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Phosphorylation of ERK in neurokinin 1 receptor-expressing neurons in laminae III and IV of the rat spinal dorsal horn following noxious stimulation.

Polgár E, Campbell AD, MacIntyre LM, Watanabe M, Todd AJ - Mol Pain (2007)

Bottom Line: We found that virtually all of the lamina III/IV NK1r-immunoreactive neurons contained pERK after each of these stimuli and that in the great majority of cases there was internalisation of the NK1r on the dorsal dendrites of these cells.In addition, we also saw neurons in lamina III that were pERK-positive but lacked the NK1r, and these were particularly evident in animals that had had the pinch stimulus.Our results demonstrate that lamina III/IV NK1r-immunoreactive neurons show receptor internalisation and ERK phosphorylation after mechanical, thermal or chemical noxious stimuli.

View Article: PubMed Central - HTML - PubMed

Affiliation: Spinal Cord Group, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. e.polgar@bio.gla.ac.uk

ABSTRACT

Background: There is a population of large neurons with cell bodies in laminae III and IV of the spinal dorsal horn which express the neurokinin 1 receptor (NK1r) and have dendrites that enter the superficial laminae. Although it has been shown that these are all projection neurons and that they are innervated by substance P-containing (nociceptive) primary afferents, we know little about their responses to noxious stimuli. In this study we have looked for phosphorylation of extracellular signal-regulated kinases (ERKs) in these neurons in response to different types of noxious stimulus applied to one hindlimb of anaesthetised rats. The stimuli were mechanical (repeated pinching), thermal (immersion in water at 52 degrees C) or chemical (injection of 2% formaldehyde).

Results: Five minutes after each type of stimulus we observed numerous cells with phosphorylated ERK (pERK) in laminae I and IIo, together with scattered positive cells in deeper laminae. We found that virtually all of the lamina III/IV NK1r-immunoreactive neurons contained pERK after each of these stimuli and that in the great majority of cases there was internalisation of the NK1r on the dorsal dendrites of these cells. In addition, we also saw neurons in lamina III that were pERK-positive but lacked the NK1r, and these were particularly evident in animals that had had the pinch stimulus.

Conclusion: Our results demonstrate that lamina III/IV NK1r-immunoreactive neurons show receptor internalisation and ERK phosphorylation after mechanical, thermal or chemical noxious stimuli.

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Phosphorylation of ERK in the ipsilateral dorsal horn after different types of noxious stimulus. pERK-immunostaining in confocal images of parasagittal sections through the medial part of the left dorsal horn from rats that were perfused with fixative 5 mins after the end of different types of noxious stimulus: a repeated pinching of the skin of the foot for 1 min (pinch), b immersion of the hindpaw in water at 52°C for 1 min (heat), c injection of 100 μl 2% formaldehyde into the hindpaw (form). These sections were also immunostained for PKCγ (not shown) and the two dashed lines, which outline lamina IIi, were drawn from the location of the plexus of PKCγ-immunoreactive dendrites (for further details see text). The positions of laminae I, IIo and III are also indicated in a. In all cases there is strong pERK-immunoreactivity that is mainly restricted to laminae I and IIo. Some pERK-immunoreactive cells were seen ventral to the IIo/IIi border, particularly after pinch. All images are projections of z-series consisting of 10 optical sections at 2 μm spacing. Scale bar = 100 μm.
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Figure 1: Phosphorylation of ERK in the ipsilateral dorsal horn after different types of noxious stimulus. pERK-immunostaining in confocal images of parasagittal sections through the medial part of the left dorsal horn from rats that were perfused with fixative 5 mins after the end of different types of noxious stimulus: a repeated pinching of the skin of the foot for 1 min (pinch), b immersion of the hindpaw in water at 52°C for 1 min (heat), c injection of 100 μl 2% formaldehyde into the hindpaw (form). These sections were also immunostained for PKCγ (not shown) and the two dashed lines, which outline lamina IIi, were drawn from the location of the plexus of PKCγ-immunoreactive dendrites (for further details see text). The positions of laminae I, IIo and III are also indicated in a. In all cases there is strong pERK-immunoreactivity that is mainly restricted to laminae I and IIo. Some pERK-immunoreactive cells were seen ventral to the IIo/IIi border, particularly after pinch. All images are projections of z-series consisting of 10 optical sections at 2 μm spacing. Scale bar = 100 μm.

Mentions: One of three types of noxious stimulus was applied to the left hindpaw of anaesthetised rats: repeated pinching of the skin for 1 minute (n = 4), immersion of the paw in water at 52°C for 1 min (n = 3), or subcutaneous injection of 100 μl 2% formaldehyde (n = 3). Five minutes after the end of each type of stimulus, numerous pERK-immunoreactive cells and processes were visible in the medial part of the ipsilateral dorsal horn in the L4 spinal cord segment (Fig. 1). In each case, these were highly concentrated in a band that occupied lamina I and the outer (dorsal) half of lamina II (lamina IIo). We have previously shown that a plexus of PKCγ-immunoreactive dendrites in the superficial dorsal horn occupies the inner (ventral) half of lamina II (IIi) [30], and the dorsal and ventral borders of this plexus were therefore used on some sections in the present study to define the boundaries between lamina IIo/IIi and lamina IIi/III, respectively. Scattered pERK-positive cells were present in the deeper laminae after each type of stimulus, and although not quantified, these appeared to be more numerous in animals that had undergone the pinch stimulus than those that had received noxious thermal or chemical stimulation (Fig. 1). A few cells with weak pERK-immunoreactivity were seen in the superficial dorsal horn in the lateral half of the ipsilateral side and on the contralateral side. However, the lamina III/IV NK1r-immunoreactive neurons in these regions were never pERK-positive.


Phosphorylation of ERK in neurokinin 1 receptor-expressing neurons in laminae III and IV of the rat spinal dorsal horn following noxious stimulation.

Polgár E, Campbell AD, MacIntyre LM, Watanabe M, Todd AJ - Mol Pain (2007)

Phosphorylation of ERK in the ipsilateral dorsal horn after different types of noxious stimulus. pERK-immunostaining in confocal images of parasagittal sections through the medial part of the left dorsal horn from rats that were perfused with fixative 5 mins after the end of different types of noxious stimulus: a repeated pinching of the skin of the foot for 1 min (pinch), b immersion of the hindpaw in water at 52°C for 1 min (heat), c injection of 100 μl 2% formaldehyde into the hindpaw (form). These sections were also immunostained for PKCγ (not shown) and the two dashed lines, which outline lamina IIi, were drawn from the location of the plexus of PKCγ-immunoreactive dendrites (for further details see text). The positions of laminae I, IIo and III are also indicated in a. In all cases there is strong pERK-immunoreactivity that is mainly restricted to laminae I and IIo. Some pERK-immunoreactive cells were seen ventral to the IIo/IIi border, particularly after pinch. All images are projections of z-series consisting of 10 optical sections at 2 μm spacing. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1803781&req=5

Figure 1: Phosphorylation of ERK in the ipsilateral dorsal horn after different types of noxious stimulus. pERK-immunostaining in confocal images of parasagittal sections through the medial part of the left dorsal horn from rats that were perfused with fixative 5 mins after the end of different types of noxious stimulus: a repeated pinching of the skin of the foot for 1 min (pinch), b immersion of the hindpaw in water at 52°C for 1 min (heat), c injection of 100 μl 2% formaldehyde into the hindpaw (form). These sections were also immunostained for PKCγ (not shown) and the two dashed lines, which outline lamina IIi, were drawn from the location of the plexus of PKCγ-immunoreactive dendrites (for further details see text). The positions of laminae I, IIo and III are also indicated in a. In all cases there is strong pERK-immunoreactivity that is mainly restricted to laminae I and IIo. Some pERK-immunoreactive cells were seen ventral to the IIo/IIi border, particularly after pinch. All images are projections of z-series consisting of 10 optical sections at 2 μm spacing. Scale bar = 100 μm.
Mentions: One of three types of noxious stimulus was applied to the left hindpaw of anaesthetised rats: repeated pinching of the skin for 1 minute (n = 4), immersion of the paw in water at 52°C for 1 min (n = 3), or subcutaneous injection of 100 μl 2% formaldehyde (n = 3). Five minutes after the end of each type of stimulus, numerous pERK-immunoreactive cells and processes were visible in the medial part of the ipsilateral dorsal horn in the L4 spinal cord segment (Fig. 1). In each case, these were highly concentrated in a band that occupied lamina I and the outer (dorsal) half of lamina II (lamina IIo). We have previously shown that a plexus of PKCγ-immunoreactive dendrites in the superficial dorsal horn occupies the inner (ventral) half of lamina II (IIi) [30], and the dorsal and ventral borders of this plexus were therefore used on some sections in the present study to define the boundaries between lamina IIo/IIi and lamina IIi/III, respectively. Scattered pERK-positive cells were present in the deeper laminae after each type of stimulus, and although not quantified, these appeared to be more numerous in animals that had undergone the pinch stimulus than those that had received noxious thermal or chemical stimulation (Fig. 1). A few cells with weak pERK-immunoreactivity were seen in the superficial dorsal horn in the lateral half of the ipsilateral side and on the contralateral side. However, the lamina III/IV NK1r-immunoreactive neurons in these regions were never pERK-positive.

Bottom Line: We found that virtually all of the lamina III/IV NK1r-immunoreactive neurons contained pERK after each of these stimuli and that in the great majority of cases there was internalisation of the NK1r on the dorsal dendrites of these cells.In addition, we also saw neurons in lamina III that were pERK-positive but lacked the NK1r, and these were particularly evident in animals that had had the pinch stimulus.Our results demonstrate that lamina III/IV NK1r-immunoreactive neurons show receptor internalisation and ERK phosphorylation after mechanical, thermal or chemical noxious stimuli.

View Article: PubMed Central - HTML - PubMed

Affiliation: Spinal Cord Group, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. e.polgar@bio.gla.ac.uk

ABSTRACT

Background: There is a population of large neurons with cell bodies in laminae III and IV of the spinal dorsal horn which express the neurokinin 1 receptor (NK1r) and have dendrites that enter the superficial laminae. Although it has been shown that these are all projection neurons and that they are innervated by substance P-containing (nociceptive) primary afferents, we know little about their responses to noxious stimuli. In this study we have looked for phosphorylation of extracellular signal-regulated kinases (ERKs) in these neurons in response to different types of noxious stimulus applied to one hindlimb of anaesthetised rats. The stimuli were mechanical (repeated pinching), thermal (immersion in water at 52 degrees C) or chemical (injection of 2% formaldehyde).

Results: Five minutes after each type of stimulus we observed numerous cells with phosphorylated ERK (pERK) in laminae I and IIo, together with scattered positive cells in deeper laminae. We found that virtually all of the lamina III/IV NK1r-immunoreactive neurons contained pERK after each of these stimuli and that in the great majority of cases there was internalisation of the NK1r on the dorsal dendrites of these cells. In addition, we also saw neurons in lamina III that were pERK-positive but lacked the NK1r, and these were particularly evident in animals that had had the pinch stimulus.

Conclusion: Our results demonstrate that lamina III/IV NK1r-immunoreactive neurons show receptor internalisation and ERK phosphorylation after mechanical, thermal or chemical noxious stimuli.

Show MeSH
Related in: MedlinePlus