PMC

Expression of SP by anterogradely labelled spinoparabrachial terminals. (A and B): A single optical section through the lateral parabrachial area reacted to reveal CTb (magenta) and substance P (SP, green). (C): A merged image. Several CTb-labelled axonal boutons are visible, and some of these are SP-immunoreactive (2 indicated with arrowheads). Scale bar: 10 μm.

Giant lamina I neurons seen in horizontal sections. (A-D): Confocal images showing a giant cell that was retrogradely labelled with CTb (green) and immunoreactive for the NK1r (grey). Note that many boutons containing VLGUT2 (red) or VGAT (blue) are associated with the cell body (*) and that these surround the dendrites (indicated by arrowheads). (E-H): Another giant cell that was not retrogradely labelled and lacked NK1r-immunoreactivity. Again, the soma (*) and dendrites (between arrowheads) are associated with numerous VGLUT2 and VGAT boutons. The 2 sets of images are projections of 5 (A-D) and 6 (E-H) optical sections at 1 μm z-spacing. Scale bar: 20 μm.

Innervation of a lamina III NK1r-negative projection neuron by CGRP- and NPY-containing axons. (A): Confocal image showing part of a transverse section through a lamina III neuron that was retrogradely labelled with CTb (blue). (B-D): The cell body is surrounded by axons that contain CGRP (red) or NPY (green). The inset in (A) shows the region through the cell body (*) stained for the NK1r, and this cell is not immunoreactive for the receptor. The images are projections of 4 optical sections at 2 μm z-spacing. Scale bar: 50 μm. CGRP, calcitonin gene-related peptide; NPY, neuropeptide Y.

Expression of NK1r and sst_2A by retrogradely labelled spinoparabrachial neurons in lamina I. Confocal images showing a single optical section through the superficial dorsal horn in the L4 segment, contralateral to the lateral parabrachial area injection site. (A) CTb labelling (red) in 4 lamina I neurons identifies these as belonging to the spinoparabrachial tract. (B-D) Immunostaining of the section for the NK1r (green) and sst_2A (blue) shows that one of these cells (marked with an arrow) expresses both receptors, while the other 3 (asterisks) are only NK1r-immunoreactive. Scale bar: 20 μm.

VGLUT2 expression by anterogradely labelled spinoparabrachial axon terminals. (A) Low-magnification image through the lateral parabrachial area (LPb) scanned to reveal CTb (red) and with dark-field optics. The LPb lies dorsal (d) and lateral (l) to the superior cerebellar peduncle (scp). (B): The region shown in the box in (A) scanned at high magnification (15 optical sections at 0.5 μm z-spacing) shows numerous CTb-labelled boutons. (C-F): Single optical section corresponding to the region shown in the box in (B), scanned to reveal CTb, VGLUT2 (green), and VGAT (blue). Four CTb-labelled boutons are indicated with arrowheads. These are all VGLUT2-immunoreactive and lack VGAT. Scale bars: (A), 200 μm; (B), 50 μm; (C-F), 10 μm.

NK1r-immunoreactive projection neurons in lamina I. (A-C) Confocal images from a horizontal section through the L5 segment of one of the mice that had received an injection of CTb into the lateral parabrachial area. The section has been scanned to reveal CTb (green) and NK1r (magenta). Six retrogradely labelled (CTb-positive) neurons are indicated with asterisks, and each of these is NK1r-immunoreactive. In addition, several smaller NK1r-immunoreactive cells that are not retrogradely labelled (CTb-negative) are visible, and 5 of these are marked with arrowheads. Projection from 5 optical sections at 2 μm z-spacing. Scale bar: 50 μm. (D): Histograms showing the numbers of lamina I NK1r-immunoreactive cells with different soma areas. The top graph shows the results for all cells (All NK1r⁺), while the middle and lower graphs show those that were not labelled (NK1r⁺ nonretrograde) or retrogradely labelled (NK1r⁺ retrograde), respectively. The dashed line corresponds to a soma area of 180 μm². Note that most of the nonretrogradely labelled neurons are smaller than this, whereas most retrogradely labelled cells are larger.

Injection sites in the 6 mice used in this study. (A) Injection sites in the 4 experiments in which CTb was injected into the lateral parabrachial area. Each vertical column represents a single experiment. The darker shaded areas show the spread of tracer, while the pale shaded area is the superior cerebellar peduncle. Numbers to the left of the drawings correspond to the position of the section posterior to the interaural plane. Drawings are based on those of Franklin and Paxinos. (B) A section through the brain from the mouse #4, corresponding to a level of ∼1.5 mm caudal to the interaural plane. The CTb injection site appears as a dark area, and near the top of the image, the pipette track can be seen passing through the inferior colliculus. (C and D) Drawings indicating the core of the injection site in the 2 mice that received intraspinal injections of CTb. (E) A section from the spinal cord of the mouse represented in (C). Scale bars: (B), 1 mm; (C), 250 μm. IC, inferior colliculus; KF, Kölliker-Fuse nucleus; LPb, lateral parabrachial area; MPb, medial parabrachial area; PAG, periaqueductal grey.