Synaptic Organization of VGLUT3 Expressing Low-Threshold Mechanosensitive C Fiber Terminals in the Rodent Spinal Cord

Validation of VGLUT3 antibodies for immunofluorescence. Antibodies raised in rabbit, guinea pig, and mouse label the same terminal-like structures in inner lamina II in the rat spinal cord. The micrographs are single deconvolved optical sections acquired with a 63×/1.4 oil immersion objective. Scale bar, 5 µm, valid for all panels.

Laminar distribution of VGLUT3 immunolabeling in the rat and mouse dorsal horn. A, VGLUT3 immunofluorescence (magenta) alone (left panels) or superimposed over darkfield micrographs of the labeled sections (right panels). In both mouse and rat spinal cord, a band of VGLUT3⁺ terminal-like structures is evident in the inner part of lamina II. The dashed lines indicate the border between lamina II and III, as assessed from the darkfield micrographs. Note that in these sections, which are from the L4 segment, the band is absent from the medial part of the dorsal horn. The micrographs were obtained with a 10×/0.3 objective. Scalebar, 100 µm, valid for all panels. B, VGLUT3 immunofluorescence relative to IB₄ binding (left panels) and PKCγ immunolabeling (right panels) in mouse and rat dorsal horn. In mouse dorsal horn, the VGLUT3⁺ band is immediately ventral to the plexus of IB₄ binding terminals, whereas in the rat, the VGLUT3⁺ band overlaps with the ventral, most intensely labeled part of the IB₄ band. By contrast, in both mouse and rat dorsal horn, the VGLUT3⁺ band overlaps with the plexus of PKCγ⁺ processes marking lamina IIi. All panels are single optical sections obtained using a 40×/1.3 oil immersion objective. Scale bar, 20 µm, valid for all panels.

Lack of colocalization of VGLUT3 and TH immunofluorescence in the rat spinal cord. TH immunoreactive processes are found throughout the spinal gray matter, including in lamina II. TH⁺ processes never colocalize with VGLUT3⁺ terminals in lamina II or in the IML in thoracic spinal cord. All panels are single deconvolved optical sections obtained using a 63×/1.4 oil immersion objective. Scale bar, 10 µm, valid for all panels.

Colocalization of VGLUT3 with VGLUT1 and VGLUT2 in the dorsal horn. A, In the rat spinal cord, VGLUT3⁺ terminals in lamina IIi are also immunoreactive for VGLUT1 and, sometimes weakly, for VGLUT2. VGLUT3⁺ terminals intermingle with IB₄ binding terminals but never bind IB₄ themselves. In the mouse, VGLUT3⁺ terminals are not VGLUT1 immunoreactive but exhibit weak-to-moderate VGLUT2 immunolabeling. Arrowheads indicate VGLUT3⁺ terminals, arrows VGLUT1⁺/VGLUT3^- terminals, and double arrowhead indicates an IB4⁺ terminal that is VGLUT2 immunoreactive but lacks VGLUT1 or VGLUT3 immunolabeling. The micrographs are single deconvolved optical sections acquired with a 63×/1.4 objective. Scale bar, 1 µm, valid for all panels. B, Overview of VGLUT3⁺ and VGLUT1⁺ immunofluorescence in rat and mouse superficial dorsal horn. VGLUT1⁺ terminals are more abundant in lamina IIi in the rat as compared to the mouse. Note also that most VGLUT1⁺ terminals are VGLUT3⁺ in the rat but not in the mouse. VGLUT3⁺ processes in lamina I, outer lamina II, and in lamina III are more common in the mouse relative to the same laminae in the rat. Dashed lines indicate the border between lamina II and III as judged from the VGLUT3 immunoreactivity. Scale bar, 20 µm, valid for all panels in B.

Size and synapse number of VGLUT3⁺ C-LTMRs. A, Examples of VGLUT3⁺ presumed C-LTMRs in lamina IIi in spinal cord sections co-immunolabeled for the excitatory synaptic marker Homer1. Each terminal is apposed to several Homer1⁺ puncta. Note the incomplete filling of the terminals with VGLUT3 immunofluorescence, partly attributed to a high prevalence of axoplasmic mitochondria. The micrographs are single deconvolved optical sections acquired with a 63×/1.4 objective. Scale bar, 1 µm, valid for all panels. B, Frequency distribution of the maximum Feret diameter of VGLUT3⁺ terminals in lamina IIi. Mouse and rat distributions were compared using the Kolmogorov–Smirnov test. C, Histogram of the number of associated Homer1⁺ puncta per VGLUT3⁺ terminal, as assessed from all optical sections occupied by a terminal. Statistical significance of the difference between mouse and rat distributions was tested using Mann–Whitney U test.

Ultrastructural identification of C-LTMR terminals in the rat. A–E, Examples of terminals showing preembedding immunoperoxidase labeling for VGLUT3 in lamina IIi. Each terminal shows characteristics of central terminals of Type II glomeruli, including a generally round outline, light axoplasm, loosely packed clear small-diameter synaptic vesicles and several mitochondria. The terminals form multiple asymmetric synapses with postsynaptic dendrites, some of which contain vesicles that likely store GABA and thus originate from inhibitory neurons. Peripheral axons form inhibitory symmetric synapses onto either the central terminal (E) or a dendrite, or both (B). In E, a Type Ia glomeruli is adjacent to a VGLUT3⁺ terminal. Note that the central terminal of the Type I glomeruli (C_Ia), presumably originating from a non-peptidergic IB₄ binding C fiber, exhibits no peroxidase labeling. D, postsynaptic dendrite lacking vesicles; V₁, postsynaptic vesicle-containing dendrite; V₂, vesicle-containing presynaptic axon. Black and white arrowheads indicate the postsynaptic aspect of asymmetric and symmetric synapses, respectively. Dashed line in C outlines the terminal for clarity. Scale bars, 500 nm (A, B, D, E) and 1 µm (C).

Ultrastructural identification of C-LTMR terminals in the mouse. A, B, Examples of terminals showing preembedding immunoperoxidase labeling for VGLUT3 in lamina IIi of the mouse. As in the rat, peroxidase-labeled terminals were morphologically identical to central terminals of Type II glomeruli. C, D, Lowicryl-embedded dorsal horn section labeled for VGLUT1 using postembedding immunogold labeling. In C, a central terminal of a Type II glomeruli (C_II) in lamina IIi devoid of VGLUT1 immunolabeling above background levels is shown. In D, another central terminal of a Type II glomeruli in lamina IIi exhibits strong VGLUT1 immunogold labeling. D, postsynaptic dendrite lacking vesicles; V₁, postsynaptic vesicle-containing dendrite; V₂, vesicle-containing presynaptic axon. Black and white arrowheads indicate the postsynaptic aspect of asymmetric and symmetric synapses, respectively. Scale bars, 500 nm (A, B) and 500 nm (C, D).

Synaptic connections between VGLUT3⁺ C-LTMRs and PKCγ neurons in rat dorsal horn. A, Overview of a portion of Lamina IIi from a lumbar spinal cord section immunolabeled for VGLUT3, PKCγ, and the excitatory synaptic marker Homer1. Many VGLUT3⁺ terminals are adjacent to PKCγ⁺ dendrites, and often apposed to Homer1⁺ puncta associated with such dendrites. Roman numerals denote Rexed’s laminae. Dashed line indicates border between Lamina II and III. Scale bar, 5 µm. B–F, Examples at higher magnification of VGLUT3⁺ terminals apposed to PKCγ⁺ dendrites. Arrowheads indicate Homer1⁺ puncta associated with PKCγ⁺ dendrites. Arrows indicate Homer1⁺ puncta associated with the VGLUT3⁺ terminals but not with PKCγ⁺ dendrites. Asterisk in e indicates a transversely cut dendritic shaft. Scale bar, 1 µm (B–F). All micrographs are single deconvolved optical sections obtained with a 63×/1.4 objective.

Synaptic connections between VGLUT3⁺ C-LTMRs and parvalbumin neurons in rat dorsal horn. A, A portion of lateral Lamina IIi from a lumbar spinal cord section immunolabeled for VGLUT3, parvalbumin, and Homer1. Many VGLUT3⁺ terminals are apposed to parvalbumin⁺ processes with Homer1⁺ puncta. Roman numerals denote Rexed’s laminae. Dashed line indicates border between lamina II and III. Dashed frame indicates the region magnified in B. Scale bar, 5 µm. B–F, Examples at higher magnification of VGLUT3⁺ terminals apposed to parvalbumin processes. Arrowheads indicate Homer1⁺ puncta, associated with parvalbumin processes, apposed to the VGLUT3⁺ terminals. Arrows indicate Homer1⁺ puncta associated with the VGLUT3⁺ terminals but not with parvalbumin⁺ processes. Scale bar, 1 µm (B–F). All micrographs are single deconvolved optical sections obtained with a 63×/1.4 objective.

Synaptic connections between VGLUT3⁺ C-LTMRs and calretinin neurons in rat dorsal horn. A, A view of lateral lamina IIi from a lumbar spinal cord section immunolabeled for VGLUT3, calretinin and Homer1. Although calretinin⁺ processes are abundant in lamina IIi, few VGLUT3⁺ terminals are in close proximity to such processes. Roman numerals denote Rexed’s laminae. Dashed line indicates border between lamina II and III. Scale bar, 5 µm. B, A VGLUT3⁺ terminal apposed to a Homer1⁺ puncta associated with a calretinin⁺ process (arrowhead). C, A VGLUT3⁺ terminal apposed to several Homer1⁺ puncta, none of which is associated with the surrounding calretinin⁺ processes. Scale bar, 1 µm (B, C). Micrographs in A–C are deconvolved single optical sections obtained with a 63×/1.4 objective.