Triggering genetically-expressed transneuronal tracers by peripheral axotomy reveals convergent and segregated sensory neuron-spinal cord connectivity

Abstract

To better understand the mechanisms through which non-painful and painful stimuli evoke behavior, new resources to dissect the complex circuits engaged by subsets of primary afferent neurons are required. This is especially true to understand the consequences of injury, when reorganization of central nervous system circuits likely contributes to the persistence of pain. Here we describe a transgenic mouse line (ZWX) in which there is Cre-recombinase-dependent expression of a transneuronal tracer, wheat germ agglutinin (WGA), in primary somatic or visceral afferent neurons, but only after transection of their peripheral axons. The latter requirement allows for both regional and temporal control of tracer expression, even in the adult. Using a variety of Cre lines to target WGA transport to subpopulations of sensory neurons, here we demonstrate the extent to which myelinated and unmyelinated "pain" fibers (nociceptors) engage different spinal cord circuits. We found significant convergence (i.e., manifest as WGA-transneuronal labeling) of unmyelinated afferents, including the TRPV1-expressing subset, and myelinated afferents to NK1-receptor-expressing neurons of lamina I. By contrast, PKCgamma interneurons of inner lamina II only receive a myelinated afferent input. This differential distribution of WGA labeling in the spinal cord indicates that myelinated and unmyelinated sensory neurons target different and spatially segregated populations of postsynaptic neurons. On the other hand, we show that neurons of deeper laminae (III-V) receive direct (i.e., monosynaptic) inputs from myelinated afferents and polysynaptic input from unmyelinated afferents. Taken together, our results indicate that peripheral sensory information is transmitted to the central nervous system both through segregated and convergent pathways.

Figure 1. Peripheral nerve injury induces lacZ transgene expression in large numbers of DRG neurons in the ZWX mouse

A : The ZWX mice express a vector consisting of a loxP flanked lacZ gene (blue) inserted upstream of genes that encode for WGA (red) and GFP-TTC (green), all of which are driven off of a CMV-enhanced/chicken β-actin promoter (CAG). In this arrangement, the neurons should produce β-galactosidase constitutively. In cells expressing Cre recombinase (+Cre), a recombination event excises the floxed lacZ cDNA and initiates expression of the WGA and GFP-TTC tracers. B: Anterograde and transneuronal transfer of the WGA tracer labels neurons (2, red) that are postsynaptic to the first-order Cre-expressing neuron (1). Retrograde and transneuronal transfer of the GFP-TTC tracer labels neurons (3, green) that are upstream of the first-order Cre-expressing neuron. C: Unexpectedly, there is no constitutive expression of the lacZ in sensory neurons of the ZWX mouse. However, peripheral nerve transection (X) triggers lacZ expression, presumably because of increased CAG promoter activity. In axotomized DRG neurons that also express Cre (yellow nucleus), both WGA and GFP-TTC expression are induced (red/green). D-F: LacZ expression in the DRG of control mice (D) or after nerve injury (E) and its transport to the dorsal horn of the spinal cord (F). Ipsilateral ventral horn motoneurons, the efferent fibers of which are transected by sciatic nerve injury, are also lacZ+ (F). Cell bodies of myelinated, N52+ (G), unmyelinated substance P+ (H) and unmyelinated IB4+ (I) neurons are lacZ+. J-R: To target expression of the tracers to subpopulations of DRG neurons, we crossed the ZWX mice with NPY-Cre mice for myelinated afferents (J-L), Nav1.8-Cre mice for unmyelinated neurons (M-O) or peripherin-Cre mice for a mixed population of unmyelinated and myelinated afferents (P-R). In the absence of peripheral nerve injury, there is no WGA in these crosses (J,M,P); nerve transection induces WGA (K,N,Q) and GFP-TTC (L,O,R). Calibration bar: 100μm for D, E; 200μm for F-R.

Figure 2. Peripheral axotomy induces expression and transport of WGA

A-C: We found that ∼50%, ∼90% and ∼11% of WGA+ neurons (red) have myelinated axons (N52+; green) in ZWX-Per (A), ZWX-NPY (B) and ZWX-Nav1.8 (C) mice, respectively. D-I: Differential patterns of anterograde transport in the 3 crosses. In ZWX-Per animals, the WGA labeling extended from laminae I to V (D, G). In ZWX-NPY mice, the WGA labeling was concentrated in medial laminae III-V (E, F). In ZWX-Nav1.8 mice, the WGA was restricted to laminae I and II (F, I). In the ZWX-Per (D) and ZWX-NPY (E) mice, we also observed WGA in ventral horn motoneurons (See text). In contrast to the high levels of WGA induced by nerve injury we never observed GFP-TTC staining in the dorsal horn of the spinal cord (data not shown). This result was expected because GFP-TTC is a retrograde tracer, and there are few, if any inputs to DRG neurons. Calibration bar: 100μm for G-I; 200μm for A-F.

Figure 3. Transneuronal WGA labeling of dorsal horn neurons

Targeting the WGA to DRG neurons with myelinated (ZWX-NPY) or unmyelinated (ZWX-Nav1.8) axons reveals discrete patterns of transneuronal labeling in the spinal cord. Thus, DRG neurons with myelinated axons transfer the WGA to PKCγ+ interneurons of lamina IIi (E) and to parvalbumin+ neurons of laminae III-V (K), but not to calbindin+ interneurons of lamina II (H). In contrast, DRG neurons with unmyelinated axons contact (and transfer WGA to) calbindin+ interneurons of lamina II (I), but neither PKCγ (F) nor parvalbumin interneurons (L). On the other hand, both myelinated and unmyelinated afferents contact projection neurons of lamina I that express the NK1 receptor (B and C, respectively). As expected each of these neurochemically-defined postsynaptic neurons is also transneuronally labeled in the ZWX-Per mice, in which both myelinated and unmyelinated axons carry the WGA (A,D,G,J). Further examples of double-labeled cells can be seen in Supplementary figures S5 and S6. Calibration bar: 50μm.

Figure 4. Expression and transneuronal transfer of the WGA from TRPV1 positive DRG neurons

Hindpaw injection of the neurotoxin capsaicin in wild type (A), but not in TRPV1 null mice (B), injures peripheral terminals of DRG neurons, manifest by nuclear ATF-3 expression in a subset of DRG neurons (red). Many, but not all of the ATF-3+ neurons express TRPV1 (C; green). In ZWX-Nav1.8 mice, capsaicin-induced nerve injury triggers expression of the WGA (red) exclusively in small caliber nociceptors (yellow corresponds to double-labeled neurons); ∼55% are TRPV1 (D); ∼36% express substance P (E) and ∼50% express P2X₃, i.e. are non-peptidergic (F). In the spinal cord, the WGA labeling is restricted to laminae I-II, and there is transneuronal transport to neurons of lamina I that express PKCγ (H) or the NK1 receptor (I) but not to PKCγ interneurons of inner lamina II (H). Note also that there are WGA-positive neurons in the neck of the dorsal horn (laminae III-V), presumably secondary to polysynaptic transfer from neurons location in the superficial dorsal horn (G, see also Figure 5). Calibration bar: 50μm for H,I; 100μm for C,G; 200μm for A,B,D-F.

Figure 5. Deep laminae of the spinal cord receive indirect inputs from unmyelinated afferents

Repeating the sciatic nerve transection in ZWX-Nav1.8 mice sustains expression of the WGA in DRG neurons and results in transneuronal labeling of neurons in deeper laminae (III-V; arrows). Compare A (single nerve cut; 1 week post-injury) with B (two cuts separated by one week; 1 week post-2^nd injury). The inability to detect deep laminae labeling after a single cut of the peripheral nerve suggests that the labeling after repeated injury resulted from transneuronal transfer of the WGA from laminae I-II neurons. Calibration bar: 150μm.

Figure 6. Regional control of WGA induction: targeting visceral afferents

A great advantage of the nerve injury inducibility of the WGA is that the subtype of peripheral nerve that is injured allows for analysis of circuits engaged by sensory neurons that innervate different peripheral organs. Here, transection of the vagus nerve in ZWX mice induces expression of nuclear ATF3 in large numbers of nodose ganglion neurons (A). As expected, the vagal nerve transection also triggered high levels of lacZ expression (B). When the vagus is cut in ZWX-Nav1.8 mice (C), the WGA is induced and transported anterogradely by vagal axons (inset in C) to the nucleus of the solitary tract (NTS; D), where it is transneuronally transferred to NTS neurons (arrows). Calibration bar: 100μm for D; 200μm for A-C.

Figure 7. Convergent and segregated spinal cord inputs arise from myelinated and unmyelinated primary afferents

Lamina I (Lam I) projection neurons that express the NK1 receptor (light blue) and presumptive interneurons that express PKCγ (pink)- or calbindin (orange) receive convergent inputs from unmyelinated peptidergic (Substance P, TRPV1 and Nav1.8+) and myelinated (presumably Aδ, NPY+) primary afferent neurons. In contrast, the inputs from these subpopulations of afferents to other laminae remain segregated. Thus, the major input to the outer part of laminae II (Lam IIo) is from unmyelinated peptidergic afferents; unmyelinated non-peptidergic DRG neurons (that bind IB4 and express Nav1.8) terminate in the most dorsal part of inner lamina II (d-Lam IIi) where they contact calbindin-expressing interneurons. By contrast, myelinated afferents (either Aδ and/or Aβ, which express NPY after peripheral nerve injury) target the most ventral part of inner lamina II (v-Lam IIi), where they contact PKCγ-expressing interneurons. These same afferents also monosynaptically contact parvalbumin-positive (yellow) and other undefined neurons (black) of laminae III-V Note that a population of non-PKCγ interneurons of the innermost part of lamina II (v-LamIIi) also receive unmyelinated inputs. Whether that input is direct or indirect remains to be determined. In addition to monosynaptic inputs from myelinated afferents, neurons located in deep laminae (III-V) also receive polysynaptic inputs from unmyelinated (including TRPV1+) afferents, presumably via interneurons of laminae I-II.