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Tactile allodynia can occur in the spared nerve injury model in the rat without selective loss of GABA or GABA(A) receptors from synapses in laminae I-II of the ipsilateral spinal dorsal horn.

Polgár E, Todd AJ - Neuroscience (2008)

Bottom Line: We found no difference in the intensity of immunolabeling for any of these markers on the two sides of the superficial dorsal horn.These results suggest that there is no significant loss of GABAergic boutons from the denervated area after SNI (which is consistent with the finding that neuronal death does not occur in this model) and that there is no depletion of GABA or GABA(A) receptors at GABAergic synapses within this region.An alternative explanation for disinhibition after nerve injury is that it results from reduced excitatory drive to GABAergic dorsal horn neurons following loss of primary afferent input to these cells.

View Article: PubMed Central - PubMed

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

ABSTRACT
Although there is evidence that reduced inhibition in the spinal dorsal horn contributes to neuropathic pain, the mechanisms that underlie this are poorly understood. We have previously demonstrated that there is no loss of neurons from laminae I-III in the spared nerve injury (SNI) model [Polgár E, Hughes DI, Arham AZ, Todd AJ (2005) Loss of neurons from laminas I-III of the spinal dorsal horn is not required for development of tactile allodynia in the SNI model of neuropathic pain. J Neurosci 25:6658-6666]. In this study we investigated whether there was a difference between ipsilateral and contralateral sides in the levels of GABA, the vesicular GABA transporter (VGAT), or the beta3 subunit of the GABA(A) receptor at synapses in the medial part of the superficial dorsal horn in this model. Tissue from rats that had undergone SNI 4 weeks previously was examined with an electron microscopic immunogold method to reveal GABA, following pre-embedding detection of GABA(A) beta3 to allow identification of GABAergic terminals. Assessment of labeling for the GABA(A) beta3 subunit and VGAT was performed by using immunofluorescence and confocal microscopy. We found no difference in the intensity of immunolabeling for any of these markers on the two sides of the superficial dorsal horn. These results suggest that there is no significant loss of GABAergic boutons from the denervated area after SNI (which is consistent with the finding that neuronal death does not occur in this model) and that there is no depletion of GABA or GABA(A) receptors at GABAergic synapses within this region. An alternative explanation for disinhibition after nerve injury is that it results from reduced excitatory drive to GABAergic dorsal horn neurons following loss of primary afferent input to these cells.

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Confocal images of immunostaining for the GABAA receptor β3 subunit and VGAT in the superficial dorsal horn. (a) Following antigen retrieval with pepsin, GABAA receptor β3 subunit-immunoreactivity (green) appears as small puncta scattered throughout the neuropil. Some of these are indicated with arrows. (b) Before pepsin treatment the section had been reacted with antibody to VGAT which was revealed with a TSA method (red). (c) A merged image shows that most of the GABAA receptor β3 subunit-immunoreactive puncta are adjacent to VGAT-immunoreactive axonal boutons. Images were obtained from single optical sections scanned through a 60× oil immersion lens. Scale bar=5 μm.
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fig3: Confocal images of immunostaining for the GABAA receptor β3 subunit and VGAT in the superficial dorsal horn. (a) Following antigen retrieval with pepsin, GABAA receptor β3 subunit-immunoreactivity (green) appears as small puncta scattered throughout the neuropil. Some of these are indicated with arrows. (b) Before pepsin treatment the section had been reacted with antibody to VGAT which was revealed with a TSA method (red). (c) A merged image shows that most of the GABAA receptor β3 subunit-immunoreactive puncta are adjacent to VGAT-immunoreactive axonal boutons. Images were obtained from single optical sections scanned through a 60× oil immersion lens. Scale bar=5 μm.

Mentions: In preliminary tests with conventional immunofluorescence reactions, we observed only very weak labeling for the GABAA receptor β3 subunit, and this presumably reflects the lower sensitivity of fluorescent labeling compared with that of the avidin/biotin/peroxidase method that was used for electron microscopy. We therefore carried out antigen retrieval with pepsin, since this has been used successfully on perfusion-fixed tissue to reveal other ionotropic receptors at synapses with confocal microscopy (Watanabe et al., 1998; Nagy et al., 2004; Polgár et al., 2008). We found that following pepsin treatment, it was possible to visualize strong punctate labeling for the GABAA receptor β3 subunit (Fig. 3a). To confirm that this labeling was located at GABAergic synapses, we analyzed sections that had also been immunostained for VGAT from two of the rats, and found that in both cases 94% of GABAA receptor β3 subunit-immunoreactive puncta were in contact with a VGAT-positive bouton, while 96–97% of VGAT-positive boutons were adjacent to at least one receptor-labeled punctum (Fig. 3).


Tactile allodynia can occur in the spared nerve injury model in the rat without selective loss of GABA or GABA(A) receptors from synapses in laminae I-II of the ipsilateral spinal dorsal horn.

Polgár E, Todd AJ - Neuroscience (2008)

Confocal images of immunostaining for the GABAA receptor β3 subunit and VGAT in the superficial dorsal horn. (a) Following antigen retrieval with pepsin, GABAA receptor β3 subunit-immunoreactivity (green) appears as small puncta scattered throughout the neuropil. Some of these are indicated with arrows. (b) Before pepsin treatment the section had been reacted with antibody to VGAT which was revealed with a TSA method (red). (c) A merged image shows that most of the GABAA receptor β3 subunit-immunoreactive puncta are adjacent to VGAT-immunoreactive axonal boutons. Images were obtained from single optical sections scanned through a 60× oil immersion lens. Scale bar=5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553186&req=5

fig3: Confocal images of immunostaining for the GABAA receptor β3 subunit and VGAT in the superficial dorsal horn. (a) Following antigen retrieval with pepsin, GABAA receptor β3 subunit-immunoreactivity (green) appears as small puncta scattered throughout the neuropil. Some of these are indicated with arrows. (b) Before pepsin treatment the section had been reacted with antibody to VGAT which was revealed with a TSA method (red). (c) A merged image shows that most of the GABAA receptor β3 subunit-immunoreactive puncta are adjacent to VGAT-immunoreactive axonal boutons. Images were obtained from single optical sections scanned through a 60× oil immersion lens. Scale bar=5 μm.
Mentions: In preliminary tests with conventional immunofluorescence reactions, we observed only very weak labeling for the GABAA receptor β3 subunit, and this presumably reflects the lower sensitivity of fluorescent labeling compared with that of the avidin/biotin/peroxidase method that was used for electron microscopy. We therefore carried out antigen retrieval with pepsin, since this has been used successfully on perfusion-fixed tissue to reveal other ionotropic receptors at synapses with confocal microscopy (Watanabe et al., 1998; Nagy et al., 2004; Polgár et al., 2008). We found that following pepsin treatment, it was possible to visualize strong punctate labeling for the GABAA receptor β3 subunit (Fig. 3a). To confirm that this labeling was located at GABAergic synapses, we analyzed sections that had also been immunostained for VGAT from two of the rats, and found that in both cases 94% of GABAA receptor β3 subunit-immunoreactive puncta were in contact with a VGAT-positive bouton, while 96–97% of VGAT-positive boutons were adjacent to at least one receptor-labeled punctum (Fig. 3).

Bottom Line: We found no difference in the intensity of immunolabeling for any of these markers on the two sides of the superficial dorsal horn.These results suggest that there is no significant loss of GABAergic boutons from the denervated area after SNI (which is consistent with the finding that neuronal death does not occur in this model) and that there is no depletion of GABA or GABA(A) receptors at GABAergic synapses within this region.An alternative explanation for disinhibition after nerve injury is that it results from reduced excitatory drive to GABAergic dorsal horn neurons following loss of primary afferent input to these cells.

View Article: PubMed Central - PubMed

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

ABSTRACT
Although there is evidence that reduced inhibition in the spinal dorsal horn contributes to neuropathic pain, the mechanisms that underlie this are poorly understood. We have previously demonstrated that there is no loss of neurons from laminae I-III in the spared nerve injury (SNI) model [Polgár E, Hughes DI, Arham AZ, Todd AJ (2005) Loss of neurons from laminas I-III of the spinal dorsal horn is not required for development of tactile allodynia in the SNI model of neuropathic pain. J Neurosci 25:6658-6666]. In this study we investigated whether there was a difference between ipsilateral and contralateral sides in the levels of GABA, the vesicular GABA transporter (VGAT), or the beta3 subunit of the GABA(A) receptor at synapses in the medial part of the superficial dorsal horn in this model. Tissue from rats that had undergone SNI 4 weeks previously was examined with an electron microscopic immunogold method to reveal GABA, following pre-embedding detection of GABA(A) beta3 to allow identification of GABAergic terminals. Assessment of labeling for the GABA(A) beta3 subunit and VGAT was performed by using immunofluorescence and confocal microscopy. We found no difference in the intensity of immunolabeling for any of these markers on the two sides of the superficial dorsal horn. These results suggest that there is no significant loss of GABAergic boutons from the denervated area after SNI (which is consistent with the finding that neuronal death does not occur in this model) and that there is no depletion of GABA or GABA(A) receptors at GABAergic synapses within this region. An alternative explanation for disinhibition after nerve injury is that it results from reduced excitatory drive to GABAergic dorsal horn neurons following loss of primary afferent input to these cells.

Show MeSH
Related in: MedlinePlus