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.
Highlights
The dorsal horn of the spinal cord receives sensory information from peripheral nerves, modulates this by means of complex local circuits, and transmits it to the brain through projection neurons[1,2]
TdTomato-positive cells were relatively numerous in lamina I, and were present at lower density in deeper dorsal horn laminae (III–VI), the lateral spinal nucleus (LSN) and the area around the central canal
Our main findings are: (1) that the Phox2a::Cre line captures 50–60% of lamina I spinoparabrachial cells, and that expression is essentially restricted to projection neurons in this lamina; (2) that the pattern of expression is not random, since larger cells and those with strong neurokinin 1 receptor (NK1r)-immunoreactivity are less likely to be Phox2a-positive; (3) that all of the Phox2a cells in lamina I possessed dendritic spines, these accounted for well under half of the Homer puncta on individual cells, and spine synapses are likely to represent a minor component of the excitatory input to these cells
Summary
The dorsal horn of the spinal cord receives sensory information from peripheral nerves, modulates this by means of complex local circuits, and transmits it to the brain through projection neurons[1,2]. It was subsequently shown that changes in the response properties of lamina I ALS neurons recorded in vivo matched the increased reflex responses seen in neuropathic pain m odels[16,17] Both electrophysiological mapping of the axons of lamina I projection c ells[18] and retrograde tracing s tudies[19] have shown that individual lamina I neurons in the rat spinal cord send collateral branches to more than one brainstem target. Roome et al.[7] have recently developed a BAC transgenic mouse line in which Cre is expressed under control of the promoter for the transcription factor Phox2a (Phox2a::Cre) After crossing this with a Cre-dependent tdTomato reporter line, they showed that tdTomato labelling was virtually restricted to ALS neurons in the spinal cord, and captured around half of those in lamina I. We have used this approach to investigate the distribution of excitatory synapses on these cells
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