The distribution of neurons expressing calcium-permeable AMPA receptors in the superficial laminae of the spinal cord dorsal horn

Abstract

The superficial dorsal horn is a major site of termination of nociceptive primary afferents. Fast excitatory synaptic transmission in this region is mediated mainly by release of glutamate onto postsynaptic AMPA and NMDA receptors. NMDA receptors are known to be Ca2+-permeable and to provide synaptically localized Ca2+ signals that mediate short-term and long-term changes in synaptic strength. Less well known is a subpopulation of AMPA receptors that is Ca2+-permeable and has been shown to be synaptically localized on dorsal horn neurons in culture (Gu et al., 1996) and expressed by dorsal horn neurons in situ (Nagy et al., 1994; Engelman et al., 1997). We used kainate-induced cobalt uptake as a functional marker of neurons expressing Ca2+-permeable AMPA receptors and combined this with markers of nociceptive primary afferents in the postnatal rat dorsal horn. We have shown that cobalt-positive neurons are located in lamina I and outer lamina II, a region strongly innervated by nociceptors. These cobalt-positive neurons colocalize with afferents labeled by LD2, and with the most dorsal region of capsaicin-sensitive and IB4- and LA4-positive afferents. In contrast, inner lamina II has a sparser distribution of cobalt-positive neurons. Some lamina I neurons expressing the NK1 receptor, the receptor for substance P, are also cobalt positive. These neurons are likely to be projection neurons in the nociceptive pathway. On the basis of all of these observations, we propose that Ca2+-permeable AMPA receptors are localized to mediate transmission of nociceptive information.

Fig. 1.

Kainate-induced cobalt loading in the dorsal horn: antagonism by CNQX, GYKI 53655, and JsTx. A, B, Kainate-induced cobalt loading in a P9 rat spinal cord slice is shown. Slices were treated with 250 μm kainate (A) or 250 μm kainate plus 50 μm CNQX (B). White lines in A delineate our approximations of dorsal horn laminae. A subpopulation of neurons in the most dorsal laminae, lamina I (LI) and outer lamina II (LII_o), show strong evidence of kainate-induced cobalt loading (black cells), with fewer cobalt-positive cells in inner lamina II (LII_i), and more cobalt-positive cells in the more ventral laminae (e.g., lamina III). Awhite arrowhead points to a cobalt-positive cell in the lateral spinal nucleus. CNQX blocked kainate-induced cobalt loading of cells throughout the spinal cord. C, D, Kainate-induced cobalt loading in a P10 rat spinal cord slice is shown. Slices were treated with 250 μm kainate (C) or 250 μm kainate plus 100 μm GYKI 53655 (D). E, F, Kainate-induced cobalt loading in a P10 rat spinal cord slice is shown. Slices were treated with 250 μm kainate (E) or 250 μm kainate plus 5 μm JsTx (F). A few sparse cells are labeled in the presence of GYKI 53655 (D) or JsTx (F). Scale bars, 40 μm.

Fig. 2.

GluR1 and GluR2 distribution in spinal cord dorsal horn: double labeling with kainate-induced cobalt uptake.A–C, GluR1 staining (red, A) and GluR2 staining (green, B) are shown in the same transverse section from a P10 rat spinal cord. Images of GluR1 and GluR2 staining are superimposed in C. GluR1 is present in many cells throughout the spinal cord and forms a laminar pattern in the superficial dorsal horn, strongest in outer lamina II. GluR2 is also found in lamina II and is strongest slightly ventral to the area of highest GluR1 expression. D–F, Kainate-induced cobalt loading (D) and GluR1 staining (E) were performed sequentially on the same sections using tissue from a P8 rat spinal cord slice (see Materials and Methods). The GluR1 (E) and kainate (D) patterns have been superimposed inF to reveal that the area of high GluR1 expression corresponds to that with kainate-induced cobalt loading.G–I, Kainate-induced cobalt loading (G) and GluR2 staining (H) were performed sequentially using tissue from a P10 rat spinal cord slice (see Materials and Methods). The GluR2 (H) and kainate-induced cobalt (G) patterns have been superimposed inI to reveal that the area of high GluR2 expression corresponds to that of the gap in kainate-induced cobalt loading in the superficial dorsal horn. Scale bars, 40 μm.

Fig. 3.

Capsaicin-induced cobalt entry serves as a marker for nociceptive DRG neurons and their nerve terminals in the dorsal horn. Kainate-induced cobalt-loaded cells are found in the region of capsaicin- responsive nociceptive afferents. A, Capsaicin (10 μm) caused cobalt loading of a subpopulation of small neurons in the DRG (A) of a P6 rat. B, C, Capsaicin-induced cobalt loading also labeled the central processes of DRG neurons (B, C) in slices from a P9 rat. The central terminals of the capsaicin-sensitive primary afferents are seen to innervate laminae I and II of the spinal cord. The boxed area in B is magnified inC, where presumptive afferent axons and terminals are seen in a punctate pattern. D, E, Kainate- and capsaicin-induced cobalt loading in the same P6 slice. Kainate-induced cobalt-positive neurons (black spots in the area of the superficial dorsal horn) are present in the region of capsaicin-sensitive nociceptive fibers (fine black speckling) (D). In another section from the same spinal cord, the kainate-induced cobalt loading was blocked with 50 μm CNQX, leaving the capsaicin-induced staining pattern more apparent (E). Scale bars: A, B, D, E, 40 μm; C, 10 μm.

Fig. 4.

The primary afferent marker, IB4, overlaps with GluR1- and GluR2-positive areas in the superficial dorsal horn.A, B, Staining with the lectin IB4 (A) and antibodies to GluR1 (B) are shown from the same transverse section from fixed P10 rat spinal cord. The area of IB4-positive terminals has been outlined in white dashes to compare the distribution of IB4-positive terminals with that of GluR1. Note that IB4 spans the outer and inner regions of lamina II in this preparation. GluR1 staining is seen in the outermost region of IB4 staining.C, D, Staining with the lectin IB4 (C) and antibodies to GluR2 (D) are shown in this transverse section from fixed P10 rat spinal cord. The area of IB4-positive terminals has been outlined in white dashes to compare the distribution of IB4-positive terminals with that of GluR2. GluR2 staining is seen in the innermost region of IB4 staining. Scale bars, 40 μm.

Fig. 5.

IB4-positive terminals and kainate-induced cobalt loading overlap in the outermost region of IB4 staining. A, B, Kainate-induced cobalt loading is seen in this section from a P12 rat spinal cord slice (A). IB4 labeling of the same section is shown in B. A box is placed around a 120 × 100 μm area at the most lateral edge of IB4 staining that was used for the analysis shown in C.C, Plots of IB4 and cobalt intensity over the 120 μm from the lateral edge of IB4 staining for the section displayed inA and B. Intensity values are averaged pixel values over 100 μm at each distance from the lateral border of IB4 staining. Values have been normalized (see Materials and Methods).D, Plots of IB4 and cobalt intensity over the 120 μm from the lateral edge of IB4 staining for average values of 10 sections from multiple slices of the same preparation as A andB (data not shown). Error bars represent the SEM for normalized cobalt and IB4 staining over this region. In bothC and D, it can be seen that the peak of the kainate-induced cobalt signal in superficial dorsal horn falls in the outermost region of IB4 staining. Scale bars, 40 μm.

Fig. 6.

Lactoseries carbohydrate antigens LD2 and LA4 mark subsets of small-diameter DRG neurons and their terminals in the dorsal horn. Kainate-induced cobalt-positive neurons coincide with the area of LD2-positive afferents (presumptive LII_o), but are sparser in the ventral area of LA4-positive afferents.A, The monoclonal antibody LD2 stains a subpopulation of small-diameter DRG neurons. A section from a P13 rat DRG is shown.B, LD2-positive primary afferents project primarily to LII_o. This section is from a P11 slice that underwent kainate-induced cobalt loading. The border of LA4 staining is predicted using an alternate section from the same slice stained for LA4 (shown in E) and is marked off with white dashes. Comparison of the staining for LD2 versus LA4 reveals that LD2 distribution is restricted to the most dorsal region of LA4 staining. C, The pattern of kainate-induced cobalt loading is shown for the same section as in B. The predicted border for the LA4 stain is outlined with black dashes. The area labeled by LD2 directly overlaps with that of cobalt-positive dorsal horn cells in what we presume to be LII_o (compare with B). D, The monoclonal antibody LA4 also stains a subpopulation of small-diameter DRG neurons in P13 rat DRG. E, LA4-positive afferents project throughout LII in this alternate section from the same P11 slice as in B and C (note the wider band of staining for LA4 as compared with LD2 in B).Dashed lines mark the border of the LA4 staining here and in F. F, The pattern of kainate-induced cobalt loading is shown for the same section as in E. The inner LA4-positive region of the dorsal horn from another section of the same spinal cord has fewer cobalt-positive dorsal horn neurons than the outermost region. Scale bars, 40 μm.

Fig. 7.

Some NK1-positive neurons also exhibit kainate-induced cobalt loading. A, B, Kainate-induced cobalt loading is shown in this P8 slice (A). Thearrowhead marks a lamina I neuron that projects in the transverse plane and is double-labeled for the NK1 receptor (B). C, D, Kainate-induced cobalt loading is shown in a neuron of the lateral spinal nucleus (arrowhead), which is seen below the border of the dorsal horn (C). This neuron is also NK1 positive (D). Scale bars, 20 μm.