from chick embryos that allows detailed study of the functional properties of developing sensory neurons innervating skin. Functionally single sensory afferents were isolated by recording from their axons in microdissected filaments of the cutaneous femoralis medialis nerve, which innervates skin of the thigh. A total of 157 single neurons were characterized from embryos [embryonic days 17-21 (E17-E21), n = 115] and hatchlings up to 3 wk old (n = 42). Neurons were initially classified on the basis of their conduction velocity; those conducting below 1.0 m/s were being classified as C fibers and faster conducting fibers as A fibers. The proportions of A and C fibers encountered in embryonic and hatchling preparations were not very different, indicating that myelination and axon growth proceeds quite slowly over the period studied. Afferent fibers that could subserve nociceptive and nonnociceptive functions were identified in the time period studied. Subpopulations of low-threshold myelinated afferent units exhibited rapidly or slowly adapting discharges to constant force stimuli and could have tactile functions. Many afferent fibers responded to noxious heat and were excited and sensitized by exposure to inflammatory mediators, suggesting that they are nociceptors. The behavior of these units changed in several respects over the period studied. The discharge of C fibers to noxious heat increased with age as did their mechanical thresholds. A substantial population of heat-responsive neurons (34% of the A fibers) present in embryos were not encountered in hatchling chicks. This indicates that substantial changes in the physiological response properties of sensory afferents occur after hatching. We conclude that this new preparation can be used for quantitative assessment of the receptive properties of developing sensory neurons and has considerable potential for the investigation of factors, such as neurotrophins, that specify and influence the functional phenotype of sensory neurons during embryonic development in vivo.

Ca2+ and Na+ permeability of high-threshold Ca2+ channels and their voltage-dependent block by Mg2+ ions in chick sensory neurones.

1. The Mg2+ block of Na+ and Ca2+ currents through high-voltage activated (HVA; L- and N-type) Ca2+ channels was studied in chick dorsal root ganglion neurones. 2. In low extracellular [Ca2+] (< 10(-8) M) and with Na+o and Cs+i as the main charge carriers (120 mM), HVA Na+ currents started to activate at -40 mV, reached inward peak values near 0 mV and reversed at about +40 mV. 3. Addition of 30-500 microM Mg2+ to the bath caused a strong depression of inward Na+ currents that was voltage and dose dependent (KD = 39 microM in 120 mM Na+ at -10 mV). The block was maximal at negative potentials (< -70 mV) and decreased with increasing positive potentials, suggesting that Mg2+ cannot escape to the cell interior. 4. Block of Ca2+ currents by Mg2+ was also voltage dependent, but by three orders of magnitude less potent than with Na+ currents (KD = 24 mM in 2 mM Ca2+ at -30 mV). The high concentration of Mg2+ caused a prominent voltage shift of channel gating kinetics induced by surface charge screening effects. To compensate for this, Mg2+ block of inward Ca2+ currents was estimated from the instantaneous I-V relationships on return from very positive potentials (+100 mV). 5. Inward Na+ and Ca2+ tail currents following depolarization to +90 mV were markedly depressed, suggesting that channels cleared of Mg2+ ions during strong depolarization are quickly re-blocked on return to negative potentials. The kinetics of re-block by Mg2+ was too fast (< 100 microseconds) to be resolved by our recording apparatus. This implies a rate of entry for Mg2+ > 1.45 x 10(8) M-1 S-1 when Na+ is the permeating ion and a rate approximately 3 orders of magnitude smaller for Ca2+. 6. Mg2+ unblock of HVA Na+ currents at +100 mV was independent of the size of outward currents, whether Na+, Cs+ or NMG+ were the main internal cations. 7. Consistent with the idea of a high-affinity binding site for Ca2+ inside the channel, micromolar amounts of Ca2+ caused a strong depression of Na+ currents between -40 and 0 mV, which was effectively relieved with more positive as well as with negative potentials (KD = 0.7 microM in 120 mM Na+ at -20 mV). In this case, the kinetics of re-block could be resolved and gave rates of entry and exit for Ca2+ of 1.4 x 10(8) M-1 S-1 and 2.95 x 10(2) s-1, respectively. 8. The strong voltage dependence and weak current dependence of HVA channel block by divalent cations and the markedly different KD values of Na+ and Ca2+ current block by Mg2+ can be well described by a previously proposed model for Ca2+ channel permeation based on interactions between the permeating ion and the negative charges forming the high-affinity binding site for Ca2+ inside the pore (Lux, Carbone & Zucker, 1990).

Cytomechanics of axonal development.

Mechanical tension is a robust regulator of axonal development of cultured neurons. We review work from our laboratory, using calibrated glass needles to measure or apply tension to chick sensory neurons, chick forebrain neurons, and rat PC12 cells. We survey direct evidence for two different regimes of tension effects on neurons, a fluid-like growth regime, and a nongrowth, elastic regime. Above a minimum tension threshold, we observe growth effects of tension regulating four phases of axonal development: 1. Initiation of process outgrowth from the cell body; 2. Growth cone-mediated elongation of the axon; 3. Elongation of the axon after synaptogenesis, which normally accommodates the skeletal growth of vertebrates; and 4. Axonal elimination by retraction. Significantly, the quantitative relationship between the force and the growth response is surprisingly similar to the simple relationship characteristic of Newtonian fluid mechanical elements: elongation rate is directly proportional to tension (above the threshold), and this robust linear relationship extends from physiological growth rates to far-above-physiological rates. Thus, tension apparently integrates the complex biochemistry of axonal elongation, including cytoskeletal and membrane dynamics, to produce a simple "force input/growth output" relationship. In addition to this fluid-like growth response, peripheral neurons show elastic behaviors at low tensions (below the threshold tension for growth), as do most cell types. Thus, neurites could exert small static forces without diminution for long periods. In addition, axons of peripheral neurons can actively generate modest tensions, presumably similar to muscle contraction, at tensions near zero. The elastic and force-generating capability of neural axons has recently been proposed to play a major role in the morphogenesis of the brain.

Targeted Transduction of CNS Neurons with Adenoviral Vectors Carrying Neurotrophic Factor Genes Confers Neuroprotection That Exceeds the Transduced Population

Application of neurotrophic factors (NFs) to the cut stump of motor nerves of neonatal rats confers neuroprotection from trauma-induced neuronal death. To test whether motoneurons are capable of responding to endogenously produced NFs, facial motoneurons were genetically modified in vivo to express several NFs and then tested for their response to peripheral nerve damage. Replication-defective adenoviral vectors [Adv. Rous sarcoma virus (RSV)-nf] representing three families of NFs were constructed that carried genes for brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor. Media from cultured cells transduced with Adv. RSV-nf contained NFs that supported the survival of cultured chick sensory neurons in the same manner as recombinant NF standards. When Adv.RSV-nf or an adenoviral vector containing the beta-galactosidase gene (Adv.RSV-beta-gal) were injected into the facial muscles of neonatal rats the vectors were retrogradely transported to the facial nucleus where the NFs or beta-gal were expressed. A fraction (approximately 10%) of the neurons were transduced as demonstrated by reverse transcriptase-PCR, histochemistry, and immunocytochemistry. In the case of Adv.RSV-BDNF, Adv.RSV-CNTF, and Adv.RSV-GDNF, a significant portion of the facial nucleus neurons was protected, 16.5, 18.2, and 53.3%, respectively, from death after axotomy, showing that neurons are capable of transporting the Adv. RSV-nf, expressing the recombinant NF genes, and responding to the NFs. In the case of Adv.RSV-GDNF, a greater number of facial nucleus motoneurons survived than were transduced, indicating that neighboring untransduced neurons were protected by the GDNF expressed by the transduced neurons by a paracrine mechanism.