Lamina-specific synaptic activation causes domain-specific alterations in dendritic immunostaining for MAP2 and CAM kinase II - PubMed (original) (raw)

Lamina-specific synaptic activation causes domain-specific alterations in dendritic immunostaining for MAP2 and CAM kinase II

O Steward et al. J Neurosci. 1999.

Abstract

Polyribosomal complexes are selectively localized beneath postsynaptic sites on neuronal dendrites; this localization suggests that the translation of the mRNAs that are present in dendrites may be regulated by synaptic activity. The present study tests this hypothesis by evaluating whether synaptic activation alters the immunostaining pattern for two proteins whose mRNAs are present in dendrites: the dendrite-specific cytoskeletal protein MAP2 and the alpha-subunit of CAMKII. High-frequency stimulation of the perforant path projections to the dentate gyrus, which terminate in a discrete band on the dendrites of dentate granule cells, produced a two-stage alteration in immunostaining for MAP2 in the dendritic laminae. Five minutes of stimulation (30 trains) caused a decrease in MAP2 immunostaining in the lamina in which the activated synapses terminate. After more prolonged periods of stimulation (1-2 hr), there was an increase in immunostaining in the sideband laminae just proximal and distal to the activated band of synapses. The same stimulation paradigm produced a modest increase in immunostaining for alpha-CAMKII in the activated laminae, with no detectable changes in the sideband laminae. The alterations in immunostaining for MAP2 were diminished, but not eliminated, by inhibiting protein synthesis; the increases in CAMKII were not. These findings reveal that patterned synaptic activity can produce domain-specific alterations in the molecular composition of dendrites; these alterations may be caused in part by local protein synthesis and in part by other mechanisms.

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Figures

Fig. 1.

Immunostaining pattern for MAP2 in the dentate gyrus after 5 min of high-frequency stimulation of the medial perforant path. A, Control immunostaining pattern contralateral to the stimulation (MAP2 Con); B, immunostaining pattern on the side in which the perforant path had received 5 min of high-frequency stimulation (MAP2 Stim); C, D, higher magnification views. Note the discrete band of decreased immunostaining in the middle molecular layer (arrows) corresponding exactly to the band of synapses that would have been activated. CA1, CA1 region of the hippocampus; DG, dentate gyrus.

Fig. 2.

Immunostaining pattern for MAP2 in the dentate gyrus after 2 hr of high-frequency stimulation of the medial perforant path. A, Control immunostaining pattern contralateral to the stimulation (MAP2 Con); B, immunostaining pattern on the side in which the perforant path had received 2 hr of high-frequency stimulation (MAP2 Stim);C, D, higher magnification views. Note the trilaminar staining pattern in the molecular layer in which the central (activated) band was bounded by two thin bands of increased immunostaining (arrows). CA1, CA1 region of the hippocampus; DG, dentate gyrus.

Fig. 3.

Immunostaining pattern for CAMKII in the dentate gyrus after 2 hr of high-frequency stimulation of the medial perforant path. A, Control immunostaining pattern contralateral to the stimulation (CAMKII Control); B, immunostaining pattern on the side in which the perforant path had received 2 hr of high-frequency stimulation (CAMKII Stim);C, D, higher magnification views. Note the discrete band of increased immunostaining in the middle molecular layer (arrows) corresponding exactly to the band of synapses that would have been activated. CA1, CA1 region of the hippocampus; DG, dentate gyrus. _E_and F compare immunostaining patterns for pan-α CAMKII (monoclonal antibody 6G9) and phosphoepitope-specific CAMKII (monoclonal antibody 22B1). These sections are from a different case than the one illustrated in A–D.

Fig. 4.

Time course of alterations in immunostaining for MAP2 and CAMKII after high-frequency stimulation of the medial perforant path. A and B illustrate the control pattern of immunostaining for MAP2 (A;MAP2 Con) and CAMKII (B; CAMKII Con) in the dorsal blade of the dentate gyrus;C, E, and G illustrate the pattern of immunostaining for MAP2 after 30 min, l hr, and 2 hr of high-frequency stimulation. D, F, and_H_ illustrate the pattern of immunostaining for CAMKII after 30 min, l hr, and 2 hr of stimulation. _Arrows_indicate bands of increased immunostaining.

Fig. 5.

Quantitative assessment of immunostaining for MAP2 and CAMKII after 2 hr of high-frequency stimulation. The_graphs_ illustrate the average optical density (OD) of labeling across the molecular layer on the control side contralateral to the stimulation (A,MAP2, Control; D, CAMKII,Control) and on the side of the stimulation (B, MAP2, EC Stim;E, CAMKII, EC Stim). Bars indicate plus or minus 1 SD of the five measurements at each level of the molecular layer. C and F are expanded versions of the graphs in B and_D_, which illustrate the method of quantification for MAP2 and CAMKII, respectively. For further details, see Results. HF, Hippocampal fissure.

Fig. 6.

Local blockade of NMDA receptors blocks the induction of LTP after high-frequency stimulation of the perforant path. The graph plots the slope of population EPSPs recorded via an MK801-filled electrode and a distant saline-filled control electrode. Single test pulses were delivered at a rate of one every 10 sec to determine baseline response amplitude, then a series of three series of trains of 400 Hz stimuli (10 pulses per train, 10 trains) were delivered, collecting 10 test responses between each train. Note the increase in response amplitude at the control site and the absence of any change in response amplitude at the MK801 site. After this testing paradigm was completed, trains of stimuli were delivered for an additional 2 hr, after which the rat was perfused for immunocytochemistry. The pattern of immunostaining in this case is illustrated in Figure 7.

Fig. 7.

Stimulation-induced alterations in immunostaining for MAP2 and CAMKII are blocked by NMDA receptor antagonists.A, Immunostaining for c-fos protein after 2 hr of high-frequency stimulation of the perforant path. A micropipette filled with MK801 was positioned in the dorsal blade of the dentate gyrus near the level of this section. Note the virtually complete blockade of c-fos induction in part of the dorsal blade between the_arrows_. B illustrates the pattern of immunostaining for c-fos on the control side contralateral to the stimulation. C and D illustrate nearby sections stained for MAP2; E and F_illustrate sections stained for CAMKII. Note the absence of alterations of immunostaining in the dorsal blade in approximately the same area in which c-fos induction is blocked. Abbreviations are as for Figure 1. The lower case letters (a–i) indicate the areas in which OD measurements were taken for the graphs of Figure8_A–I, respectively.

Fig. 8.

Quantitative analysis of stimulation-induced alterations in immunostaining for c-fos, MAP2, and CAMKII in the presence of NMDA receptor antagonists. The _graphs_illustrate the average OD of labeling across the molecular layer in the areas indicated by lower case letters a–i in Figure 7. Error bars indicate ±1 SD of the five measurements at each level of the molecular layer. Note that at the MK801 site, there is complete blockade of c-fos induction (B), an elimination of the trilaminar staining pattern for MAP2 (E), and an elimination of the discrete band of increased immunostaining for CAMKII.

Fig. 9.

Systemic injections of cycloheximide block the alterations in immunostaining for MAP2 but not the increases in immunostaining for CAMKII. A, Induction of expression of c-fos protein after 2 hr of high-frequency stimulation of the perforant path as revealed by immunocytochemistry (C-fos Stim).B, Pattern of immunostaining for c-fos on the control side contralateral to the stimulation. C and_D_ illustrate sections from an animal that received cycloheximide (20 mg/kg, i.p.) just before the initiation of the stimulation (C-fos Con). C, Immunostaining pattern for c-fos on the side of the stimulation (C-fos Stim + CHX); D, contralateral side (C-fos Con + CHX). Note that the lack of induction of c-fos protein on the stimulated side as a consequence of inhibiting protein synthesis during the stimulation period. E and F illustrate the immunostaining patterns for MAP2 on the stimulated and control sides of the same animal illustrated in C and D. There is a hint of the trilaminar staining pattern in one location, but throughout most of the dentate gyrus, the pattern of MAP2 immunostaining resembles that on the nonstimulated side.G and H illustrate the immunostaining patterns for CAMKII on the stimulated and control sides of the same animal illustrated in C and D. Note that the discrete band of increased staining appears comparable to what is seen in animals that did not receive protein synthesis inhibitors.

Fig. 10.

Local inhibition of protein synthesis blocks the alterations in immunostaining for MAP2 but not the increases in immunostaining for CAMKII. In this experiment, a recording micropipette filled with cycloheximide (CHX) (20 mg/ml in saline) was present on the stimulated side. _A_illustrates the immunostaining pattern for c-fos on the stimulated side (c-fos Stim+CHX). The diffusion of cycloheximide from the pipette produced an area several hundred micrometers in diameter in which protein synthesis was inhibited, as documented by the absence of induced expression (arrows). In areas distant from the micropipette, there was still strong induction of c-fos protein expression. B illustrates the immunostaining pattern for c-fos on the control side. _C_illustrates the immunostaining pattern for MAP2 in the area in which protein synthesis had been inhibited (MAP2 Stim+CHX). The trilaminar staining pattern is much less evident than in areas distant from the site of protein synthesis inhibition. D illustrates a region distant from the CHX-containing micropipette. E and _F_illustrate the immunostaining pattern for CAMKII in the area in which protein synthesis had been inhibited. Note that the discrete band of increased immunostaining appears comparable to what is seen in animals that did not receive protein synthesis inhibitors.

Fig. 11.

Quantitative assessment of the effects of local blockade of protein synthesis. To document the effects of local blockade of protein synthesis, OD measurements were taken across the molecular layer in three representative cases in which CHX was present in the recording micropipette. Measurements were taken at the CHX site and in a distant location. A single quantitative measure of the alteration in immunostaining was then determined as in Figure5_C_,F, enabling us to calculate the average change in immunostaining at the site of CHX application in comparison to distant sites. A, The strong induction of c-fos that normally occurs as a consequence of the stimulation is completely blocked in the area near the CHX-filled micropipette.B, The alteration in immunostaining for MAP2 is diminished, but not eliminated, in the area near the CHX-filled micropipette. C, The increase in immunostaining for CAMKII is only slightly diminished in the area near the CHX-filled micropipette.

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Lamina-specific synaptic activation causes domain-specific alterations in dendritic immunostaining for MAP2 and CAM kinase II - PubMed (original) (raw)