Expression of AMPA Receptor Subunits at Synapses in Laminae I–III of the Rodent Spinal Dorsal Horn

Abstract

Background Glutamate receptors of the AMPA type (AMPArs) mediate fast excitatory transmission in the dorsal horn and are thought to underlie perception of both acute and chronic pain. They are tetrameric structures made up from 4 subunits (GluR1-4), and subunit composition determines properties of the receptor. Antigen retrieval with pepsin can be used to reveal the receptors with immunocytochemistry, and in this study we have investigated the subunit composition at synapses within laminae I–III of the dorsal horn. In addition, we have compared staining of AMPArs with that for PSD-95, a major constituent of glutamatergic synapses. We also examined tissue from knock-out mice to confirm the validity of the immunostaining. Results As we have shown previously, virtually all AMPAr-immunoreactive puncta were immunostained for GluR2. In laminae I–II, ~65% were GluR1-positive and ~60% were GluR3-positive, while in lamina III the corresponding values were 34% (GluR1) and 80% (GluR3). Puncta stained with antibody against the C-terminus of GluR4 (which only detects the long form of this subunit) made up 23% of the AMPAr-containing puncta in lamina I, ~8% of those in lamina II and 46% of those in lamina III. Some overlap between GluR1 and GluR3 was seen in each region, but in lamina I GluR1 and GluR4 were present in largely non-overlapping populations. The GluR4 puncta often appeared to outline dendrites of individual neurons in the superficial laminae. Virtually all of the AMPAr-positive puncta were immunostained for PSD-95, and 98% of PSD-95 puncta contained AMPAr-immunoreactivity. Staining for GluR1, GluR2 and GluR3 was absent in sections from mice in which these subunits had been knocked out, while the punctate staining for PSD-95 was absent in mice with a mutation that prevents accumulation of PSD-95 at synapses. Conclusion Our results suggest that virtually all glutamatergic synapses in laminae I–III of adult rat spinal cord contain AMPArs. They show that synapses in laminae I–II contain GluR2 together with GluR1 and/or GluR3, while the long form of GluR4 is restricted to specific neuronal populations, which may include some lamina I projection cells. They also provide further evidence that immunostaining for AMPAr subunits following antigen retrieval is a reliable method for detecting these receptors at glutamatergic synapses.

Pain after Discontinuation of Morphine Treatment Is Associated with Synaptic Increase of GluA4-Containing AMPAR in the Dorsal Horn of the Spinal Cord

“…An association was observed between GluA4 and GluA2 and between GluA4 and GluA3, but we could not detect a GluA4-GluA1 interaction. This is in agreement with previous studies showing that GluA1 and GluA4 are expressed by different neuronal populations in the spinal cord (Polgar et al, 2008b;Todd et al, 2009). Next, we examined the effects of morphine on the association of GluA4 with GluA2 and GluA3.…”

“…Our immunohistochemical analyses show that morphine treatment resulted in an increase of GluA4 expression in laminae III-V. Interestingly, Polgar et al (2008b) reported that GluA4-immunoreactive neurons are GluA1-negative, suggesting that GluA1 and GluA4 are present in different neuronal populations. The observed lack of association between GluA4 and GluA1 revealed by our co-immunoprecipitation analyses is in agreement with their findings.…”

“…In addition, expression and phosphorylation levels of GluA4 are significantly increased in the postsynaptic density at the same time point. Previous studies have suggested that GluA4-GluA2 heteromers are highly abundant in laminae III-V (Polgar et al, 2008b;Todd et al, 2009). In agreement with these studies, our immunohistochemical analyses localized GluA4-containing AMPAR in laminae III-V (Figure 4d).…”

“…AMPAR are tetramers consisting of dimers of four different subunits, GluA1-4, all of which are expressed in the dorsal horn (Polgar et al, 2008b). GluA2-containing AMPAR have low Ca 2 þ permeability, whereas GluA2-lacking AMPAR are readily permeable to Ca 2 þ (Malinow and Malenka, 2002).Trafficking of AMPAR is partially regulated by phosphorylation mechanisms.…”

Neuroplasticity of ascending and descending pathways after somatosensory system injury: reviewing knowledge to identify neuropathic pain therapeutic targets

“…The explained plastic changes in spinal nociceptive neurons are known as 'central sensitization'. 26,27 The aforementioned neurotransmitters interact with N-methyl-D-aspartate receptor (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), metabotropic receptors of glutamate (mGluR), neurokinin-1 receptor (NK1R) and purinergic receptors (P2X) of spinal nociceptive projection neurons [28][29][30][31][32][33][34][35] causing their depolarization and the generation of painful signals to be scattered throughout the nociceptive spinothalamic pathway. In turn, this chemical neurotransmission causes an influx of calcium ions in the spinal nociceptive neurons of second order, 36 a process that activates calcium-dependent intracellular cascades, inducing phosphorylation sensitization 37 and ionic channel and membrane receptor overexpression (Table 3).…”