Richard Zigmond, PhD

 Lab Research History

In one of the very early examples of activity dependent gene expression, we showed that stimulation of the cervical sympathetic trunk led to an increase in the activity of protein level, and mRNA level of tyrosine hydroxylase, the enzyme that catalyzes the rate-limiting step in the synthesis of the neurotransmitter norepinephrine in the superior cervical ganglion (SCG). Interestingly, antidromic stimulation of the superior cervical ganglion did not mimic the effect of synaptic stimulation.

Fig. 1. Time course of the effects of preganglionic nerve stimulation on tyrosine hydroxylase (TH), dopamine beta hydroxylase (DBH) and dopa decarboxylase (DDC) in the SCG. The cervical sympathetic trunk (CST) was stimulated for 90 min with trains of 40 Hz for 250 out of every 750 msec. TH and DBH activity reached a maximum at 3 days. Neither DDC nor total protein changed significantly. The data are the means +/- S.E.M. from 8-10 SCG except for day 5 on which N=5.

In studies on a second mechanism of TH regulation, namely the acute activation of this enzyme by phosphorylation, we made the unexpected discovery that synapses in the rat SCG use a second transmitted in addition to acetlylcholine (Fig. 2), and we presented evidence suggesting that this molecule could be a peptide of the secretin-glucagon family (Fig. 3). Currently evidence suggests that the transmitter is pituitary adenylate cyclase activating peptide (PACAP).

Fig 2. The preganglionic trunk of the SCG was stimilated at 10 Hz for 30 min. Atropine, Hexamethonium (Hex) or both antagonists were added 10 min prior to the beginning of stimulation. Hatched bars stimulated ganglia, open bars control ganglia. 

Fig. 3. Concentration-response curve for the stimulation of TH activity by secretin and VIP. Ganglia were preincubated with peptide for 60 min prior to addition of a dopa decarboxylase inhibitor. Dopa accumulation was measured during the subsequent 15 min.

An exciting consequence of the discovery that there is cotransmission in this ganglion was the finding that the extent to which transmission in the SCG is cholinergic or non-cholinergic (peptidergic) depends on the pattern of impulse activity (Fig. 4).

Fig. 4. Effect of cholinergic antagonists on the increase in TH activity produced by preganglionic nerve stimulation. Stimulation was for 30 min at 10 Hz continuously or at 10 Hz for 1 out of every 6 sec. Hexamethonium (3mM) and atropine (6µM) were added to some groups prior to stimulation. The presence of a cholinergic component in the transsynaptic effect depends on the stimulation pattern.

In a second neuronal system, we showed that the 40-fold circadian rhythm in activity of pineal serotonin N-acetyltransferase (NAT), a regulatory enzyme in the pathway for melatonin synthesis, could be mimicked by electrical stimulation of the sympathetic innervation of the gland (producing a night time effect) followed by cessation of stimulation (producing a day time effect). While studying the pineal gland, we recognized that the rhythm in NAT provided a unique bioassay which could be used for measuring recovery of function following lesions in the sympathetic nervous system. These studies lead us to two important discoveries. The first finding was that of a novel mechanism of neural plasticity, which we termed “heteroneuronal uptake”, in which a nerve ending (even one that is itself electrically silent) can take up transmitter released by another ending and thereby modulate the latter’s postsynaptic efficacy (Fig. 5).

Fig. 5. A model to account for the recovery of pineal NAT activity after unilateral denervation (cutting the postganglionic internal carotid nerve) but not after unilateral decentralization (cutting the preganglionic CST). Two sympathetic nerves, one from the left and one from the right SCG are pictured innervating the same pinealocyte. In the normal animal, both neurons are electrophysiologically active and are releasing NE as shown by the white arrows. In addition, both neurons take up NE as shown by the black arrows. The situation in unilaterally decentralized pineal glands is depicted in the middle panel. In this diagram, a neuron from one SCG is shown as electrophysiologically inactive and therefore as not releasing NE. However the uptake capacity of this neuron is intact. Therefore the net effect of such a neuron is to inhibit the postsynaptic effectiveness of the active neurons from the contralateral ganglion. In the right panel, the situation in the unilaterally denervated pineal gland is shown. Sometime between 9 and 32 h after cutting one postganglionic trunk, the terminals of these nerve degenerate, thus decreasing the number of varicosities taking up NE. The loss of these NE uptake sites increases the postsynaptic effective of the neurons from the contralateral intact ganglion.

The second finding was that, although preganglionic sympathetic nerves can regenerate after a lesion and can effectively reinnervate neurons in the SCG, normal pineal function is not restored (Fig. 6). This finding conflicts with the prediction from a classical study by John Langley in the early 1900’s on regeneration of these neurons. We hypothesize that many of the neurons that reinnervate neurons in the SCG that project to the pineal gland are different from the original neurons and do not get the needed input from the circadian pacemaker in the suprachiasmatic nucleus of the hypothalamus which directs the normal rhythm in NAT activity (Fig. 7).

Fig. 6. Peak night pineal NAT activity after crushing both CSTs and the effect of a double lesion on this recovered activity.

Fig. 7. Model of misrouting of fibers during reinnervation of the SCG.

Another unexpected discovery was the finding that the snake venom from which bungarotoxin is purified contains a second toxin (“neuronal bungarotoxin”) which blocks synaptic transmission in autonomic ganglia. After purifying this toxin, we used it to quantify the density of nicotinic receptors at ganglionic synapses. Autoradiographic grains from 125 I-labeled neuronal bungarotoxin were concentrated at synapses in the chick ciliary ganglion as shown in Fig. 8. We also used this labeled toxin to localize a class of nicotinic receptors on SCG neurons in culture and in the rat brain.

Fig. 8. Preganglionic nerve terminals (nt) and post-ganglionic cell bodies (cb) in the chick ciliary ganglion. The black autoradiographic grains from radioactive neuronal bungarotoxin are concentrated at synapses within the ganglion.

In 1989, I moved to Case Western Reserve University to join its newly formed Department of Neurosciences. During the first year, we made a serendipitous discovery that when the SCG is placed in organ culture levels of vasoactive intestinal peptide (VIP) increase dramatically (i.e., ~30-fold; Fig. 9), rather than decreasing as we expected, since we assumed the peptide was present in preganglionic nerve terminals.

Fig. 9. Increase in VIP-like immunoreactivity in adult SCG maintained in organ culture.

This finding led to the second major theme of our research, namely, the molecular and cellular responses of neurons to injury. When we started these studies, neuropeptide regulation in postganglionic SCG neurons was thought to be regulated primarily by afferent nerve activity, which is abolished in organ culture, the idea being that activity suppressed the peptide phenotype. However, we demonstrated in vivo that it was axotomy of the SCG, not deafferentation, which mimicked the effects seen in organ culture. A trivial reason for this finding would have been that axotomy, by blocking transport of the peptide, led to a buildup in the cell body. However, we found that axotomy also leads to an increase in VIP mRNA. We went on to find that the cytokine leukemia inhibitory factor (LIF) is induced in non-neuronal cells in the ganglion both after axotomy and after explantation. Conditioned medium from ganglion non-neuronal cells stimulates VIP expression and this effect is blocked by an antibody to LIF (Fig. 10).

LIF, but not CNTF, from non-neuronal cells increase neuronal VIP expression.

Fig. 10. VIP levels in dissociated SCG neurons with and without addition of ganglion non-neuronal cell conditioned medium (GNCM).

In vivo the axotomy-induced increase in VIP is reduced, but not completely abolished, in LIF knockout mice (Fig. 11). This finding was the first demonstration of an effect of LIF on the nervous system in vivo. Also we found that galanin and substance P were regulated similarly.

Fig. 11. Axotomy-induced VIP expression in the SCG is reduced in LIF -/- mice.

Interestingly, unlike the case with the SCG, in the dorsal root ganglion (DRG), LIF was not induced after axotomy; however, the cytokine was induced in the lesioned sciatic nerve. We believe this is the first demonstration of an oft hypothesized relationship between changes in cytokine and growth factor expression in the distal segment of a lesioned nerve and the expression of certain genes by the axotomized neurons. In addition, using galanin knockout mice, we demonstrated that the induction of galanin after axotomy plays an important role in the conditioning lesion response of sensory neurons.

We have evidence that LIF is only one of a number of molecules that influence the response of sympathetic and sensory neurons to axotomy. LIF is a member of a family of cytokines, called the gp130 family. Also, we have shown that removal of the influence of the target derived neurotrophic factor nerve growth factor (NGF) is an important signaling event. Injection of animals with a nerve growth factor antiserum produces an increase in galanin expression in both the SCG and DRG and triggers a conditioning lesion-like effect in sympathetic neurons.

It has long been known that macrophages play a role in neural regeneration after axotomy; however, this influence has been viewed as occurring solely via an effect on Wallerian degeneration in the distal nerve segment. Some years ago, we discovered that, in addition to accumulating in the distal nerve, macrophages also accumulate around axotomized neuronal cell bodies. We have examined macrophage infiltration into DRGs and SCGs in two mutant strains of mice: the slow Wallerian degeneration mouse (Wlds) and a knockout for the receptor CCR2, the receptor on which the macrophage chemokine CCL2 acts. In both mouse strains, macrophage infiltration into the DRG was abolished and macrophage infiltration into the SCG was diminished. We then demonstrated that these macrophages within peripheral ganglia play an important role in peripheral nerve regeneration by activating neurite out-growth in response to a conditioning lesion (Fig. 12).

Fig. 12. Neurite outgrowth from explants of adult L5 DRGs ipsilateral (black bars) and contralateral (white bars) to a unilateral sciatic nerve transection 7 days previously in wild type (WT) and CCR2-/- mice. Growth of the 20 longest neurites was measured at 24 h (left hand histogram) and 48 h (right hand histograms) after placement in culture on a growth permissive substrate. A conditioning lesion effect was seen in the contralateral but not the ipsilateral ganglia.

Another quite unexpected finding from these experiments came when we looked at Wallerian degeneration in the two mouse strains 7 days after unilateral transection using luxol fast blue staining. As expected the clearance of myelin in the Wlds mice was severely impeded; however, quite dramatically the results for the CCR2-/- mice were indistinguishable from controls, in spite of the fact that we found no infiltration of macrophages into the sciatic nerve in these animals (Fig. 13). These results raise the question of what cell type is the predominant phagocyte in the absence of infiltrating macrophage. Current work in the lab indicate an important role for neutrophils.

Fig. 13. (a). Quantitation of the myelin in the distal sciatic nerve after unilateral transection 7 days earlier and in the contralateral nerve. The micrographs represent sections from the ipsilateral (e– g) and contralateral (b– d) nerves from WT, Wlds, and CCR2-/-mice. *p<0.05, **p<0.001. Scale bar, 20 µm.

We next examined whether over expression of the macrophage chemokine CCL2 stimulated neurite outgrowth. Control (uninjured) mice were injected intrathecally with an adeno-associated virus coding for CCL2. The virus infected neurons in the dorsal root ganglia and caused them to express CCL2. As a consequence, macrophages invaded the ganglia in spite of the fact that they had not been injured. 

Fig. 14. Wild type mice were inject intrathecally with AAV5-CCL2 or a control virus coding for YFP. Three weeks later when CCL2 expression and macrophage accumulation were at their maximum, DRGs were removed and placed in organ culture (A-C) or dissociated cell culture(D-F). Neurite outgrowth was enhanced neurons from AAC5-CCL2 treated mice. Addition of recombinant CCL2 directly to control neurons produced no effect on neurite outgrowth (G-L)

Three weeks later when the ganglia were placed in organ culture or dissociated cell culture, neurite outgrowth was enhanced (Fig. 14). When the same protocol was performed in CCR2 knockout animals, no effect on neurite outgrowth occurred. Addition of CCL2 recombinant protein to control cultures also produced no effect on neurite outgrowth (Fig. 14), suggesting that the viral stimulation of neurite outgrowth depended on macrophage accumulation in the ganglia.