Subcellular localization of CO-stained mitochondria in the barrel cortex of WT mice. (A) Low-magnification electron micrograph showing the frequent localization of CO-stained mitochondria in neuronal cell bodies (colored in light yellow), as well as their relative distribution in the dendritic (light blue), axonal (light green), and glial (light pink) elements of the neuropil at P10. Scale bar = 1 μm. (B) A higher magnification of the field framed in A shows a dendritic branch that contains 5 CO-positive mitochondria (d+) and a nearby dendrite with a CO-negative mitochondrion (white asterisk; d- and a-, dendritic and axonal elements without CO-positive mitochondria). Scale bar = 0.5 μm. (C) CO-stained mitochondria are located in 2 dendritic branches (d+), one of which is making a synaptic contact with an axon terminal, also at P10. Scale bar = 0.5 μm. (D) Number of CO-stained mitochondria in dendritic, axonal, and glial elements per 10³ μm² of neuropil surface. (E) Dendritic, axonal, and glial labeling indices displayed as percentages of CO-positive mitochondria over total numbers of mitochondria in each type of structure.

Golgi analysis of WT and HZ barrel neurons at P5 and P7. Golgi impregnation was performed in WT and HZ cortices followed by cresyl violet counterstaining (A, black lines denote location of cortical barrels). Both WT and HZ barrel neurons displayed similar qualitative features to those previously described (Pasternak and Woolsey 1975). Directionally oriented cells were found in both genotypes (white asterisks, boxed images in B–E magnified in F-1). Barrel boundaries were estimated from cresyl violet counterstaining (gray ellipses denote approximate barrel boundaries in B–E to aid in visualization). Magnified images show retained septal-oriented dendrites in GAP-43 HZ cells (compare black arrowhead in F (WT) to G (HZ) for P5 and black arrow in H (WT) to I (HZ) for P7). Quantitative comparisons of Golgi-impregnated dendrites at P5 and P7 in WT and HZ mice (J–O). Several dendritic parameters were analyzed. Randomly selected layer IV stellate barrel cortex cells were measured for longest versus shortest dendrites (J, K), variations in branch bifurcations (L, M), and percent of septal-oriented dendrites (N, O). At P5, longest and shortest primary branches were significantly longer in WT (white bars, P < 0.01 and P < 0.05, Student’s t-test) than in HZ neurons (black bars; J). While primary branches were longer overall in P5 WT mice, the number of primary branches was not significantly different. However, HZ mice showed significantly more secondary branches at P5 (K, P < 0.05, Student’s t-test). By P7, longest and shortest branch lengths do not significantly differ (P = 0.06, Student’s t-test; K). There were also no significant differences in primary or secondary bifurcation counts at P7 (M). WT and HZ mice show oriented dendrites at P5 and P7. However, WT cells displayed fewer septal-oriented dendrites at P5—23% of WT cells compared with 65% in HZ (N, P < 0.001, Student’s t-test). By P7, 35% of WT and 43% of HZ cells display septal-oriented dendrites; these results do not differ significantly (O, P = 0.322, Student’s t-test). These findings suggest a partial restoration of HZ dendritic arbors to WT phenotype between P5 and P7. Scale bar = 50 μm. a = axon.

NMDA and AMPA expression delineates barrels in somatosensory cortex at early ages. Ontogeny of GluR1 and NR1 pattern formation begins early during critical period development. Cortices flattened tangential to the plane of S1 showed a negative barrel pattern (labeling primarily barrel septi) following GluR1 immunohistochemistry at P4 (A), P5 (B), and P7 (C). In contrast, NR1 immunohistochemistry demonstrated a positive barrel pattern (labeling primarily in barrel hollows) at P4 (D), P5 (E), and P7 (F). WT cortex showed a tightly clustered barrel pattern when immunostained for NR1, silver stained, and viewed under dark-field microscopy (G). GAP-43 HZ cortex displayed barrels that are less compact with poorly defined barrel/septal boundaries (H). GAP-43 KO cortex failed to show an NR1-mediated barrel pattern (I). PMBSF, posterior medial barrel subfield; SW, small whiskers. Scale bar = 100 μm.

Schematic representation of GAP-43 effects on postsynaptic differentiation. (A) At P5 WT cortical neurons display dendrites oriented toward the barrel hollow and a large number of immature synapses that are functionally silent due to the presence of NMDA but not AMPA receptors. In contrast, GAP-43 HZ mice display cortical deficits in the development of dendritic and synaptic architecture at these ages. HZ cortical neurons display shorter average dendrites, increased secondary branches, and increased retention of septal-oriented dendrites possibly due to the sparser, less confined TCAs that are present at these ages (McIlvain et al. 2003). HZ cortical dendrites have fewer NR1 and GluR1 puncta and show higher colocalized NR1/GluR1 sites indicative of functional synapses. (B) Between P5 and P7, WT neurons undergo a switch in synaptic glutamate receptor composition in an activity-dependent manner, whereby silent synapses are converted into functional synapses through the insertion of AMPA receptors (Petralia et al. 1999). Interestingly, this does not occur in GAP-43 HZ animals. In fact, there is a rapid addition of synaptic sites such that synaptic density increases as compared with WT, and AMPA/NMDA colocalization is reduced as compared with HZ P5 animals but is comparable to WT P7 animals. This suggests that homeostatic mechanisms may be employed to compensate for the deficits in early development in HZ cortex. These mechanisms increase synaptic number and restore the AMPA/NMDA balance that is crucial for developmental plasticity. Dendritic parameters also normalize at P7 in HZ cortex with reestablishment of dendritic length and arborization and pruning of septal-oriented dendritic branches.

CO-reactivity differs in WT and HZ barrel cortex. Digital photomicrographs of CO-reacted barrel cortex were analyzed using National Institutes of Health image for average density measurements within the core barrel field (B1-3, C1-3, D1-3; outlined in A). Representative examples of CO-reacted barrel cortex at P5 and P7 in GAP-43 HZ and WT mice showing visible qualitative differences in CO-reactive intensity between HZ and WT cortices (A–D). Nissl-stained sections at P7 show no visible changes in barrel field cytoarchitecture in WT (E) compared with GAP-43 HZ mice (F). Quantitative analysis of mean core barrel field density among P4–P5, P6–P14, and P21 demonstrate that HZ core barrels become significantly more reactive between P6 and P14, returning to WT levels at P21 (G) (P < 0.05, Student’s t-test). Scale bar = 500 μm. Density scale bar = white (least reactive), black (most reactive). Data reported as mean ± standard error of the mean. *P < 0.05.

Indistinct and variable HZ barrel patterning of MAP-2 immunostaining at P5. WT cortex, immunostained with anti MAP-2 antibodies, shows clear barrel boundaries when sectioned coronally (A–C) or tangentially (G). By contrast, a variety of pattern phenotypes were found in identically processed GAP-43 HZ (D–F) cortices. In HZ coronal sections, MAP-2 immunostaining varied from no patterning (D), to slight patterning (E), to near normal patterning (F). In HZ tangential sections, MAP-2 immunostained barrel patterns were not clear (H). These results suggest incomplete barrel segregation and a failure of appropriate dendritic development. Scale bar = 100 μm (A–F), 50 μm (G–H). Arrowheads in (A–F) indicate barrels. In G and H, asterisks mark visible and presumptive barrels, while arrows point to presumptive barrel septa.

Dendritic protrusion density is increased on layer 4 neurons in GAP-43 HZ animals. Dendritic protrusion density was low and highly variable between dendrites at P5 in both WT and HZ animals (A, B). The majority of protrusions at these ages had immature morphologies with thin stalks and no delineated head. By P7, protrusion density had increased and appeared more uniform between dendrites on a single cell (C, D). Mature spines were visible along with thin, immature protrusions. Dendrites from GAP-43 HZ animals had increased protrusion density as compared with WT dendrites (*P < 0.05; E). Scale bar = 10 μm.

Contrasting expression patterns of NR1 and GluR1 puncta in C3 barrels of WT and GAP-43 HZ mice. Representative images of glutamate receptor expression in the C3 barrel show that at P5 expression of NR1 puncta in GAP-43 HZ barrels (D) appears to be reduced relative to WT (A). Although GluR1 puncta densities appear comparable (B, WT; E, HZ), NR1/GluR1 colocalized sites appear to be more numerous in GAP-43 HZ C3 barrels (F, white arrowheads in inset) than in WT (C, white arrowheads in inset). (G) NR1 and GluR1 puncta densities (normalized values to WT) are significantly reduced in GAP-43 HZ (white bars) C3 barrels compared with WT (dashed line) from P3 to P5 (*P < 0.05, Student’s t-test, comparing HZ normalized to WT), while GAP-43 HZ (black bars) NR1 and GluR1 puncta densities are significantly higher by P6–P7 (*P < 0.05, Student’s t-test, comparing HZ normalized to WT). (H) Colocalization coefficients in GAP-43 HZ C3 barrels (black bars) are significantly higher from P3 to P5 (P < 0.05, Student’s t-test) but return to WT levels by P6–P7. *P < 0.05. Inset = ×300 magnification. Scale bar = 50 μm (16.6-μm for inset).