Characterisation of lamina I anterolateral system neurons that express Cre in a Phox2a-Cre mouse line

Abstract

A recently developed Phox2a::Cre mouse line has been shown to capture anterolateral system (ALS) projection neurons. Here, we used this line to test whether Phox2a-positive cells represent a distinct subpopulation among lamina I ALS neurons. We show that virtually all lamina I Phox2a cells can be retrogradely labelled from injections targeted on the lateral parabrachial area (LPb), and that most of those in the cervical cord also belong to the spinothalamic tract. Phox2a cells accounted for ~ 50–60% of the lamina I cells retrogradely labelled from LPb or thalamus. Phox2a was preferentially associated with smaller ALS neurons, and with those showing relatively weak neurokinin 1 receptor expression. The Phox2a cells were also less likely to project to the ipsilateral LPb. Although most Phox2a cells phosphorylated extracellular signal-regulated kinases following noxious heat stimulation, ~ 20% did not, and these were significantly smaller than the activated cells. This suggests that those ALS neurons that respond selectively to skin cooling, which have small cell bodies, may be included among the Phox2a population. Previous studies have defined neurochemical populations among the ALS cells, based on expression of Tac1 or Gpr83. However, we found that the proportions of Phox2a cells that expressed these genes were similar to the proportions reported for all lamina I ALS neurons, suggesting that Phox2a is not differentially expressed among cells belonging to these populations. Finally, we used a mouse line that resulted in membrane labelling of the Phox2a cells and showed that they all possess dendritic spines, although at a relatively low density. However, the distribution of the postsynaptic protein Homer revealed that dendritic spines accounted for a minority of the excitatory synapses on these cells. Our results confirm that Phox2a-positive cells in lamina I are ALS neurons, but show that the Phox2a::Cre line preferentially captures specific types of ALS cells.

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

“…The distribution of Phox2a cells in the L2 spinal cord segments of Phox2a::Cre;Ai9 mice was similar to that reported previously 5 , 25 ( Figures 1 and 2 ). Within the dorsal horn these cells were concentrated in lamina I, present at a lower density in the deeper laminae of the dorsal horn (III-V) and largely absent from lamina II.…”

“…This contrasts with ALS neurons in lamina I, the LSN and deeper laminae, since many of these are not Phox2a-positive. 5 , 25 …”

“…One was the BAC transgenic line (Phox2a::Cre), in which Cre recombinase is expressed under control of the Phox2a promoter. 5 , 25 The other two were reporter lines (Ai9, Ai32), in which Cre-mediated excision of a STOP cassette drives expression of either tdTomato (Ai9) or yellow fluorescent protein (YFP) fused to channelrhodopsin (Ai32). The Phox2a::Cre line was crossed with each of these reporters to generate mice in which cells that expressed Phox2a at any point during development were permanently marked by the presence of tdTomato or YFP, with the latter being targeted to the plasma membrane.…”

“…To determine the proportion of Phox2a-positive antenna cells that were retrogradely labelled from LPb, and the proportion of retrogradely labelled antenna cells that were Phox2a-positive, we used transverse sections from the L2 segments of 6 Phox2a::Cre;Ai9 mice that had been used in a previous study (experiments 1–6 in reference 25 ). In each case, the animals had received an injection of 300 nL 1% cholera toxin B subunit (CTb) targeted on the left LPb, and in two cases, an equivalent injection was also made into the right LPb.…”

“…The Ai32 reporter line (instead of Ai9) was used for these experiments because the membrane-associated YFP expression is more suitable for neuronal reconstruction. 25 The sections had been reacted with antibodies against green fluorescent protein (GFP, this antibody also detects YFP), CGRP (rabbit antibody), VGLUT2 and the postsynaptic density protein Homer. The cell bodies and dendritic trees (including dendritic spines) were initially drawn using Neurolucida.…”