Heterogeneity of Ca2+-permeable AMPA/kainate channel expression in hippocampal pyramidal neurons: fluorescence imaging and immunocytochemical assessment - PubMed (original) (raw)
Heterogeneity of Ca2+-permeable AMPA/kainate channel expression in hippocampal pyramidal neurons: fluorescence imaging and immunocytochemical assessment
Fumio Ogoshi et al. J Neurosci. 2003.
Abstract
The presence of Ca2+-permeable AMPA/kainate (Ca-A/K) channels on hippocampal pyramidal neurons (HPNs) has been controversial, although they are present on many forebrain GABAergic neurons. We combined high-resolution fluorescence Ca2+ imaging with surface AMPA receptor (AMPAR) subunit immunocytochemistry to examine the expression of functional Ca-A/K channels in dissociated hippocampal neurons at the subcellular level. In GABAergic neurons [identified by glutamate decarboxylase (GAD) immunocytochemistry], focal application of AMPA induced large dendrosomatic intracellular [Ca2+] ([Ca2+]i) rises, consistent with their known strong Ca-A/K channel expression. Surface immunostaining for the AMPAR subunits GluR1 and GluR2 revealed abundant dendritic GluR1 puncta containing little or no GluR2, which, when present, was limited to diffuse staining in the soma and proximal dendrites. In contrast, the majority of HPNs (putatively identified by morphological criteria and lack of GAD labeling) showed little or no AMPA-induced [Ca2+]i rise. Correspondingly, most HPNs showed strong dendritic labeling for both GluR1 and GluR2 that colocalized extensively. A subpopulation of HPNs, however, displayed noticeable [Ca2+]i rises that began and often reached their highest levels in discrete dendritic regions. In these HPNs, levels of GluR1 relative to GluR2 were higher, and GluR1 was often present without overlying GluR2. The present studies, which are the first to directly examine the relationship between the local complement of cell surface AMPAR and the presence of dendritic Ca-A/K channels, clearly indicate that considerable cell surface GluR2 does not preclude the presence of Ca-A/K channels and further show that HPNs display considerable heterogeneity in terms of apparent Ca-A/K channel expression.
Figures
Figure 1.
Kainate-stimulated Co2+ uptake labeling and immunohistochemical characterization of cultured hippocampal neurons. A, B, Co2+ labeling of GABAergic neurons and putative HPNs. Hippocampal cultures were immunolabeled for GAD and subjected to kainate-stimulated Co2+ labeling. After photographing the GAD stain, the Co2+ stain was developed, and the cells were rephotographed. A representative GAD(+) neuron (A, top) with characteristic large soma and smooth, sparsely branching dendrites, is strongly and uniformly Co2+(+) (bottom). Although most GAD(-) neurons were also Co2+(-), a subset did show Co2+ labeling (B), which when present was often most evident in the distal dendrites and often extended into apparent dendritic spines (inset, arrows). C. CaMKIIα labeling of GABAergic neurons and putative HPNs. A culture was double-immunolabeled for GAD (red) and CaMKIIα (green). Note that CaMKIIα is strongly present in GAD(-) putative HPNs but is mostly absent from the GAD(+) neuron (see overlay, right). Scale bars, 10 μm.
Figure 2.
Detection of Ca-A/K channels using fluorescence Ca2+ imaging. A, Pseudocolor images. Neurons were loaded with the low-affinity fluorescent Ca2+ probe fluo-4ff and exposed to a brief pulse (1 sec) of AMPA (25 μ
m
) in the presence of MK-801 and Gd3+ (both at 10 μ
m
). Images represent Δ_F_ in a responding neuron at the times indicated after onset of the exposure (i-iii) and bright-field appearance of the neuron after Co2+ labeling (iv). Most neurons (82%; n = 33) that (like this one) displayed substantial AMPA-induced somatic [Ca2+]i rises (Δ_F_, >2.0) were Co2+(+). The color calibration bar shows pseudocolor mapping of Δ_F_ (applies to all pseudocolor Ca2+ images). Scale bars, 10 μm. B, Reversible block of [Ca2+]i rises by Ca-A/K channel and AMPAR antagonists: traces of representative single-cell responses. After an initial AMPA pulse, selected responding neurons were exposed to a second AMPA pulse in the presence of the Ca-A/K channel blocker NAS (300 μ
m
; top), or the selective AMPAR antagonist GYKI 52466 (100 μ
m
; bottom), followed by a third pulse of AMPA alone.
Figure 3.
Colocalization of Ca2+ hot spots with synaptophysin and GluR1 puncta. A, Colocalization with synaptophysin. After AMPA exposure and generation of pseudocolor images of Δ_F_ (left), neurons were stained for the synaptic marker synaptophysin (right); bottom panels show blowups of areas in rectangles. Note the strong overlap between Ca2+ hot spots and synaptophysin puncta (arrows). B, Colocalization with GluR1. Similarly, after imaging (pseudocolor image on left), this culture was stained for cell surface GluR1; right panels show blowups of the area indicated, showing a Δ_F_ pseudocolor image (top) and GluR1 labeling (bottom). Note the overlap between Ca2+ hot spots and GluR1 puncta (arrows). Scale bars, 10 μm.
Figure 4.
Distinct pattern of AMPA-induced [Ca2+]i rises in GABAergic neurons and putative HPNs. Cells were puffed with AMPA for 1 sec (i, ii, pseudocolor images of Δ_F_ at the times indicated after onset of the exposure) followed by Co2+ loading, GAD immunocytochemistry (iii, inset), and development of the Co2+ stain (iii) as described. Note that the GAD(+) neuron (A) showed strong dendrosomatic [Ca2+]i rises and correspondingly strong Co2+ labeling, whereas the GAD(-) putative HPN (B) displayed prominent localized dendritic responses that corresponded to areas of dendritic Co2+ labeling (B, ii, iii, arrows), before onset of substantial somatic [Ca2+]i rises.
Figure 5.
Surface expression patterns of AMPAR subunits in hippocampal neurons. Neurons were triple-labeled for surface GluR1 (green), surface GluR2 (red), and GAD (blue) and examined for differences in the pattern of GluR staining. Right panels show blowup of areas in rectangles, displaying GluR1 (top), GluR2 (middle), and overlap (bottom). A, GAD(+) neurons typically displayed strong and abundant dendritic GluR1 puncta along the dendritic shaft, whereas GluR2 labeling was generally limited to the soma and proximal dendritic shafts. B, Most putative HPNs possessed abundant dendritic GluR1 and GluR2 puncta that colocalized extensively and was most evident at a distance from the shaft on apparent dendritic spines. C, A subset of putative HPNs, however, displayed large numbers of puncta in which GluR1 labeling was predominant. Scale bar, 10 μm.
Figure 6.
Correlation between AMPA-induced [Ca2+]i rises and AMPAR subunit surface expression in a GABAergic neuron. Top, The pseudocolor image displays peak [Ca2+]i rises after a 1 sec AMPA application. Bottom, After imaging, the cell was triple-labeled for surface GluR1 (green), surface GluR2 (red), and GAD (blue). Note the strong [Ca2+]i rise throughout the cell corresponding to strong punctate dendritic GluR1 labeling, with little GluR2. Also note that the atypical GAD(-) putative HPN in the top left corner, which displays prominent dendritic GluR2 with little GluR1, did not display any detectable [Ca2+]i rise on AMPA application, presumably because of an absence of Ca-A/K channels. Scale bar, 10 μm.
Figure 7.
Correlation between AMPA-induced [Ca2+]i rises and AMPAR subunit surface expression in putative HPNs. Hippocampal neurons were imaged after a 1 sec AMPA exposure (top panels), followed by triple immunofluorescence labeling for GluR1, GluR2, and GAD (bottom panels). In a Ca-A/K(-) HPN (A), GluR1 (green) and GluR2 (red) puncta colocalize extensively. In contrast, in a Ca-A/K(+) HPN (B), GluR1 is strongly present in many puncta along with little or no GluR2. Scale bars, 10 μm.
Figure 8.
Ca-A/K(+) HPNs have greater dendritic GluR1 relative to GluR2 than Ca-A/K(-) HPNs: quantitative assessments. A, Averaged dendritic GluR1 and GluR2 fluorescence. Neurons were imaged followed by triple immunolabeling, and average raw fluorescence intensity of GluR1 and GluR2 was measured in the dendrites of each neuron. Bars show mean GluR1/GluR2 fluorescence ratios in Ca-A/K(+) and Ca-A/K(-) HPNs. *Fluorescence ratio greater than that in Ca-A/K(-) neurons; p < 0.05 (_n_ = 11 Ca-A/K(-), 7 Ca-A/K(+) HPNs). _B_. Counting and characterization of puncta. After imaging, GluR1 and GluR2 labeling patterns in the same set of neurons were also assessed by counting all evident discrete puncta in the dendrite of each neuron and categorizing each one as GluR1-predominant (GluR1 > GluR2) or as showing labeling for GluR2 at least as strong as for GluR1 (GluR2 ≥ GluR1). *Percent GluR1 > GluR2” puncta significantly greater than in Ca-A/K(-) HPNs; p < 0.0001.
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