Multiple effects of artemin on sympathetic neurone generation, survival and growth

To define the role of artemin in sympathetic neurone development, we have studied the effect of artemin on the generation, survival and growth of sympathetic neurones in low-density dissociated cultures of mouse cervical and thoracic paravertebral sympathetic ganglia at stages throughout embryonic and postnatal development. Artemin promoted the proliferation of sympathetic neuroblasts and increased the generation of new neurones in cultures established from E12 to E14 ganglia. Artemin also exerted a transient survival-promoting action on newly generated neurones during these early stages of development. Between E16 and P8, artemin exerted no effect on survival, but by P12, as sympathetic neurones begin to acquire neurotrophic factor independent survival, artemin once again enhanced survival, and by P20 it promoted survival as effectively as nerve growth factor (NGF). During this late period of development, artemin also enhanced the growth of neurites from cultured neurones more effectively than NGF. Confirming the physiological relevance of the mitogenic action of artemin on cultured neuroblasts, there was a marked reduction in the rate of neuroblast proliferation in the sympathetic ganglia of mice lacking the GFRα3 subunit of the artemin receptor. These results indicate that artemin exerts several distinct effects on the generation, survival and growth of sympathetic neurones at different stages of development.

Nerve Growth Factor Regulates Sodium But Not Potassium Channel Currents in Sympathetic B Neurons of Adult Bullfrogs

The TTX-sensitive and -resistant components of the voltage-gated Na+ current (TTX-s I Na and TTX-r I Na) are increased within 2 wk of cutting the axons of B-cells in bullfrog paravertebral sympathetic ganglia (BFSG). Axotomy also increases the noninactivating, voltage-activated K+ current (M current I M), whereas delayed rectifier K+ current ( I K) is reduced. We found that similar effects were produced when BFSG B cells were dissociated from adult bullfrogs and maintained in a defined-medium, neuron-enriched, low-density, serum-free culture. Thus the density of TTX-s I Na, TTX-r I Na, and I M were transiently increased, whereas I K density was decreased. Reduction in voltage-sensitive, Ca2+-dependent K+ current ( I C) was attributed to previously documented decreases in Ca2+ channel current ( I Ca). To test whether axotomy- or culture-induced changes in ion channel function reflect loss of retrograde influence of nerve growth factor (NGF), we examined the effect of murine β-NGF on TTX-s I Na, TTX-r I Na, I K, and I M. Culture of neurons for 15 days in the presence of NGF (200 ng/ml), more than doubled total I Na density but did not enhance neurite outgrowth. The TTX-r I Nadensity was increased about threefold and the TTX-s I Na density increased 2.4-fold. NGF did not affect the activation or inactivation kinetics of the total Na+ conductance. Effects of NGF were blocked by the transcription inhibitors, cordycepin (20 μM) and actinomycin D (0.01 μg/ml). I K and I M were unaffected by NGF, and although I C was enhanced, this likely reflected the known effect of NGF on I Ca in BFSG neurons. Na+ channel synthesis and/or expression in adult sympathetic neurons is therefore subject to selective regulation by NGF. Despite this, the increase in I Na and I M as well as the decrease in I K seen in BFSG neurons in culture or after axotomy cannot readily be explained in terms of alterations in the availability of target-derived NGF.

Interaction of vasomotor and exocrine neurons in bullfrog paravertebral sympathetic ganglia

A 2 min sample of an intracellular recording of in vivo synaptic activity from a vasomotor C-neuron in a bullfrog sympathetic ganglion was converted to a series of stimulus pulses. This physiologically derived activity was used to stimulate preganglionic C-fibres of similar ganglia studied in vitro. Intracellular recordings were made from exocrine B-cells within the ganglia. Although they do not receive fast, nicotinic synaptic input from preganglionic C-fibres, B-cell excitability was profoundly increased by stimulation of C-fibres with physiologically derived activity. Also, subthreshold depolarizing current pulses that failed to generate action potentials in B-cells under control conditions almost always generated action potentials whilst C-fibres were activated. These effects were attenuated or prevented by the luteinizing hormone releasing hormone antagonist, [D-pyro-Glu1,D-Phe2,D-Trp3,6]-LHRH (70 µM). The physiological release of luteinizing hormone releasing hormone from C-fibres therefore causes an interaction between vasomotor and exocrine outflow within a paravertebral sympathetic ganglion.Key words: ganglionic transmission, hypertension, autonomic nerve, m-current, neuropeptide.

On the role of muscarinic and peptidergic receptors in ganglionic transmission in bullfrogs in vivo

Synaptic activity of individual B and C cells in the paravertebral sympathetic ganglia of urethan-anesthetized bullfrogs was monitored with intracellular electrodes. Postganglionic activity from the B and C fiber populations was monitored with suction electrodes. Intravenous infusion of muscarine (0.1 ml of 8 microM) excited individual B cells and increased the amplitude and rate of spontaneous, postganglionic B fiber population discharges. Muscarine also increased the number of action potentials (APs) within each burst of synaptic activity in individual C cells. Because atropine (0.1 ml of 0.1 microM) had little or no effect on postganglionic population B or C fiber activity, the muscarinic slow inhibitory postsynaptic potentials and slow excitatory postsynaptic potentials (EPSPs) are unlikely to be involved in the transmission, modulation, or integration of postganglionic outflow in vivo. Atropine did, however, decrease the number of APs per burst in individual C cells, an effect that could be explained if excitatory presynaptic muscarinic receptors exist on C fiber terminals. Stimulation of preganglionic C fibers at "physiological" frequencies evoked a lasting afterdischarge in postganglionic B fibers that was blocked by a combination of atropine and [D-pyro-Glu1,D-Phe2,D-Trp3,6]-luteinizing hormone-releasing hormone (LHRH). Release of LHRH from C fiber terminals and activation of the peptidergic, late-slow EPSP mechanism in B cells may therefore play a role in ganglionic transmission in vivo.

The expression of neuropeptide Y immunoreactivity in the avian sympathoadrenal system conforms with two models of coexpression development for neurons and chromaffin cells

We have studied the expression and development of neuropeptide Y-like immunoreactivity (NPY-LI) in the sympathoadrenal system of the chicken using single and double immunocytochemical techniques and radioimmunoassay. NPY-LI is expressed by neurons of the paravertebral sympathetic ganglia and by chromaffin cells of the adrenal gland in embryonic and adult chickens. The peptide is coexpressed with catecholaminergic properties in neurons. In chromaffin cells, it is also expressed with immunoreactivity to somatostatin and serotonin. We have used the expression of NPY-LI to analyze how cells that coexpress two or more neuroactive substances arrive at their final phenotype. Our results suggest that the ontogeny of coexpression in neurons of the avian paravertebral sympathetic ganglia occurs in a sequential pattern, where the expression of the peptide follows the initial expression of the “classical neurotransmitter”. In contrast, in chromaffin cells, expression of the peptides occurs concomitantly with expression of catecholaminergic properties or soon after. Initially, coexpression of several neuroactive substances occurs, but this is followed by further specialization where the expression of one peptide prevails over the other. We believe that the two models of coexpression shown by our results can be used to describe the ontogeny of coexpression in other cells of the nervous system.