Compartment-specific regulation of plasma membrane calcium ATPase type 2 in the chick auditory brainstem

Abstract

Calcium signaling plays a role in synaptic regulation of dendritic structure, usually on the time scale of hours or days. Here we use immunocytochemistry to examine changes in expression of plasma membrane calcium ATPase type 2 (PMCA2), a high-affinity calcium efflux protein, in the chick nucleus laminaris (NL) following manipulations of synaptic inputs. Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Deprivation of the contralateral projection from NM to NL leads to rapid retraction of ventral, but not the dorsal, dendrites of NL neurons. Immunocytochemistry revealed symmetric distribution of PMCA2 in two neuropil regions of normally innervated NL. Electron microscopy confirmed that PMCA2 localizes in both NM terminals and NL dendrites. As early as 30 minutes after transection of the contralateral projection from NM to NL or unilateral cochlea removal, significant decreases in PMCA2 immunoreactivity were seen in the deprived neuropil of NL compared with the other neuropil that continued to receive normal input. The rapid decrease correlated with reductions in the immunoreactivity for microtubule-associated protein 2, which affects cytoskeleton stabilization. These results suggest that PMCA2 is regulated independently in ventral and dorsal NL dendrites and/or their inputs from NM in a way that is correlated with presynaptic activity. This provides a potential mechanism by which deprivation can change calcium transport that, in turn, may be important for rapid, compartment-specific dendritic remodeling.

Figure 1

Western blot assay of anti-PMCA2 in tissues from chicks, wild type (WT) mice, and dfw^2J mice. Molecular weight standards (left) were used to determine relative sizes of labeled protein. The lanes 1 and 2 were loaded with approximate 5 μg of total protein. Chicken reactivity is nearly identical to that in the wild type mouse with respect to the molecular weights at ~ 130 kDa. The dfw^2J lane was loaded with 12.5 μg of total protein and showed no evidence of PMCA2 protein of the correct molecular weight.

Figure 2

PMCA2 immunoreactivity in the NL and NM under control condition. A: A low-magnification photomicrograph showing the overall pattern of PMCA2 immunoreactivity in NM and NL. B–C: High-magnification photomicrographs showing the perisomatic staining of PMCA2 in the caudal NL (B) and NM (C). Sections were double-labeled with PMCA2 (green) and a nuclear marker DAPI (blue). Red and white arrows indicate unstained and stained glial cells, respectively. The inset in B was taken from the ventral glial layer of NL. D–F: Double labeling of PMCA2 (green) and a somatodendritic marker MAP2 (magenta) in NL. Merged image in F. G–I: Double labeling of PMCA2 (green) and DAPI (blue) in NL. Merged image in I. White dashed lines in H-I indicate the borders between NL neuropil region and the adjacent glial layers. Scale bar = 200 μm in A; 20 μm in B and C; 10 μm in the inset in B; 50 μm in I (applies to D–I).

Figure 3

Low- (A–B) and high-magnification (C–H) photomicrographs of sections double-labeled with PMCA2 (green) and a presynaptic maker, SNAP-25 (magenta) in NL (A, and F–H) and NM (B–E). E and H are merged images of NM neuron soma and NL neuopil region, respectively. White arrows and arrowheads indicate the subcellular components with double labeling or with single labeling for PMCA2, respectively. Scale bar = 50 μm in B (applies to AB); 5 μm in E (applies to C–E); 7.5 μm in H (applies to F–H).

Figure 4

Transmission electron micrograph of PMCA2 immunoreactivity in NL neuropil region. Gold particles are distributed in a membrane-associated manner in both dendritic (d) and axonal (a) structures. Arrows and arrowheads point out the gold particles in a dendritic and an axonal structure, respectively. Scale bar = 1 μm.

Figure 5

Differential innervation of NL dendrites and the consequences of the surgeries. A and B: Schematic drawings of the chick brainstem in the coronal plane illustrate the affected dendritic fields following XDCT transection (A) and unilateral cochlea removal (B). Red color indicates the surgical site for each manipulation and deafferented axons and dendrites influenced by each manipulation. C: A Low-magnification photomicrograph illustrating a complete XDCT transection (black arrow). Note the significant difference in PMCA2 staining intensity between the dorsal and ventral neuropil regions of NL, bilaterally, 3 hours after the transection. Image photomontages were performed in C using Adobe Photoshop (Adobe Systems Inc., Mountain View, CA). Abbreviations: NA, nucleus angularis. Scale bar = 500 μm in C.

Figure 6

PMCA2 immunoreactivity in NL following XDCT transection. A was taken from an un-operated case and B–F from the animals that survived 0, 0.5, 3, 6, and 14h following the surgery, respectively. Note that staining intensities between the dorsal and ventral neuropil domains appear similar in A–B and different in C–F. The ventral neuropil exhibited a lower density of staining compared to the dorsal neuropil 0.5 to 14h following the surgery. Scale bar = 50 μm in F (applies to A–F).

Figure 7

Time course of the average density of PMCA2 immunoreactivity in the deprived NL neuropil following manipulations. A: The average percent difference (PD) across the whole NL following XDCT transection or unilateral cochlea removal. PDs were averaged from two sides of the brain following XDCT transection. B: PDs were measured from the caudal, intermediate, and rostral portions of the nucleus following XDCT transection. All error bars indicate standard deviation. The asterisk (*) and n.s. below individual bars indicate that the PD of a specific survival group or position is significantly smaller (p < 0.05) than or non-significant (p > 0.05) from the corresponding un-operated control, respectively.

Figure 8

Four individual examples demonstrating changes in PMCA2 immunoreactivity as a function of the distance from the NL cell bodies. Animals survived 6 hours following XDCT transection. PD values were calculated from a single section selected from the intermediate portion of NL where the dendrites extend about 35 μm dorsally and ventrally to the neuronal cell bodies. Decreases in PMCA2 immunoreactivity were detected in both the proximal and distal portions of NL neuropil.

Figure 9

PMCA2 immunoreactivity in NL at 3h (A–D) and 14h (E–F) following unilateral cochlea removal. A, C, and E were taken from the side contralateral to the operated ear, while B, D, and F from the side ipsilateral to the operated ear. Staining intensities between the dorsal and ventral neuropil domains are clearly different on the contralateral side and appear comparable on the ipsilateral side. Scale bar = 200 μm in A (applies to A–B); 50 μm in F (applies to C–F).

Figure 10

PMCA2 immunoreactivity in NL at 3h following bilateral cochlea removal. Staining intensity in the ventral neuropil (V) is notably lower than in the dorsal neuropil (D). Scale bar = 20 μm.

Figure 11

Correlation of changes in PMCA2 and MAP2 following unilateral cochlea removal. The average percent difference (PD) was calculated as the change in the density of the immunoreactivity in deprived neuropil domain relative to the intact domain. CR 3h, CR 6h, and CR 14h represent 3h, 6h, or 14h following unilateral cochlea removal. Note changes in PMCA2 immunoreactivity in deprived ventral domain (A) are larger than those in deprived dorsal domain (B). Each data point represents an individual case. The animal with a larger change in PMCA2 immunoreactivity tended to have a larger change in MAP2 immunoreactivity.

Figure 12

PMCA2 immunoreactivity in NM at 3h (A and B) and 14h (C and D) following unilateral cochlea removal. A and C were taken from the side contralateral to the operated ear, while B and D from the side ipsilateral to the operated ear. Pattern and intensity of the staining in NM are similar between two sides at both survival times. Scale bar = 50 μm in D (applies to AD).