Characterisation of deep dorsal horn projection neurons in the spinal cord of the Phox2a::Cre mouse line

Abstract

Projection neurons belonging to the anterolateral system (ALS) underlie the perception of pain, skin temperature and itch. Many ALS cells are located in laminae III-V of the dorsal horn and the adjacent lateral white matter. However, relatively little is known about the excitatory synaptic input to these deep ALS cells, and therefore about their engagement with the neuronal circuitry of the region. We have used a recently developed mouse line, Phox2a::Cre, to investigate a population of deep dorsal horn ALS neurons known as "antenna cells", which are characterised by dense innervation from peptidergic nociceptors, and to compare these with other ALS cells in the deep dorsal horn and lateral white matter. We show that these two classes differ, both in the density of excitatory synapses, and in the source of input at these synapses. Peptidergic nociceptors account for around two-thirds of the excitatory synapses on the antenna cells, but for only a small proportion of the input to the non-antenna cells. Conversely, boutons with high levels of VGLUT2, which are likely to originate mainly from glutamatergic spinal neurons, account for only ∼5% of the excitatory synapses on antenna cells, but for a much larger proportion of the input to the non-antenna cells. VGLUT1 is expressed by myelinated low-threshold mechanoreceptors and corticospinal axons, and these innervate both antenna and non-antenna cells. However, the density of VGLUT1 input to the non-antenna cells is highly variable, consistent with the view that these neurons are functionally heterogeneous.

Keywords: Anterolateral system; Tac1; antenna cell; low-threshold mechanoreceptor; peptidergic nociceptor; substance P.

Figure 1.

Phox2a cells and their relationship to axons immunoreactive for CGRP, SP or NPY. (a), A transverse section through the dorsal horn from the L2 segment of a Phox2a::Cre;Ai9 scanned to reveal tdTomato in Phox2a cells. Two antenna cells (arrows) are present in this section, together with four deep dorsal horn Phox2a cells that are not antenna cells (arrowheads) and a lamina I cell (double arrowhead). (b) The same section showing immunoreactivity for SP (green), CGRP (red) and NPY (blue). Axons containing these peptides are concentrated in the superficial dorsal horn, but also form bundles in deeper laminae. (c) Staining for tdTomato (grey) has been superimposed on that for the neuropeptides, revealing the association between bundles of peptidergic axons and the antenna cells. The area in the box is shown as an inset at higher magnification. All images are projections of 16 confocal optical sections at 2 μm z-spacing. Scale bar = 200 μm. CGRP, calcitonin gene-related peptide; NPY, neuropeptide Y; SP, substance P.

Figure 2.

The distribution of different classes of Phox2a cells. (a–c), Plots showing the locations of Phox2a cells in the L2 segment in 3 Phox2a::Cre;Ai9 mice. All Phox2a cells (apart from those in the ventral horn) that were present on either side in 26, 23 and 19 transverse sections (respectively) were plotted onto an outline of the grey matter. Approximate laminar boundaries are shown, together with the position of the lateral spinal nucleus (LSN). Note that lamina VI is not present in the L2 spinal segment, and therefore laminae V and VII are directly adjacent. Cells were assigned to three main classes: lamina I (orange), antenna (green) and deep non-antenna (blue). The latter were subdivided into those near the central canal (light blue), those in the lateral white matter (dark blue) and the remainder (mid-blue), which were mainly present in the lateral part of lamina V but with some extension dorsally and ventrally. (d) A confocal image showing one of the sections from the animal represented in (c). A lamina V antenna cell is indicated with an arrow, and this has a dorsal dendrite that extends into the superficial dorsal horn. This cell corresponds to the one marked with the arrow in (c). The image in (d) is a projection of 17 optical sections at 2 μm z-spacing. Scale bar = 50 μm.

Figure 3.

Retrograde labelling of Phox2a antenna cells. (a) Two Phox2a antenna cells (arrows) revealed by expression of tdTomato (tdTom) in a transverse section through the dorsal horn from the L2 segment of a mouse that had received an injection of cholera toxin B subunit (CTb) into the lateral parabrachial area on the opposite side. (b) immunostaining for calcitonin gene-related peptide (CGRP) shows the association of peptidergic afferent boutons with the dendrites of these two cells. (c, d) both of the Phox2a cells are retrogradely labelled with CTb (blue). Images are projections from seven optical sections at 2 μm z-spacing. Scale bar = 100 μm.

Figure 4.

Reconstructions of deep dorsal horn Phox2a cells. Neurolucida reconstructions of antenna and non-antenna cells in sagittal sections from Phox2a::Cre;Ai32 mice. Antenna cells are shown in red, and the non-antenna cells in either green (for those located in the grey matter) or blue (for those in the white matter). The boxes on cells 4 and 9 indicate regions of these two cells that are illustrated in Figure 5. Scale bar = 100 μm. D, dorsal; V, ventral; RC, rostrocaudal.

Figure 5.

Contacts on deep dorsal horn Phox2a cells from boutons that are immunoreactive for CGRP and/or VGLUT2. (a) Part of the dorsal dendrite of an antenna cell (cell #4 in Figure 4) from a Phox2a::Cre;Ai32 mouse labelled to reveal YFP (blue) and Homer (red). Several Homer puncta can be seen on the dendritic shaft. (b, c) These images show immunostaining for either CGRP or VGLUT2 in green. Several of the Homer puncta are in contact with profiles that are CGRP-immunoreactive, and some of these also show weak VGLUT2-immunoreactivity (examples indicated with arrowheads). The arrow points to a Homer punctum that is not associated with CGRP. In this case, there is very weak VGLUT2-immunoreactivity adjacent to the Homer. The double arrow in (a) indicates a Homer punctum on a dendritic spine. (d) Part of the dendrite of a non-antenna cell that was located in the white matter (cell #9 in Figure 4) from a Phox2a::Cre;Ai32 mouse labelled to reveal YFP (blue) and Homer (red). Again, several Homer puncta can be seen on the dendritic shaft. (e, f) These images show immunostaining for either CGRP or VGLUT2 in green, although there is very little CGRP in this region. Several of the Homer puncta are in contact with VGLUT2-immunoreactive boutons (some shown with arrows). The arrowhead points to a Homer punctum that is not associated with VGLUT2. The double arrow in (d) indicates a Homer punctum on a dendritic spine. Images in (a)–(c) are from a single optical section, those in (d)–(f) are projected from three optical sections at 0.5 μm z-spacing. Scale bar: 5 μm. CGRP, calcitonin gene-related peptide; YFP, yellow fluorescent protein.

Figure 6.

Contacts on deep dorsal horn Phox2a cells from VGLUT1-immunoreactive boutons. (a, b) Part of a dendrite of an antenna cell from a Phox2a::Cre;Ai32 mouse labelled to reveal YFP (blue), Homer (red) and VGLUT1 (green). The dendrite contains several Homer puncta, one of which is marked by an arrow. This punctum is associated with a VGLUT1 bouton that contacts several other Homer puncta, and is therefore likely to originate from a myelinated low-threshold mechanoreceptive (A-LTMR) primary afferent. (c, d) Part of a dendrite belonging to a non-antenna Phox2a cell that was located in the deep part of the dorsal horn. Again, a Homer punctum (arrow) in the dendrite is associated with a VGLUT1 bouton that contacts several other Homer puncta. (e, f) A dendrite from a different non-antenna deep dorsal horn Phox2a cell has a Homer punctum (arrow) that is contacted by a small VGLUT1 profile. This profile is not associated with multiple Homer puncta, and is therefore likely to originate from a corticospinal axon. Images are projections of 2 (a, b, e, f) or 3 (c, d) confocal optical sections at 0.5 μm z-spacing. Scale bar = 5 μm. YFP, yellow fluorescent protein.

Figure 7.

Density of synapses from VGLUT1 profiles on non-antenna cells. Scatter plot showing the density of contacts onto Homer puncta on spines/shafts of non-antenna cells at which a VGLUT1-immunoreactive bouton was present. The cells were located either in the deep grey matter (n = 10, green circles) or in the lateral white matter (n = 8, blue circles). VGLUT1 boutons contacting these cells were classified as corticospinal if they were only adjacent to one Homer punctum, and as A-low threshold mechanoreceptor (A-LTMR) if they were adjacent to two or more Homer puncta.

Figure 8.

Expression of Tac1 mRNA by an antenna cell. (a) Immunostaining for tdTomato (tdTom) in a Phox2a cell (asterisk) in lamina III. (b) The cell is associated with numerous boutons that are immunoreactive for CGRP (blue), confirming that it is an antenna cell. (c) fluorescent in situ hybridisation for Tac1 mRNA reveals strong labelling of the antenna cell, and also the presence of Tac1 in three other cells nearby (marked with arrowheads). (d) A merged image showing expression of Tac1 in the tdTomato cell. All images are projections of 11 confocal optical sections at 1 μm z-spacing. Scale bar = 20 μm. CGRP, calcitonin gene-related peptide.