Retrograde factors in peripheral nerves.

The relationship between the neuron and its target is explored and the possible mechanisms for achieving correct connections are analysed. The most plausible mechanism is the presence of a retrograde intra-axonal message from the target to the neuronal cell body. The molecular form of the message and the mechanisms to achieve this signal transduction are discussed and it is proposed that there are two types of neurotrophic factors. One has a short-acting second messenger, itself incapable of surviving for the time required for transport to the cell body and thus requiring the transport of the message-generating complex to the cell body. The other has a long-lasting second messenger complex which is well able to survive the transport to the cell body so that there is no need for the transport of the neurotrophic factor itself. Thus all neurotrophic factors do not themselves require retrograde axonal transport and such non-transportable factors may generate intricate messages due to associations of signal transduction molecules via binding sites such as phosphorylated tyrosines and the src homology domain 2.

Gi alpha and Gi beta are part of a signalling complex in Balb/c3T3 cells: phosphorylation of Gi beta in growth-factor-activated fibroblasts.

Stimulation of division of Balb/c3T3 cells by epidermal growth factor (EGF) and/or insulin is inhibited by pertussis toxin. The G-protein involvement in this response includes the growth factor receptor-induced translocation of the alpha-subunit of Gi (Gi alpha) to the nucleus, where Gi alpha binds specifically to chromatin of dividing cells. This paper reports the first data of studies on the mode of interaction of tyrosine kinase growth factor receptors with Gi alpha, and the mechanism by which Gi affects cell proliferation. When Gi alpha was immunoprecipitated from Triton X-100 extracts of Balb/c3T3 cells, several other proteins were co-precipitated. The major proteins, of 110,000, 60,000 and 36,000 M(r), were not directly recognized by the Gi alpha antibody, showing that Gi alpha was in a complex with these proteins. The 36,000 M(r) protein was recognized by G beta-common antiserum, so confirming its identity as Gi beta. The 36,000 M(r) protein was phosphorylated in cells activated for 20 h with platelet-derived growth factor, epidermal growth factor and insulin, but not after 3 min or 1 h of stimulation. Both Gi alpha and G beta-common antibodies precipitated the phosphorylated 36,000 protein. Gi beta phosphorylation was similarly observed in response to activation by EGF alone for 20 h, but to a lesser extent. Phosphotyrosine antibodies also precipitated a 36,000 M(r) phosphorylated protein from growth factor-activated cells, suggesting that Gi beta may be phosphorylated on tyrosine. Therefore, Gi beta phosphorylation appears to represent a late event after activation of cells by tyrosine kinase growth factor receptors. We are currently examining the role of this event in signal transduction, particularly in relation to control of nuclear responses.

Mammalian motoneuron development: effect of peripheral deprivation on motoneuron numbers in a marsupial.

In nonmammalian vertebrates, the survival of developing motoneurons is dependent on their contacting appropriate target cells. It is generally accepted that developing mammalian motoneurons have a similar dependency on their target, but as yet there is little experimental evidence to support this contention. This is mainly because of the difficulty of experimenting on eutherian embryos. We have, therefore, been studying neuronal development in the tammar (an Australian marsupial) as its nervous system is immature at birth. Radical or partial removal of hindlimb buds from newborn tammars resulted in an increased motoneuron cell death. The motoneurons which survived in the operated tammars did so by innervating muscle remnants. In the instances where a group of muscles was totally removed, the corresponding motonuclei appeared to be totally lost. This study supports the hypothesis that mammalian motoneurons must contact their appropriate muscle in order to survive through the period of natural neuronal cell death.

Deletion of guanine nucleotide binding protein alpha z subunit in mice induces a gene dose dependent tolerance to morphine.

The mechanism underlying the development of tolerance to morphine is still incompletely understood. Morphine binds to opioid receptors, which in turn activates downstream second messenger cascades through heterotrimeric guanine nucleotide binding proteins (G proteins). In this paper, we show that G(z), a member of the inhibitory G protein family, plays an important role in mediating the analgesic and lethality effects of morphine after tolerance development. We blocked signaling through the G(z) second messenger cascade by genetic ablation of the alpha subunit of the G protein in mice. The Galpha(z) knockout mouse develops significantly increased tolerance to morphine, which depends on Galpha(z) gene dosage. Further experiments demonstrate that the enhanced morphine tolerance is not caused by pharmacokinetic and behavioural learning mechanisms. The results suggest that G(z) signaling pathways are involved in transducing the analgesic and lethality effects of morphine following chronic morphine treatment.

Differences in sensitivity to nerve growth factor of axon formation and tyrosine hydroxylase induction in cultured sympathetic neurons.

Superior cervical ganglia from 2-day-old and 3-week-old rats were maintained in vitro for up to 2 weeks in the presence of a range of concentrations of nerve growth factor up to 100 micrograms/ml. Nerve fibre length and density were measured and tyrosine hydroxylase activity of these cultures assayed after various times. Ganglia were also examined for catecholamines and neuronal numbers using fluorescence histochemistry and histology respectively. In cultures maintained without nerve growth factor, or in those containing low concentrations of nerve growth factor (3 ng/ml), tyrosine hydroxylase decreased to 5-10% of the initial levels by 14 days in vitro. The presence of the high concentration of 1 microgram/ml nerve growth factor in the culture medium or the addition of such a concentration during the culture period did not prevent an initial decrease in tyrosine hydroxylase but subsequently increased the enzyme activity. The maximal effect of nerve growth factor on nerve fibre density was at low concentrations whereas its maximal effect on neuronal survival, tyrosine hydroxylase activity or nerve fibre elongation was at high concentrations. After 2 days in culture, maximum neurite production occurred in cultures containing 10 ng/ml, while maximum nerve fibre elongation and tyrosine hydroxylase activity occurred in cultures containing 100 micrograms/ml nerve growth factor. We conclude that low concentrations of nerve growth factor, as occur in plasma, cause maximum axon formation while high concentrations of nerve growth factor, as occur in effector organs, induce maximum tyrosine hydroxylase activity and cell survival. The former process may be mediated via cell surface receptors and the latter via retrograde axonal transport of nerve growth factor to the cell body, following uptake by the terminal regions of the axons.

Intraspinal release of substance P and calcitonin gene-related peptide during opiate dependence and withdrawal.

The antibody microprobe technique was used to study the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of anaesthetized spinalized cats pretreated twice daily for 3.5 days with increasing doses of morphine hydrochloride (2-20 mg/kg, i.p.). Both peptides were released in the region of the substantia gelatinosa during noxious cutaneous thermal stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of naloxone increased the nociceptive excitation of lumbar dorsal horn neurons, but did not alter the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial gray matter dorsal to these neurons. In addition, the release of both peptides was not significantly different to that detected under similar experimental conditions in opioid-naive cats. The results suggest that alterations in neuropeptide release from the central terminals of nociceptive primary afferent neurons do not occur during states of opiate dependence and withdrawal, and thus do not contribute to the characteristic signs of these phenomena in dependent animals.

Transforming growth factor-beta 2 is anterogradely and retrogradely transported in motoneurons and up-regulated after nerve injury.

The survival of motoneurons is dependent on them receiving continual trophic support from muscle fibres and various other cell types. Numerous putative survival factors have been identified and a set of criteria established by which these candidates can be assessed. These criteria include the need for the factor and its receptors to be in appropriate locations and for the factor or its second message to be retrogradely transported. In this paper, we demonstrate that a multifunctional cytokine, transforming growth factor-beta 2, appears to meet these criteria. The locations of the transforming growth factor-beta 2 and its receptors in the neuromuscular system were determined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Motoneurons were shown to synthesize the three proteins involved in transforming growth factor-beta 2 signalling (types I and II transforming growth factor-beta receptor and betaglycan) and to transport them anterogradely, where they were inserted into the axonal membrane and nerve terminal. Transforming growth factor-beta 2 was detected in the synaptic portions of muscle fibres, motoneurons and in injured nerves, indicating that motoneurons may be exposed to multiple and potentially redundant sources of transforming growth factor-beta 2. Double-ligation experiments were used to demonstrate that motoneurons transport transforming growth factor-beta 2 up and down their axons. The anterograde transport of both transforming growth factor-beta 2 and its receptors, coupled with the fact that most of a motoneuron's mitochondria are located in the axon, raises the issue of whether the repression of the initiation of apoptosis is restricted to the cell body or occurs along the entire length of a neuron.

Anterograde and retrograde transport of active extracellular signal-related kinase 1 (ERK1) in the ligated rat sciatic nerve.

Neurons are one of the most polarized cells and often the nerve terminals may be located long distances from the cell body, thus signal transduction in neurons unlike other cells may need to be conducted over large distances. The mitogen-activated protein/extracellular signal-regulated kinases (MAP kinases or ERKs) regulate a diverse array of functions and in neurons, the ERK signalling pathways appear to have an important role in activity-dependent regulation of neuronal function. Using the ligated rat sciatic nerve as an experimental model we previously showed that the ERK1/2, MAP/ERK kinase (MEK1/2) and the p110 catalytic subunit of PI3-kinase are transported in the rat sciatic nerve. We have extended these findings to determine if these proteins are transported in the active state using antibodies that specifically detect the active form of ERK1/2, MEK1/2 and AKT which is activated downstream of PI3-kinase. We show significant accumulation of active ERK1 on the proximal and distal sides of a nerve ligation after 16 h. Active ERK2 also appeared to be accumulating at the ligature, however this did not reach statistical significance. In contrast there was not any significant accumulation of active MEK1/2 or active AKT. A component of both active ERK1 and active ERK2 is present in between the two ligations suggesting they are also present in the surrounding Schwann cells and are activated in response to nerve injury. Taken together our results suggest that a component of the accumulation of active ERK1 on the distal and proximal side of the nerve ligations results from transport in the anterograde and retrograde direction in the rat sciatic nerve.

Targeted disruption of the mouse Gz-alpha gene: a role for Gz in platelet function?

Gz is one of nine G proteins identified in platelets and its role in these cells is unknown. Our laboratory has generated a mouse deficient in the Gz-alpha gene in the hope of determining its in vivo function. Bleeding times from the tail tip of Gzalpha deficient mice was significantly longer than wild type mice. Platelet aggregation and ATP secretion did not differ between wild type and Gzalpha deficient mice. When mice were presented with a thromboembolism challenge no differences were observed in the survival or mortality of wild type or Gzalpha deficient mice, however a strain difference was observed. Ignoring the genetic background of a mutant mouse might lead to a misinterpretation of results and thus it is absolutely critical to take the genetic background into account when assessing any aspect of a mutant mouse.

Involvement of pertussis toxin-sensitive and -insensitive mechanisms in alpha-adrenoceptor modulation of noradrenaline release from rat sympathetic neurones in tissue culture.

1. Sympathetic neurones derived from superior cervical ganglia of neonatal rats and maintained in tissue culture were used to investigate the modulation of neurotransmitter release by presynaptic receptors. Three week old cultures of neurones were loaded with [3H]-noradrenaline to label endogenous neurotransmitter stores. Release of noradrenaline was evoked by depolarization with raised extracellular K+ in the presence of desipramine and corticosterone to prevent uptake of released catecholamine. 2. Potassium (55 mmol l-1) depolarization for 30 s caused more than a four fold increase in 3H overflow from basal levels but this increase was reduced by up to 40% in the presence of exogenous noradrenaline (1 mumol l-1). The inhibition by noradrenaline of depolarization-evoked overflow was blocked by the alpha 1/alpha 2-adrenoceptor antagonist, phentolamine. Phentolamine alone did not increase K(+)-evoked 3H overflow. 3. The alpha 2-adrenoceptor antagonist, yohimbine, produced a concentration-dependent block of the inhibition by noradrenaline of K(+)-evoked overflow, while the alpha 1-adrenoceptor antagonist, prazosin, was without effect at concentrations up to 0.1 mumol l-1. 4. The beta-adrenoceptor antagonist, propranolol, neither reduced K(+)-evoked overflow nor increased the degree of inhibition caused by the addition of 1 mumol l-1 noradrenaline. 5. The alpha 2-adrenoceptor agonist, clonidine (1 mumol l-1) was less effective than noradrenaline at inhibiting K(+)-evoked overflow, while the alpha 1-adrenoceptor agonist, phenylephrine (1 mumol l-1) had no significant effect. 6. The L-channel calcium blocker, nicardipine (1 mumol l-1) significantly inhibited 3H overflow evoked by K+. In the presence of L-channel block, however, noradrenaline still inhibited residual evoked overflow.7. In the presence or absence of nicardipine, pertussis toxin pretreatment (1 nmol 1-1) reduced, but did not prevent, the effect of noradrenaline (1 micromol 1-1). Pertussis toxin alone caused a significant enhancement of K+-evoked 3H overflow.8. The data indicate that on postganglionic neurones of cultured rat sympathetic ganglia there are alpha 2-adrenoceptors that modulate neurotransmitter release, but no functional beta-adrenoceptors that mediate an enhancement of transmitter release. The data suggest further that in this preparation the mechanism of alpha2-adrenoceptor modulation may involve pertussis toxin sensitive and insensitive G-proteins and effects on calcium channels other than L-type.

Axonal transport of neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta in the adult rat sciatic nerve.

Neuronal calcium sensor-1 (NCS-1) and its putative substrate phosphatidylinositol 4-kinase beta (PtdIns 4-kinase beta) both indirectly regulate synaptic vesicle exocytosis and are located in DRG neurites. In this study we have tested whether NCS-1 and PtdIns 4-kinase beta are transported in axons using the analysis of double ligation approach in the adult rat sciatic nerve. We show that NCS-1 accumulates on both the distal and proximal side of the nerve ligation indicating that this protein undergoes bidirectional transport in axons. In contrast, PtdIns 4-kinase beta accumulated on the distal side which suggests that it undergoes retrograde axonal transport and unlike NCS-1 was also present in non-neuronal cells.

Transport of endosomal early antigen 1 in the rat sciatic nerve and location in cultured neurons.

Early endosomal antigen 1 (EEA1) is known to be a marker of early endosomes and in cultured hippocampal neurons it preferentially localizes to the dendritic but not the axonal compartment. We show in cultured dorsal root ganglia and superior cervical ganglia neurons that EEA1 localizes to the cell bodies and the neurites of both sensory and sympathetic neurons. We then show in vivo using a ligated rat sciatic nerve that EEA1 significantly accumulates on the proximal side and not on the distal side of the ligation. This suggests that EEA1 is transported in the anterograde direction in axons either as part of the homeostatic process or to the nerve ligation site in response to nerve injury.

Release of immunoreactive somatostatin in the spinal dorsal horn of the cat.

Antibody microprobes were used to investigate the possible release of immunoreactive somatostatin (irSS) within the lumbar spinal cord of anaesthetized cats. A basal release of irSS was detected in the region of the substantia gelatinosa of the dorsal horn. By comparison with in vitro standards the concentration of SS detected in this region was estimated at 10(-7) M. This release of irSS was not significantly altered by electrical stimulation of large myelinated primary afferent fibres but was increased when unmyelinated afferents were additionally stimulated. Release of irSS was also detected at the spinal cord surface. The results support a role for somatostatin in nociceptive transmission in the spinal cord.

Analysis of antibody microprobe autoradiographs by computerized image processing.

A computerized digitizing method to analyze the autoradiographs derived from the antibody microprobe technique is described. The method enables the averaging of groups of microprobes that have been placed in similar locations in the central nervous system during the same physiological stimulation. Control and experimental groups of microprobes can be compared, and a level of significance for the release of a neuropeptide obtained. In addition estimates can be made of the concentration of peptide present in regions of release.

The preparation and use of antibody microprobes.

A new method of detecting release of neuropeptides in the central nervous system is described. Glass micropipettes are treated with gamma-aminopropyltriethoxysilane resulting in a fine outer coating of a siloxane polymer containing free amino groups. Glutaraldehyde is then used to covalently couple protein A which in turn binds antibodies to a particular peptide. Following use in the central nervous system, microprobes are incubated in a radiolabelled form of the peptide being studied and release is detected on autoradiographs as localized zones of inhibition of binding of the labelled peptide. The spatial resolution of the method is at least 100 micron. Necessary tests of the validity of the technique are also described.

Development and characterization of human and mouse specific antibodies to CuZn-superoxide dismutase (SOD1).

Mutations in the copper/zinc superoxide dismutase (SOD1) gene are associated with 15-20% of the familial forms of motor neuron disease. Mice where a transgene has been incorporated that encodes for the human SOD1 mutation develop a form of motor neurone disease that closely resembles human forms of this disease. We have produced and characterized species-specific antibodies to epitopes in the SOD1 protein, amino acids 25-37, a region that distinguishes between the human and the mouse species of SOD1. The antisera generated were unable to immunoprecipitate the mouse or the human forms of SOD1 from tissue extracts unless the homodimeric complex of SOD1 was denatured. As SOD1 exists as a homodimeric complex in the cytoplasm of cells, this suggests that amino acids in position, 25-37 are close to the dimeric interface of SOD1.

Effects of axotomy on the trans-synaptic regulation of enzyme activity in adult rat superior cervical ganglia.

The effects of surgical transection of the postganglionic nerve trunk of the superior cervical ganglion on the total protein content and levels of the enzymes tyrosine hydroxylase, DOPA decarboxylase and choline acetyltransferase have been studied in the adult rat. There is a minor decrease in the total activities of these 3 enzymes accompanied by a large increase in the total protein content of the ganglion. The trans-synaptic induction of the enzyme tyrosine hydroxylase by reserpine is not affected by postganglionic axotomy. Increased activity mediated by reserpine caused no change in the total activities of either DOPA decarboxylase or choline acetyltransferase. Previously observed effects of postganglionic axotomy on preventing transmission through the ganglion are compared with these results and the possible mechanisms by which trans-synaptic induction may occur are discussed.

The response of adrenergic neurones to axotomy and nerve growth factor.

Division of the axons of adrenergic neurones by crushing the postganglionic nerve trunks of rat superior cervical ganglia (SCG) at 6 days of age resulted in a permanent atrophy of the SCG reflected by a persistent decrease in the total protein content and in the activities of the enzymes tyrosine hydroxylase and DOPA decarboxylase. Administration of nerve growth factor (NGF) to rats with unilateral axotomy at a dose of 10 mug/g/day for the period 7-21 days of age resulted in hypertrophy of both normal and axotomised SCG. There was a progressive rise in the total protein content and in the activities of the two enzymes till the end of the treatment period in both SCG. After treatment ceased there was a progressive fall in the total protein content and activities of the two enzymes reaching a stable level after 4 weeks. The level reached for treated unoperated SCG remained elevated when compared to untreated control SCG. Axotomised treated SCG had approximately the same biochemical parameters as untreated control SCG and very much elevated over untreated axotomised SCG. These final levels persisted for at least 56 days after treatment had ceased. Animals showed a persistent ptosis after axotomy at 6 days of age but treatment with NGF resulted in a functional recovery by 11 weeks of age. It is suggested that there is normally a retrograde transfer of a factor durind development from the target cell to the perikarya of the neurone permitting survival if the appropriate connections are made. Failure to make such a contact results in cedd death. The cell death occurring normally, and the cell death resulting from axotomy, can both be prevented by NGF treatment leading to an hypertrophy of both SCG. This consistent with the hypothesis than NGF is the retrograde trophic agent for the sympathetic nervous system in the developing animal.

The effects of axotomy on the development of the rat superior cervical ganglion.

The effects of division of the postganglionic axons of the adrenergic neurones in the superior cervical ganglion of the rat were examined with regard to the total ganglionic protein content and tyrosine hydroxylase and DOPA decarboxylase activities. Axotomy before the twelfth postnatal day results in a marked atrophy of the ganglion and a reduction in the total enzyme content of the ganglion. Axotomy after postnatal day 21 results in the normal adult ganglion response with a large increase in the total protein content of the ganglion and only minor changes in the total enzyme content of the ganglion. Axotomy between the postnatal days 12 and 21 results in an intermediate response. Thus it can be concluded there is a critical period during the development of the ganglion during which the adrenergic neurones undergo a maturation governed by their contact with the peripheral target cell via axons. These results suggest a trophic role of the end organ on the adrenergic neurone.

The effect of the retrograde axonal transport of nerve growth factor on the morphology of adrenergic neurones.

An injection of nerve growth factor (NGF) into one eye of neonatal rats results in an increase in the tyrosine hydroxylase activity of the ipsilateral superior cervical ganglion. This effect was seen maximally after the intraocular injection of a depot preparation of NGF linked to cellulose. The sympathetic neurones that innervate the eye can be identified by autoradiography after the retrograde axonal transport of either NGF or tetanus toxin labelled with [125I]iodine. It was only those cells having their terminals in the vicinity of the depot preparation. This demonstrates that NGF transported from the periphery to the cell bodies is effectively retained within the transporting cell and is not released to act on extracellular receptors within the ganglion. It is suggested that this specificity of action for NGF reaching the ganglion in this fashion is important during normal development in determining the survival of adrenergic neurones.