Synaptic connectivity of the TRPV1-positive trigeminal afferents in the rat lateral parabrachial nucleus

Abstract

Recent studies have shown a direct projection of nociceptive trigeminal afferents into the lateral parabrachial nucleus (LPBN). Information about the synaptic connectivity of these afferents may help understand how orofacial nociception is processed in the LPBN, which is known to be involved primarily in the affective aspect of pain. To address this issue, we investigated the synapses of the transient receptor potential vanilloid 1-positive (TRPV1+) trigeminal afferent terminals in the LPBN by immunostaining and serial section electron microscopy. TRPV1 + afferents arising from the ascending trigeminal tract issued axons and terminals (boutons) in the LPBN. TRPV1+ boutons formed synapses of asymmetric type with dendritic shafts and spines. Almost all (98.3%) TRPV1+ boutons formed synapses with one (82.6%) or two postsynaptic dendrites, suggesting that, at a single bouton level, the orofacial nociceptive information is predominantly transmitted to a single postsynaptic neuron with a small degree of synaptic divergence. A small fraction (14.9%) of the TRPV1+ boutons formed synapses with dendritic spines. None of the TRPV1+ boutons were involved in axoaxonic synapses. Conversely, in the trigeminal caudal nucleus (Vc), TRPV1+ boutons often formed synapses with multiple postsynaptic dendrites and were involved in axoaxonic synapses. Number of dendritic spine and total number of postsynaptic dendrites per TRPV1+ bouton were significantly fewer in the LPBN than Vc. Thus, the synaptic connectivity of the TRPV1+ boutons in the LPBN differed significantly from that in the Vc, suggesting that the TRPV1-mediated orofacial nociception is relayed to the LPBN in a distinctively different manner than in the Vc.

Keywords: lateral parabrachial nucleus; nociceptive; synaptic connectivity; trigeminal; ultrastructure.

FIGURE 1

Light micrographs showing immunostaining for TRPV1 in the lateral parabrachial nucleus (LPBN: A,B) and the trigeminal ganglion (TG: C,D), the TRPV1+ axons and terminals in the LPBN arising from the ascending trigeminal tract (E–K), and examples of TRPV1+ axon terminals in the LPBN that were further analyzed by electron microscopy (L,M). (A–D) Immunohistochemical staining for TRPV1 in the LPBN (A,B) and TG (C,D). The TRPV1 immunostaining in the axons in the LPBN and neurons in the TG was completely abolished by pre-adsorption with a blocking peptide (10 μg/ml), confirming the specificity of the TRPV1 antibody. (E–K) Light micrographs showing that the TRPV1+ axons and terminals in the LPBN (E–G) arise from the ascending trigeminal tract (Vtr: K). These axons (K) course along the medial edge of the middle cerebellar peduncle (mcp: J) and the dorsal border of the trigeminal principal nucleus (Vp: H,I) and issue axon collaterals and terminals in the LPBN (F,G). (L,M) Examples of TRPV1+ axons and terminals in the LPBN that were further studied by electron microscopy. (F–K,M) Are enlargements of the boxed areas in panels (E,L), respectively. Arrowheads indicate TRPV1+ axons and terminals. scp, superior cerebellar peduncle. Scale bars = 10 μm in panels (A,B,F–K,M), 50 μm in panels (C,D) and 500 μm in panels (E,L).

FIGURE 2

Electron micrographs of adjacent thin sections (A and B, C and D, E and F, each pair about 200 nm apart) in the lateral parabrachial nucleus (LPBN) showing TRPV1+ boutons (asterisks) forming synapse with one dendrite (d in panels A,B), two dendrites (d1 and d2 in panels C,D) and with a dendritic spine (ds in panels E,F). The TRPV1+ bouton (asterisk) can be identified by the presence of electron-dense immunoreaction product (arrow) within the axoplasm. Arrowheads indicate synapses. Scale bars = 500 nm in panel (B) (also applies to panel A) and (F) (also applies to panels C–E).

FIGURE 3

Electron micrographs of adjacent thin sections (A and B, C and D, each pair about 200 nm apart) in the trigeminal caudal nucleus (Vc). (A,B) A TRPV1+ bouton (asterisk) makes a synaptic contact with 3 dendritic shafts (d1–d3) and one dendritic spine (ds). (C,D) A TRPV1+ bouton (asterisk) makes a synaptic contact with one dendritic shaft (d) and receives an axoaxonic synapse from a presynaptic ending containing pleomorphic vesicles (p). Arrows indicate electron dense TRPV1 immunoreaction product. Arrowheads indicate synapses. TRPV1+ boutons and presynaptic ending are outlined with a dashed line. Scale bar = 500 nm in panel (D) (also applies to panels A–C).