Sciatic nerve transection triggers release and intercellular transfer of a genetically expressed macromolecular tracer in dorsal root ganglia

Abstract

We recently developed a genetic transneuronal tracing approach that allows for the study of circuits that are altered by nerve injury. We generated transgenic (ZW-X) mice in which expression of a transneuronal tracer, wheat germ agglutinin (WGA), is induced in primary sensory neurons, but only after transection of their peripheral axon. By following the transneuronal transport of the tracer into the central nervous system (CNS) we can label the circuits that are engaged by the WGA-expressing damaged neurons. Here we used the ZW-X mouse line to analyze dorsal root ganglia (DRG) for intraganglionic connections between injured sensory neurons and their neighboring "intact" neurons. Because neuropeptide Y (NPY) expression is strongly induced in DRG neurons after peripheral axotomy, we crossed the ZW-X mouse line with a mouse that expresses Cre recombinase under the influence of the NPY promoter. As expected, sciatic nerve transection triggered WGA expression in NPY-positive DRG neurons, most of which are of large diameter. As expected, double labeling for ATF-3, a marker of cell bodies with damaged axons, showed that the tracer predominated in injured (i.e., axotomized) neurons. However, we also found the WGA tracer in DRG cell bodies of uninjured sensory neurons. Importantly, in the absence of nerve injury there was no intraganglionic transfer of WGA. Our results demonstrate that intraganglionic, cell-to-cell communication, via transfer of large molecules, occurs between the cell bodies of injured and neighboring noninjured primary afferent neurons.

Figure 1

Axotomy induced expression of the ZW transgene in NPY-ZW-X transgenic mice. A: ZW-X transgenic mice carry a DNA construct in which both the LacZ (β-galactosidase-neomycin resistance fusion protein) and WGA cDNAs are under the control of the ubiquitous cytomegalovirus enhanced, chicken β-actin promoter (CβA). Under normal conditions, only the LacZ gene is expressed. After Cre recombination the loxP-flanked LacZ is excised, and the WGA is transcribed. B: Due to random insertion of the ZW transgene in the genome of ZW-X mice, under normal conditions sensory neurons do not express the lacZ cDNA. C: In contrast, after nerve injury (in this example, sciatic nerve transection), there is a very strong induction of lacZ, ipsilateral to the axotomy. D: In a similar vein, under normal conditions, neuropeptide Y (NPY) is not expressed in DRG neurons. E: However, nerve injury induces expression of NPY in large numbers of sensory neurons, most of which are of medium-to-large diameter. F: In NPY-ZW-X double transgenic animals, in the absence of peripheral nerve injury, there is no expression of the transneuronal tracer WGA in sensory neurons. G: However, nerve injury induces strong expression of WGA in large number of sensory neurons of NPY-ZW-X mice. Scale bar = 100 µm for B,D,F; 200 µm for A,C,E.

Figure 2

WGA induction in myelinated sensory neurons is Cre-mediated (in NPY-ZW-X mice). After axotomy, sensory neurons that express WGA (A,D,G) also express NPY (B,C), the neurofilament 200 marker of cell bodies with myelinated axons (NF200; E,F) and ATF3 (H,I). This illustrates that axotomy-induced expression of Cre recombinase in NPY-positive DRG neurons led to excision of the floxed lacZ cDNA and expression of WGA in myelinated (NF200-positive), injured (ATF3-positive) sensory neurons. Note, however, that some WGA-immunoreactive neurons did not express NPY or ATF3 (arrows in C,I, respectively), indicating that the WGA did not arise from Cre-mediated excision in these neurons but rather was transneuronally transferred from injured (WGA+/NPY+/ATF3+) to uninjured (WGA+/NPY−/ATF3−) sensory neurons (see also Fig. 3). Insets contain high magnifications of WGA/marker double-labeled cells. Scale bar = 100 µm for A–C; 200 µm for D–I.

Figure 3

Intraganglionic transfer of WGA from injured to uninjured sensory neurons. Because DRG neurons cannot express simultaneously the lacZ and WGA cDNAs (see plasmid construct in Fig. 1), the presence of both WGA (red) and β-gal (green) in DRG neurons (arrows) indicates that the WGA must have been transneuronally transferred from injured (ATF3- and NPY-positive, but β-gal-negative) to uninjured (β-gal-positive and ATF3-negative) neurons, establishing intraganglionic transneuronal transfer of WGA. Insets contain high magnifications of WGA/marker double labeled cells. Scale bar = 100 µm.

Figure 4

Intraganglionic transfer of WGA from injured sensory neurons to surrounding satellite cells. The presence of WGA (red) in satellite cells (arrows in A,B) that surround sensory neurons further demonstrates that there is intraganglionic communication between the cell bodies of injured sensory neurons and nonneuronal cells following nerve injury. Nuclei are stained blue (DAPI). These results were confirmed at the electron microscopic level. C,D: A neuron that is WGA-immunoreactive lies adjacent to a WGA-immunoreactive satellite cell. The rectangle in C is magnified in D and highlights the satellite cell (outlined in black) and DAB immunoreaction product (arrows). Note also that an adjacent neuron (asterisk in C) is unlabeled. Scale bar = 100 µm for A; 20 µm for B; 6.2 µm for C.

Figure 5

In the absence of nerve injury, there is no intraganglionic transfer of WGA. A–C: In Per-ZW mice, under normal conditions (WT), the WGA tracer (red) is expressed in a wide variety of sensory neurons, none of which express β-gal (green). D–F: In contrast, after peripheral nerve injury (transection of the sciatic nerve; SNC), we observed a small number of WGA+ neurons that costained for β-gal (arrows and insets), reflecting thus intraganglionic transfer of WGA. Scale bar = 100 µm.