Specific targeting of ganglion cell sprouts provides an additional mechanism for restoring peripheral motor circuits in pelvic ganglia after spinal nerve damage

Abstract

The pelvic ganglia contain both sympathetic and parasympathetic neurons and provide an interesting model in which to study the effects of a distributed spinal nerve lesion. Previous animal studies have suggested that after either lumbar or sacral nerve injury, some functional connections are restored between preganglionic and postganglionic neurons. It has been proposed that this is because of intact preganglionic axons sprouting collaterals to supply denervated ganglion cells. However, this has never been demonstrated, and our study has investigated whether the ganglion cells themselves contribute to axogenesis and restoration of peripheral circuitry. We have monitored the growth of axons from pelvic ganglion cells after lumbar or sacral nerve injury (partial decentralization), or a combination of the two (total decentralization). These new processes were distinguished from intact preganglionic terminals by their immunoreactivity for substances present only in pelvic ganglion neurons (vasoactive intestinal peptide, neuropeptide Y, and tyrosine hydroxylase). The proportion of pelvic neurons surrounded by these immunostained fibers was then assessed. Complete removal of preganglionic terminals provides the biggest stimulus for growth of new axon processes (sprouts), which grow profusely within just a few days. These arise from each of the main chemical classes of pelvic neurons but grow at different rates and have different distributions. Importantly, some chemical classes of sprouts preferentially supply neurons of dissimilar histochemistry, suggesting the presence of very specific targeting mechanisms rather than random growth. These sprouts are transient, however, those formed after partial decentralization appear to be maintained. Moreover, after lesion of either lumbar or sacral spinal nerves, many sprouts arise from neurons with intact spinal connections and innervate neurons that have lost their preganglionic inputs. This provides a very different alternative mechanism to reestablish communication between preganglionic and postganglionic neurons. In conclusion, we have demonstrated a rapid and selective axogenesis within the pelvic ganglion after spinal nerve injury. This may allow the development of novel strategies by which autonomic nerve pathways can be experimentally manipulated, to facilitate more rapid return of appropriate peripheral reflex control.

Fig. 1.

Sprout formation in pelvic ganglia after total decentralization. VIP sprout formation in the dorsal region of the pelvic ganglion in (a) a control (intact) animal and after total decentralization, (b) 2 d, and (c) 4 d after nerve transections.d–f, Baskets formed 8 d after total decentralization are shown at higher magnification. d, A VIP basket (arrow) encapsulates a VIP-negative neuron in the ventral region of the ganglion. e, An NPY basket is associated with an NPY neuron (arrow), whereas a nearby group of NPY-negative neurons is also surrounded by NPY baskets.e, TH baskets are always associated with TH-negative neurons. Scale bar applies to all micrographs: a, 50 μm (a–c); 25 μm (d–f).

Fig. 2.

Time course of basket formation after total decentralization of the pelvic ganglion. Data are expressed as mean ± SE (n = 4 animals for each time point). Where error bars are not shown, these are smaller than the symbols.

Fig. 3.

Time course of basket formation after total or partial decentralization of the pelvic ganglion. Time course of (A) TH, (B) NPY, or (C) VIP basket formation from totally decentralized ganglia 0, 1, 2, 4, 8, and 12 d after nerve lesions (data are the same as those shown in Fig. 2). Partially decentralized ganglia were only examined 0, 4, 8, and 12 d after nerve transection. Data are expressed as mean ± SE (n = 4 animals for each time point). Where error bars are not shown, these are smaller than the symbols. From each animal, 438–1528 neurons were examined for the presence of baskets.

Fig. 4.

Histochemical features of pelvic neurons and their associated baskets that have formed after partial or total decentralization. TH baskets do not supply TH neurons, and NPY baskets are not formed above control values 8 or 12 d after sympathetic decentralization. Data are expressed as mean ± SE, at which four animals were analyzed for each point.

Fig. 5.

Association of sprout baskets with neurons of known target organs. a, A fluorogold-labeled pelvic neuron supplying the prostate gland (arrow) is encapsulated by an NPY basket (b) 4 d after total decentralization. c, A fast blue-labeled neuron innervating the vas deferens (arrow) is supplied by a VIP basket (d). Scale bar: a, 25 μm (applies to all micrographs).

Fig. 6.

Target neurons of sprout baskets formed after total decentralization of the pelvic ganglia. The percentage of pelvic ganglion cells retrogradely labeled from each organ that are supplied by baskets 4 d after total decentralization. For each type of sprout basket (TH, NPY, or VIP), no statistical differences were associated with pelvic neurons projecting to any of the pelvic organs. From each animal, between 154–1755 retrogradely labeled neurons were analyzed (n = 3 animals for each point).

Fig. 7.

Synaptophysin immunoreactivity in decentralized pelvic ganglia (sacral nerve lesion, 12 d).a, A VIP basket (arrow) supplies a decentralized neuron, as demonstrated by its lack of bright synaptophysin-positive terminals that surround nearby neurons (b); SIF cells are also synaptophysin-immunoreactive. c, Two VIP baskets (arrows) are brightly immunoreactive for synaptophysin (d), and all bright synaptophysin varicosities contain VIP. e, A group of synaptophysin-immunoreactive SIF cells (arrow) are supplied by VIP sprouts (f). Scale bar: a, 25 μm (refers to all micrographs).

Fig. 8.

Proposed mechanisms of reorganization of neural pathways in the partially decentralized pelvic ganglion. The top diagram represents the intact pelvic ganglion, with equal proportions of sympathetic (S) and parasympathetic (P) neurons. Mechanism 1 (preganglionic sprouting) is the currently held view of how remodelling occurs in the pelvic ganglion after transection of parasympathetic preganglionic axons. Here, sympathetic preganglionic neurons form collaterals that make connections with decentralized neurons. Conversely, if sympathetic preganglionic axons were transected, parasympathetic spinal neurons would sprout collaterals to innervate denervated neurons. The present study has demonstrated that the second mechanism (postganglionic sprouting) occurs after partial decentralization. Here, postganglionic neurons with intact spinal connections sprout collaterals to supply decentralized neurons. Both mechanisms will result in activation of decentralized neurons after stimulation of the intact spinal nerves.