Developmental and adult GAP-43 deficiency in mice dynamically alters hippocampal neurogenesis and mossy fiber volume.

Growth-associated protein-43 (GAP-43) is a presynaptic protein that plays key roles in axonal growth and guidance and in modulating synapse formation. Previous work has demonstrated that mice lacking one allele of this gene (GAP-43+/- mice) exhibit hippocampal structural abnormalities, impaired spatial learning and stress-induced behavioral withdrawal and anxiety, behaviors that are dependent on proper hippocampal circuitry and function. Given the correlation between hippocampal function, synaptic connectivity and neurogenesis, we tested if behaviorally naïve GAP-43+/- mice had alterations in either neurogenesis or synaptic connectivity in the hippocampus during early postnatal development and young adulthood, and following behavior testing in older adults. To test our hypothesis, we examined hippocampal cell proliferation (Ki67), number of immature neuroblasts (doublecortin, DCX) and mossy fiber volume (synaptoporin) in behaviorally naïve postnatal day 9 (P9) and P26, and behaviorally experienced 5- to 7-month-old GAP-43+/- and +/+ littermate mice. P9 GAP-43+/- mice had fewer Ki67+ and DCX+ cells compared to +/+ mice, particularly in the posterior dentate gyrus, and smaller mossy fiber volume in the same region. In young adulthood, however, male GAP-43+/- mice had more Ki67+ and DCX+ cells and greater mossy fiber volume in the posterior dentate gyrus relative to male +/+ mice. These increases were not seen in females. In 5- to 7-month-old GAP-43+/- mice (whose behaviors were the focus of our prior publication), there was no global change in the number of proliferating or immature neurons relative to +/+ mice. However, more detailed analysis revealed fewer proliferative DCX+ cells in the anterior dentate gyrus of male GAP-43+/- mice compared to male +/+ mice. This reduction was not observed in females. These results suggest that young GAP-43+/- mice have decreased hippocampal neurogenesis and synaptic connectivity, but slightly older mice have greater hippocampal neurogenesis and synaptic connectivity. In conjunction with our previous study, these findings suggest that GAP-43 is dynamically involved in early postnatal and adult hippocampal neurogenesis and synaptic connectivity, possibly contributing to the GAP-43+/- behavioral phenotype.

Emergence of layer IV barrel cytoarchitecture is delayed in somatosensory cortex of GAP-43 deficient mice following delayed development of dendritic asymmetry.

The emergence of barrel cytoarchitecture in mouse somatosensory cortex is extremely well defined. However, mechanisms underlying the development of this cellular organization are not completely understood. While it is generally accepted that hollows emerge via passive displacement of cortical cells by dense thalamocortical afferent clusters in barrel centers, it is not known what causes cellular segregation of barrel sides and septa. Here, we hypothesized that the emergence of sides and septa is related to the progressive asymmetry of dendrites from the cells of the barrel side toward the barrel hollow during development. We tested this hypothesis in the barrel cortex of growth-associated protein-43 heterozygous mice (GAP43 (+/-) mice) that display a 2-day delay in retraction of septally oriented dendrites compared to (+/+) littermates. We predicted that this delayed retraction would result in a subsequent 2-day delay in the emergence of barrel sides and septa. Using cresyl violet staining of barrel cortex, we found that initial emergence of hollows was not different between GAP43 (+/-) mice and (+/+) littermate controls. However, the emergence of sides and septa was delayed by 2 days, supporting our hypothesis that the emergence of barrel sides and septa is related to, and perhaps reliant upon, the developmental step of dendritic orientation toward barrel hollows. This process, which is mechanistically distinct from the emergence of barrel hollows, is likely due to both active and passive events resulting from asymmetric cell orientation.

Postsynaptic deregulation in GAP-43 heterozygous mouse barrel cortex.

Formation of whisker-related barrels in primary somatosensory cortex (S1) requires communication between presynaptic thalamocortical afferents (TCAs) and postsynaptic cortical neurons. GAP-43 is crucially involved in targeting TCAs to postsynaptic S1 neurons but its influence on the interactions between these 2 elements has not been explored. Here, we tested the hypothesis that reduced early expression of presynaptic GAP-43 (GAP-43 heterozygous [HZ] mice) alters postsynaptic differentiation of barrel cells. We found a transient increase in cytochrome oxidase staining between P6 and P14 in HZ animals, indicative of increased metabolic activity in barrel cortex during this time. Golgi impregnation and microtubule-associated protein 2 immunohistochemistry showed anomalous dendritic patterning in GAP-43 HZ cortex at P5, with altered dendritic length and branching and abnormal retention of dendrites that extend into developing septa. This deficiency was no longer apparent at P7, suggesting partial recovery of dendritic pruning processes. Finally, we showed early defects in synaptogenesis from P4 to P5 with increased colocalization of NR1 and GluR1 staining in HZ mice. By P7, this colocalization had normalized to wild type levels. Taken together, our findings suggest abnormal postsynaptic differentiation in GAP-43 HZ cortex during early barrel development, followed by adaptive compensation and partial phenotypic rescue.

Pharmacological modulation of cortical plasticity following kainic acid lesion in rat barrel cortex.

OBJECT: The brain shows remarkable capacity for plasticity in response to injury. To maximize the benefits of current neurological treatment and to minimize the impact of injury, the authors examined the ability of commonly administered drugs, dextroamphetamine (D-amphetamine) and phenytoin, to positively or negatively affect the functional recovery of the cerebral cortex following excitotoxic injury. METHODS: Previous work from the same laboratory has demonstrated reorganization of whisker functional responses (WFRs) in the rat barrel cortex after excitotoxic lesions were created with kainic acid (KA). In the present study, WFRs were mapped using intrinsic optical signal imaging before and 9 days after creation of the KA lesions. During the post-lesion survival period, animals were either treated with intraperitoneal D-amphetamine, phenytoin, or saline or received no treatment. Following the survival period, WFRs were again measured and compared with prelesion data. RESULTS: The findings suggest that KA lesions cause increases in WFR areas when compared with controls. Treatment with D-amphetamine further increased the WFR area (p < 0.05) while phenytoin-treated rats showed decreases in WFR areas. There was also a statistically significant difference (p < 0.05) between the D-amphetamine and phenytoin groups. CONCLUSIONS: These results show that 2 commonly used drugs, D-amphetamine and phenytoin, have opposite effects in the functional recovery/plasticity of injured cerebral cortex. The authors' findings emphasize the complex nature of the cortical response to injury and have implications for understanding the biology of the effects of different medications on eventual functional brain recovery.

GAP-43 is critical for normal targeting of thalamocortical and corticothalamic, but not trigeminothalamic axons in the whisker barrel system.

Mice lacking the growth-associated protein GAP-43 (KO) show disrupted cortical topography and no barrels. Whisker-related patterns of cells are normal in the KO brainstem trigeminal complex (BSTC), while the pattern in KO ventrobasal thalamus (VB) is somewhat compromised. To better understand the basis for VB and cortical abnormalities, we used small placements of DiI to trace axonal projections between BSTC, VB, and barrel cortex in wildtype (WT) and GAP-43 KO mice. The trigeminothalamic (TT) pathway consists of axons from cells in the Nucleus Prinicipalis that project to the contralateral VB thalamus. DiI-labeled KO TT axons crossed the midline from BSTC and projected to contralateral VB normally, consistent with normal BSTC cytoarchitecture. By contrast, the KO thalamocortical axons (TCA) projection was highly abnormal. KO TCAs showed delays of 1-2 days in initial ingrowth to cortex. Postnatally, KO TCAs showed multiple pathfinding errors near intermediate targets, and were abnormally fasciculated within the internal capsule (IC). Interestingly, most individually labeled KO TCAs terminated in deep layers instead of in layer IV as in WT. This misprojection is consistent with birthdating analysis in KO mice, which revealed that neurons normally destined for layer IV remain in deep cortical layers. Early outgrowth of KO corticofugal (CF) axons was similar for both genotypes. However, at P7 KO CF fibers remained bundled as they entered the IC, and exhibited few terminal branches in VB. Thus, the establishment of axonal projections between thalamus and cortex are disrupted in GAP-43 KO mice.

GAP-43 heterozygous mice show delayed barrel patterning, differentiation of radial glia, and downregulation of GAP-43.

GAP-43 heterozygous (HZ) mice exhibit abnormal thalamocortical pathfinding, fasciculation, and terminal arborization at postnatal day 7 (P7). Here we tested whether these defects are correlated with delayed development of HZ cortical patterns. We assessed the rate of barrel segregation and radial glia differentiation in wild-type (WT) and HZ cortices. Since GAP-43 is involved in some forms of neural plasticity, we also compared the duration of the critical period for lesion-induced plasticity in both genotypes. Cytochrome oxidase histochemistry revealed a delay of approximately 1 day in barrel pattern formation in GAP-43 HZ mice. GAP-43 WT barrels showed complete segregation between P2-P3, while HZ barrels did not reach the same level of segregation until P3-P4. We found a similar delay in the transformation of radial glia from monopolar to multipolar phenotypes, from P5 in WT to P7 in HZ cortex. Radial glial cells represent many of the neuronal progenitors in developing cortex and aid in cell migration. Thus, the delay in radial glial differentiation may contribute to the delay in HZ barrel segregation. Interestingly, we found no change in the extent of the critical period for HZ cortical responsiveness to early peripheral damage or in the time course of the cortical response. As expected, GAP-43 expression in HZ cortex is significantly reduced early in development. However, HZ GAP-43 expression remains at maximum levels after P9, when it is normally downregulated. As a result, HZ GAP-43 expression is near-normal by P26, by which time near-normal barrel dimensions have been restored. Our findings indicate that GAP-43 deficiency leads to early delays in barrel development and suggest that these failures are followed by homeostatic responses, including prolonged GAP-43 expression. These compensatory mechanisms may rescue normal cortical reorganization in neonates and near-normal barrel morphology and GAP-43 expression in adulthood.

GAP-43 is critical for normal development of the serotonergic innervation in forebrain.

Serotonergic (5-HT) axons from the raphe nuclei are among the earliest afferents to innervate the developing forebrain. The present study examined whether GAP-43, a growth-associated protein expressed on growing 5-HT axons, is necessary for normal 5-HT axonal outgrowth and terminal arborization during the perinatal period. We found a nearly complete failure of 5-HT immunoreactive axons to innervate the cortex and hippocampus in GAP-43-null (GAP43-/-) mice. Abnormal ingrowth of 5-HT axons was apparent on postnatal day 0 (P0); quantitative analysis of P7 brains revealed significant reductions in the density of 5-HT axons in the cortex and hippocampus of GAP43-/- mice relative to wild-type (WT) controls. In contrast, 5-HT axon density was normal in the striatum, septum, and amygdala and dramatically higher than normal in the thalamus of GAP43-/- mice. Concentrations of serotonin and its metabolite, 5-hydroxyindolacetic acid, and norepinephrine were decreased markedly in the anterior and posterior cerebrum but increased in the brainstem of GAP43-/- mice. Cell loss could not account for these abnormalities, because unbiased stereological analysis showed no significant difference in the number of 5-HT dorsal raphe neurons in P7 GAP43-/- versus WT mice. The aberrant 5-HT innervation pattern persisted at P21, indicating a long-term alteration of 5-HT projections to forebrain in the absence of GAP-43. In heterozygotes, the density and morphology of 5-HT axons was intermediate between WT and homozygous GAP43-/- mice. These results suggest that GAP-43 is a key regulator in normal pathfinding and arborization of 5-HT axons during early brain development.

Abnormal thalamocortical pathfinding and terminal arbors lead to enlarged barrels in neonatal GAP-43 heterozygous mice.

GAP-43 has been implicated in axonal pathfinding and sprouting, synaptic plasticity, and neurotransmitter release. However, its effect on cortical development in vivo is poorly understood. We have previously shown that GAP-43 knockout (-/-) mice fail to develop whisker-related barrels or an ordered whisker map in the cortex. Here we used cytochrome oxidase (CO) histochemistry to demonstrate that GAP-43 heterozygous (+/-) mice develop larger than normal barrels at postnatal day 7 (P7), despite normal body and brain weight. Using serotonin transporter (5HT-T) histochemistry to label thalamocortical afferents (TCAs), we found no obvious abnormalities in other somatosensory areas or primary visual cortex of GAP-43 (+/-) mice. However, TCA projections to (+/-) primary auditory cortex were not as clearly defined. To clarify the mechanism underlying the large-barrel phenotype, we used lipophilic (DiI) axon labeling. We found evidence for multiple pathfinding abnormalities among GAP-43 (+/-) TCAs. These axons show increased fasciculation within the internal capsule, as well as abnormal turning and branching in the subcortical white matter. These pathfinding errors most likely reflect failures of signal recognition and/or transduction by ingrowing TCAs. In addition, many DiI-labeled (+/-) TCAs exhibit widespread, sparsely branched terminal arbors in layer IV, reflecting the large-barrel phenotype. They also resemble those found in rat barrel cortex deprived of whisker inputs from birth, suggesting a failure of activity-dependent synaptogenesis and/or synaptic stabilization in (+/-) cortex. Our findings suggest that reduced GAP-43 expression can alter the fine-tuning of a cortical map through a combination of pathfinding and synaptic plasticity mechanisms.

Large-scale plasticity in barrel cortex following repeated whisker trimming in young adult hamsters.

Using the 2DG/immunostaining method [McCasland, J.S., Graczyk, G.M., 2000. Metabolic mapping-Unit 1.6. In: Gerfen, C.R. (Ed.), Current Protocols in Neuroscience. Wiley, New York, pp 1.6.1-1.6.15], we have previously demonstrated large-scale plasticity in whisker/barrel fields of young adult hamsters subject to follicle ablation on postnatal day 7 (P7) [Somatosens. Motor Res. 13 (1996) 245]. This plasticity occurs after the barrel field has formed, but before neuronal differentiation and synaptogenesis are complete. The present study tested for similar large-scale plasticity following whisker deprivation in young adult hamsters, when neuronal and synaptic development are more mature. Beginning around P40, animals had all whiskers except row C trimmed on alternating days for periods ranging from 1 h to 2 weeks, after which they were administered (3)H 2DG (i.p.) and allowed to explore a fresh empty cage. Autoradiograms from these animals showed a clear expansion in the zone of heavy 2DG labeling with continued whisker trimming. Hamsters with row C spared overnight showed markedly higher labeling in the row C barrels, as expected. After 2 weeks of repeated trimming, the pattern of 2DG labeling in the barrel field ranged from complete activation of all large-whisker columns, as in a previous study of P7 follicle ablation, down to a more localized activation of rows B, C, and D. Intermediate periods of trimming produced more localized label in the region of row C. There was a clear trend toward larger areas of activation with longer periods of trimming. Because inhibitory neurons are strongly activated in all cases, this large-scale neuronal plasticity must take place in the presence of strong inhibition. The data show that simple trimming of all but a few whiskers in normally reared adults leads to abnormally widespread metabolic labeling encompassing virtually the entire barrel field. Taken together, our findings suggest that a large-scale synaptic reorganization occurs in barrel fields deprived of normal sensory input in the adult as well as during postnatal development.