Richard E. Zigmond

Leukemia inhibitory factor mediates an injury response but not a target-directed developmental transmitter switch in sympathetic neurons

Leukemia inhibitory factor (LIF; also known as cholinergic differentiation factor) is a multifunctional cytokine that affects neurons, as well as many other cell types. To examine its neuronal functions in vivo, we have used LIF-deficient mice. In culture, LIF alters the transmitter phenotype of sympathetic neurons, inducing cholinergic function, reducing noradrenergic function, and altering neuropeptide expression. In vivo, a noradrenergic to cholinergic switch occurs in the developing sweat gland innervation, and changes in neuropeptide phenotype occur in axotomized adult ganglia. We find that the gland innervation of LIF-deficient mice is indistinguishable from normal. In contrast, neuropeptide induction in ganglia cultured as explants or axotomized in situ is significantly suppressed in LIF-deficient mice. Thus, LIF plays a role in transmitter changes induced by axotomy but not by developmental interactions with sweat glands.

Involvement of Leukemia Inhibitory Factor in the Increases in Galanin and Vasoactive Intestinal Peptide mRNA and the Decreases in Neuropeptide Y and Tyrosine Hydroxylase mRNA in Sympathetic Neurons After Axotomy

Abstract: In response to axonal injury, noradrenergic sympathetic neurons of the adult superior cervical ganglion (SCG) alter their neurotransmitter phenotype. These alterations include increases in the levels of the neuropeptides, galanin, vasoactive intestinal peptide (VIP), and substance P (SP) and a decrease in the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). Previous studies have indicated that after axotomy in vivo, leukemia inhibitory factor (LIF) plays an important role in increasing the contents of galanin and VIP in the SCG. In the present study, by examining the time courses of the changes in LIF and neuropeptide mRNA and by using LIF null mutant mice, we have determined that LIF alters neuropeptide content in part by increasing levels of peptide mRNA. In addition, LIF also makes a small contribution to the axotomy‐induced down‐regulation of mRNA encoding TH and neuropeptide Y, both of which are normally expressed at high levels in the SCG. Finally, by using a LIF‐blocking antiserum, this cytokine was found to regulate SP expression in an in vitro axonal injury model. Thus, after axotomy, a single factor, LIF, participates in the down‐regulation of peptides/proteins involved in normal neurotransmission and the up‐regulation of a group of neuropeptides normally not present in the SCG that may be involved in regeneration.

LEUKAEMIA INHIBITORY FACTOR INDUCED IN THE SCIATIC NERVE AFTER AXOTOMY IS INVOLVED IN THE INDUCTION OF GALANIN IN SENSORY NEURONS

Dramatic changes occur in neuropeptide expression in sensory and sympathetic neurons following axonal injury. Based on the finding that the cytokine leukemia inhibitory factor (LIF) plays an important role in mediating these changes in sympathetic neurons, its participation in triggering changes in sensory neurons was examined. By the use of transgenic mice in which the LIF gene had been knocked out, LIF was found to contribute to the induction of galanin expression in dorsal root ganglia (DRG) after sciatic nerve lesion. On the other hand, two other neuropeptide changes that occur in DRG under these conditions, the reduction of substance P and induction of neuropeptide Y, were independent of LIF expression. In the sympathetic superior cervical ganglion, transection of the postganglionic nerves close to the ganglion resulted in a rapid induction of LIF mRNA in the ganglion and in the lesioned nerve trunk. In contrast, transection of the sciatic nerve close to or distant from the DRG caused a rapid induction of LIF mRNA in the lesioned nerve, but not in the DRG. DRG were capable of making substantial amounts of LIF mRNA when placed in explant cultures, but, in vivo, only a slight induction was found even when both central and peripheral nerve processes of these sensory neurons were transected. These latter observations suggest that, in contrast to the superior cervical ganglia, the DRG environment inhibits the lesion‐induced expression of LIF in vivo, and/or explanted DRG produce stimulatory signals not found in vivo., Together with the data on the induction of galanin, these observations provide evidence that LIF, generated at a site at some distance from the ganglion, is involved in triggering part of the cell body reaction in sensory neurons.

The number and distribution of sympathetic neurons that innervate the rat pineal gland

The sympathetic innervation of the rat pineal gland was examined using a variety of anatomical techniques. Following the injection of horseradish peroxidase into the pineal gland, approximately 250 labeled neurons were found in the ipsilateral superior cervical ganglion. No labeled neurons were found in the middle or inferior cervical ganglia. In animals whose left internal carotid nerve was lesioned prior to the injection of peroxidase, an average of only three labeled neurons was found in the ipsilateral superior cervical ganglion. These data suggest that most, if not all, of the sympathetic neurons innervating the pineal gland exit from the superior cervical ganglia via the internal carotid nerves. The distribution of sympathetic neurons innervating the pineal gland was similar, though slightly more rostrally placed, than the distribution of the entire population of superior cervical ganglion neurons which project into the internal carotid nerve. Both the small number of neurons innervating the pineal gland and their wide distribution in the rostral part of the superior cervical ganglion indicate that their study at the level of the ganglion would be difficult. Sympathetic axons reach the pineal gland via the nervi conarii. Electron microscopic studies indicate that in each nervus conarii there are about 440 axons which make contact with the surface of the pineal gland. In certain cases, bundles of axons from the left and right nervi conarii were found to fuse. Additional evidence for the intermingling of axons from the two nervi conarii was seen in orthograde transport studies with horseradish peroxidase.

Characterization of a snake venom neurotoxin which blocks nicotinic transmission in the avian ciliary ganglion

Bungarus multicinctus venom was fractionated by ion exchange chromatography and the various fractions were assayed for their ability to block synaptic transmission through the chick ciliary ganglion. alpha-Bungarotoxin purified from this venom failed to block transmission at 50 micrograms/ml. A second neurotoxin, which we designate Toxin F, blocked transmission at 1-3 micrograms/ml and also blocked ganglionic depolarizations induced by carbachol. Toxin F was clearly distinguishable from alpha-bungarotoxin on the basis of molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and isoelectric point. Binding assays revealed that 125I-labeled toxin F bound to two sites in the ciliary ganglion: one site that was shared by alpha-bungarotoxin and toxin F and another site that was recognized solely by toxin F. Carbachol and d-tubocurarine displaced only that [125I]toxin F bound to the shared site and had no effect on [125I]toxin F bound to the site recognized by toxin F alone. The results suggest that toxin F blocks synaptic transmission in the chick ciliary ganglion by a postsynaptic mechanism. Further study is required to determine whether this effect of toxin F is mediated through a direct interaction with ganglionic nicotinic receptors.

Regional distribution of tyrosine hydroxylase, norepinephrine and dopamine within the amygdaloid complex of the rat

Electrical stimulation and ablation studies suggest that the amygdaloid complex participates in a variety of autonomic, endocrine and behavioural functions 9. Within the amygdala important differences have been found concerning the participation in these functions of the phylogenetically older corticomedial nuclei and the more recently evolved basolateral nuclei 9. Histochemical studies suggest that these areas differ also in their content of cholinergic and catecholaminergic neuronal processes~,8,1:L Using sensitive radiochemical assays we are currently studying the distribution of different neurotransmitter systems in discrete parts of the amygdala. In the present report we have examined the localization within different regions of the amygdala of norepinephrine, dopamine and tyrosine hydroxylase, the enzyme that catalyzes the ratelimiting step in the synthesis of these amines. Male Wistar rats (400-500 g) were anaesthetized with Nembutal. The following dissection procedures were performed in a cold room (4 °C). The rats were perfused through the heart with ice-cold dextrose (4.3 °o)-saline (0.18 ~,). The brain was rapidly removed from the skull, placed in cold Krebs-phosphate buffer and the dura was peeled off. Frontal sections were cut using a McIlwain tissue chopper. A petri dish containing a layer of wax and several pieces of filter paper was used in place of the normal cutting stage. The brain was attached to the filter paper by its dorsal surface with a drop of Loctite Quickset Adhesive 404 (Boehringer Corp., London) and was also pinned to the layer of wax. Five hundred micron sections were cut, one at a time. Each section was placed on a glass slide and kept on ice until it was dissected. Beginning with the section where the anterior commissure first crosses the midline (A 7190 in the atlas of K6nig and Klippell°), the following areas were rapidly removed with small dissecting knives under the light microscope : anterior amygdala (AA), pyriform cortex (CPF), basolateral amygdala (BE), corticomedial amygdala (AM), central nucleus (AC), caudate putamen (CP). Sections were usually obtained up to level A 2580 where the dorsal and the ventral hippocampus are continuous. In order to dissect

Characterization of neuronal nicotinic receptors by snake venom neurotoxins

Abstract Neuronal nicotinic ACh receptors differ pharmacologically from nicotinic receptors on skeletal muscle. The use of certain snake venom neurotoxins has now led to a more complete determination of the pharmacological properties of these neuronal receptors, as well as to their ultrastructural localization. This review highlights results found using one such neurotoxin, toxin F, (also called bungarotoxin 3.1 and κ-bungarotoxin. Toxin F blocks nicotinic receptors in several neuronal preparations while having little affinity for nicotinic receptors in skeletal muscle. Autoradiographic studies using [ 125 I] toxin F indicate that nicotinic receptors in autonomic ganglia are clustered at synaptic sites, though their density is 3–30 times lower than that of nicotinic receptors at the neuromuscular junction.

Neuronal bungarotoxin blocks the nicotinic stimulation of endogenous dopamine release from rat striatum.

Nicotinic receptors in the brain are receiving increased attention due in part to the recent cloning of receptor subunits and to postmortem studies revealing alterations in receptor density associated with Alzheimers disease. The peptide neurotoxin neuronal bungarotoxin (NBT) has been shown to block nicotinic cholinergic responses in autonomic ganglia and in retinal ganglion cells. These findings suggest that NBT may be a useful probe for studying nicotinic receptors in the brain. Therefore, we have investigated the effects of NBT on the nicotine-mediated enhancement of endogenous dopamine release from rat striatal slices. It was found that the transient increase in dopamine release caused by 100 microM nicotine was completely blocked by 100 nM NBT, indicating that NBT is a functional nicotinic antagonist in this system.

Regulation of neuropeptide expression in sympathetic neurons. Paracrine and retrograde influences.

Sympathetic neurons and other peripheral neurons exhibit a great deal of plasticity in their neuropeptide phenotype in adulthood. In this review, two phenotypes have been described in detail: that of normal sympathetic neurons and that of axotomized neurons. Two factors produced by nonneuronal cells, LIF and NGF, determine which of these phenotypes is expressed. Under normal conditions, the neurons receive NGF primarily, if not exclusively, from the target tissues they innervate. Prior to surgery, the nonneuronal cells within the ganglion and nerve tract express little, if any, LIF. This milieu favors the expression of NPY and suppresses the expression of VIP, galanin, and substance P (Fig. 6). After axotomy, however, this situation is reversed. The neuronal cell bodies are deprived of target-derived NGF and are exposed to LIF both within the ganglion and at the site of the injury (Fig 6). Both the removal of NGF and the exposure to LIF inhibit NPY expression, while promoting the expression of VIP and galanin. Expression of substance P after axotomy occurs primarily, if not entirely, because of the effects of LIF, with the removal of NGF playing no obvious role in the regulation of this peptide.

The effects of α- and β-neurotoxins from the venoms of various snakes on transmission in autonomic ganglia

We have previously shown that certain commercially available lots of alpha-bungarotoxin block transmission in ciliary and choroid neurons of both pigeon and chicken ciliary ganglia at a concentration of 10 microgram/ml (1.2 microM). The blockade is antagonized by pre-incubation with 100 microM tubocurarine. Further evidence that this blockade is produced by a postsynaptic action, as one would expect of an alpha-neurotoxin, are our findings that: (a) exposure to the toxin prevents the depolarization of ganglion cells normally seen in response to the cholinergic agonist, carbachol; and (b) the blocking activity of the toxin is removed by treatment with membranes purified from Torpedo electric organ containing an excess of alpha-neurotoxin binding sites. A high affinity binding site for [125I]alpha-bungarotoxin was characterized in the chicken ciliary ganglion. However, since it is labelled equally well by lots of alpha-bungarotoxin which block transmission and those that do not, this site does not appear to be involved in the blockade of transmission. alpha-Cobratoxin (from Naja naja siamensis), the alpha-neurotoxin L.s. III (from Laticauda semifasciata) and certain lots of alpha-bungarotoxin produce a partial blockade of transmission in ciliary neurons of the pigeon ciliary ganglion at a concentration of 10 microgram/ml (1.2 microM), but have no effect on transmission in choroid neurons. Two other alpha-neurotoxins from Laticauda semifasciata, erabutoxin a and erabutoxin b, have no effect on transmission in either cell population at this concentration. None of the alpha-neurotoxins tested had any effect on transmission in either the rat superior cervical ganglion or the rat pelvic ganglion at concentrations up to 100 microgram/ml (12 microM). Collagenase treatment of these ganglia, in an attempt to increase access of the toxins to ganglion cells, did not alter these negative results. beta-Bungarotoxin (0.5 microgram/ml, 0.02 microM) produces a complex blockade of transmission in both avian ciliary ganglia and rat superior cervical ganglia. Unlike the action of alpha-bungarotoxin, the blockade of ciliary ganglion transmission by beta-bungarotoxin is irreversible and is not prevented by pretreatment with tubocurarine.