Identifying local and descending inputs for primary sensory neurons

Abstract

Primary pain and touch sensory neurons not only detect internal and external sensory stimuli, but also receive inputs from other neurons. However, the neuronal derived inputs for primary neurons have not been systematically identified. Using a monosynaptic rabies viruses-based transneuronal tracing method combined with sensory-specific Cre-drivers, we found that sensory neurons receive intraganglion, intraspinal, and supraspinal inputs, the latter of which are mainly derived from the rostroventral medulla (RVM). The viral-traced central neurons were largely inhibitory but also consisted of some glutamatergic neurons in the spinal cord and serotonergic neurons in the RVM. The majority of RVM-derived descending inputs were dual GABAergic and enkephalinergic (opioidergic). These inputs projected through the dorsolateral funiculus and primarily innervated layers I, II, and V of the dorsal horn, where pain-sensory afferents terminate. Silencing or activation of the dual GABA/enkephalinergic RVM neurons in adult animals substantially increased or decreased behavioral sensitivity, respectively, to heat and mechanical stimuli. These results are consistent with the fact that both GABA and enkephalin can exert presynaptic inhibition of the sensory afferents. Taken together, this work provides a systematic view of and a set of tools for examining peri- and extrasynaptic regulations of pain-afferent transmission.

Figure 7. Schematic drawing of extra- and peri-synaptic inputs onto sensory neurons revealed by this study.

(A) Intraganglion inputs to sensory neuron soma. Glutamate and possibly other transmitters released from the soma of sensory neurons can potentially modulate activities of neighboring sensory neurons in an extrasynaptic manner. (B) Intraspinal inputs onto sensory afferent terminals. Certain types of spinal GABAergic (some are also glycinergic) interneurons can form axo-axonic synapses with sensory afferents, and small numbers of glutamatergic interneurons have axons (or collaterals) that locate immediately adjacent to sensory afferent terminals. These spinal neurons regulate afferent transmission either through direct synaptic inputs or through spillover. (C) Supraspinal RVM–derived inputs onto sensory afferent terminals. RVM serotonergic and dual GABA/enkephalinergic neurons provide inputs primarily onto nociceptive sensory afferents.

Figure 6. Behavioral effects of silencing and activating the inputs from the dual GABAergic/enkephalinergic RVM neurons.

(A–C) Behavioral tests for the effects of silencing or ablating RVM-pre neurons compared with controls. Numbers (n) of mice in each group are shown on the graphs. (A) Hot plate paw-withdraw latency, (B) tail-flick latency; and (C) paw-withdraw threshold of von Frey test. (D and E) PENK1 in situ hybridization (ISH; red) confirmed the loss of PENK1-expressing RVM neurons after coinjection of Lenti-Penk1-Cre and AAV-FLEX-taCasp3-TEVp. Control sample (D) and caspapse-ablated sample (E). (F–H) Behavioral tests for the effects of activating RVM-pre neurons compared with controls. Numbers (n) of mice in each group are shown on the graphs. Hot plate paw-withdraw latency (F); tail-flick latency (G); and paw-withdraw threshold of von Frey test (H). Error bars represent means ± SEM. P values represent comparison to WT and control injection values (*P < 0.05). Differences were determined by Student’s t test between 2 groups, or one-way ANOVA followed by post-hoc Bonferroni test for multiple groups. n = 5~10 mice.

Figure 5. RVM-pre neurons project axons to dorsal spinal cord, and their activation evokes DRPs.

(A) Representative images of labeled RVM neurons from mice coinjected with Lenti-Penk1-Cre and AAV-FLEX-GFP. (B and C) In situ hybridization showed that RVM neurons labeled by coinjection of Lenti-Penk1-Cre and AAV-FLEX-GFP express PENK1 (B) and/or GAD2 (C). (D–F) Costaining with anti-vGlut1 (marker for touch afferents) revealed that labeled RVM axons are concentrated in 2 regions (arrows): a superficial region above the vGlut1⁺ layers and the deep vGlut1⁺ lamina V. (G–I) Costaining with IB4 (marker of lamina II) revealed that labeled RVM axons innervate both lamina I and II. (J) Representative image of ChR2 expression in RVM neurons after coinjection of Lenti-Penk1-Cre and AAV-FLEX-hChR2-eYFP in RVM. (K and L) Optogenetic stimulation of ChR2-expressing axons in spinal block–evoked DRPs. Two representative traces are shown. (M and N) Light-evoked EPSP peak amplitude and latency were shown (n = 4 mice). Scale bar is 100 μm for all images.

Figure 4. Analyses of ΔG-RV-GFP–labeled neurons in RVM.

(A and B) Representative images of ΔG-RV-GFP–labeled RVM neurons in Avil-Cre RΦGT mice. Enlarged image of labeled neurons in A is shown in B. (C–H) Representative images from in situ hybridization (ISH) analyses of ΔG-RV-GFP–labeled RVM neurons. The probes used are GAD2 (C), GlyT2 (D), TPH2 (E), PENK1 (F), TRH (G), and MOR (H). (I and J) Representative 2-color in situ experiments that examine the coexpression of PENK1 and GAD2 (I), and PENK1 and TPH2 (J) in RVM region. Scale bar for all images is 100 μm. Arrows indicate double-labeled neurons.

Figure 3. Molecular characterizations of ΔG-RV-GFP–labeled spinal cord neurons.

Representative images from the 2-color in situ hybridization (red signals) and immunostaining (green, anti-GFP) analyses of ΔG-RV-GFP–labeled spinal neurons in Avil-Cre RΦGT mice are shown. Arrows indicate some of the GFP⁺ neurons expressing the molecular marker examined. (A–F) Images from ipsilateral spinal cord are shown. The in situ probes used are GAD1 (A), GAD2 (B), GlyT2 (C), vGlut2 (D), SST (E) and PENK1 (F). Scale bar, 100 μm. (G–I) Images from contralateral spinal cord with in situ probes of GAD2 (G), GlyT2 (H), and vGlut2 (I) are shown. Scale bar, 100 μm.

Figure 2. ΔG-RV-GFP–labeled cells in DRG and the spinal cord.

(A–C) Representative images of ΔG-RV-GFP–labeled neurons in DRG (cervical segment C6 or C7) from RΦGT (control), Avil-Cre RΦGT, or Trpv1-Cre RΦGT mouse. Scale bar, 100 μm. (D–F) Representative images of ΔG-RV-GFP–labeled neurons in the cervical spinal cord from RΦGT (control), Avil-Cre RΦGT, or Trpv1-Cre RΦGT mouse. Scale bar, 100 μm. (G–I) Magnified view of the boxed areas in D–F. Asterisks indicate glial cells. (J) Representative image of EnvA-RV-GFP–labeled neurons in DRG from Trpv1-Cre RΦGT RΦtomato mouse. Arrows indicate GFP–single positive neurons.

Figure 1. Schematic drawing of strategy for tracing presynaptic inputs onto sensory afferents.

(A) Genetic crosses were used to generate Avil-Cre RΦGT and Trpv1-Cre RΦGT mice such that the rabies G protein can be selectively expressed in either all or TRPV1-lineage of DRG sensory neurons. (B) ΔG-RV-GFP was injected into the plantar skin of the right front paw of P1 pups, as illustrated. Seven days after injection, the samples are collected and analyzed. (C) Expected outcome of the viral tracing experiments: ΔG-RV-GFP will infect DRG neurons from peripheral axon terminal and will be transported back to the cell bodies, where the deficient virus will be complemented by the rabies G protein. Subsequently, some of the replicated ΔG-RV-GFP will be released at the central afferent terminals in the dorsal horn and infect their putative presynaptic partners.