James S. McCasland

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. J. Comp. Neurol. 462:252–264, 2003.

Resistance to change and vulnerability to stress: Autistic-like features of GAP43 deficient mice

There is an urgent need for animal models of autism spectrum disorder (ASD) to understand the underlying pathology and facilitate development and testing of new treatments. The synaptic growth‐associated protein‐43 (GAP43) has recently been identified as an autism candidate gene of interest. Our previous studies show many brain abnormalities in mice lacking one allele for GAP43 [GAP43 (+/−)] that are consistent with the disordered connectivity theory of ASD. Thus, we hypothesized that GAP43 (+/−) mice would show at least some autistic‐like behaviors. We found that GAP43 (+/−) mice, relative to wild‐type (+/+) littermates, displayed resistance to change, consistent with one of the diagnostic criteria for ASD. GAP43 (+/−) mice also displayed stress‐induced behavioral withdrawal and anxiety, as seen in many autistic individuals. In addition, both GAP43 (+/−) mice and (+/+) littermates showed low social approach and lack of preference for social novelty, consistent with another diagnostic criterion for ASD. This low sociability is likely because of the mixed C57BL/6J 129S3/SvImJ background. We conclude that GAP43 deficiency leads to the development of a subset of autistic‐like behaviors. As these behaviors occur in a mouse that displays disordered connectivity, we propose that future anatomical and functional studies in this mouse may help uncover underlying mechanisms for these specific behaviors. Strain‐specific low sociability may be advantageous in these studies, creating a more autistic‐like environment for study of the GAP43‐mediated deficits of resistance to change and vulnerability to stress.

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.

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.

Use-dependent plasticity in barrel cortex: Intrinsic signal imaging reveals functional expansion of spared whisker representation into adjacent deprived columns

We used optical imaging of intrinsic cortical signals, elicited by whisker stimulation, to define areas of activation in primary sensory cortex of normal hamsters and hamsters subjected to neonatal follicle ablation at postnatal day seven (P7). Follicle ablations were unilateral, and spared either C-row whiskers or the second whisker arc. This study was done to determine if the intrinsic cortical connectivity pattern of the barrel cortex, established during the critical period, affects the process of representational plasticity that follows whisker follicle ablation. Additionally, we tested the ability to monitor such changes in individual cortical whisker representations using intrinsic signal imaging. Stimulation of a single whisker yielded peak activation of a barrel-sized patch in the somatotopically appropriate location in normal cortex. In both row and arc-spared animals, functional representations corresponding to spared follicles were significantly stronger and more oblong than normal. The pattern of activation differed in the row-sparing and arc-sparing groups, in that the expansion was preferentially into deprived, not spared areas. Single whisker stimulation in row-spared cases preferentially activated the corresponding barrel arc, while stimulation of one whisker in arc-spared cases produced elongated activation down the barrel row. Since whisker deflection normally has a net inhibitory effect on neighboring barrels, our data suggest that intracortical inhibition fails to develop normally in deprived cortical columns. Because thalamocortical projections are not affected by follicle ablation after P7, we suggest that the effects we observed are largely cortical, not thalamocortical.

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.

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.

Anomalous functional organization of barrel cortex in GAP-43 deficient mice.

Growth associated protein 43 (GAP-43), found only in the nervous system, regulates the response of neurons to axon guidance signals. It is also critical for establishing normal somatotopy. Mice lacking GAP-43 (KO) show aberrant pathfinding by thalamocortical afferents, and do not form cortical whisker/barrels. GAP-43 heterozygous (HZ) mice show more subtle deficits--delayed barrel segregation and enlarged barrels at postnatal day 7. Here, we used cortical intrinsic signal imaging to characterize adult somatotopy in wildtype (WT), GAP-43 KO, and HZ mice. We found clear foci of activation in GAP-43 KO cortex in response to single-whisker stimulation. However, the KO spatial activation patterns showed severe anomalies, indicating a loss of functional somatotopy. In some cases, multiple foci were activated by single whiskers, while in other cases, the same cortical zone was activated by several whiskers. The results are consistent with our previous findings of aberrant pathfinding and clustering by thalamocortical afferent axons, and absence of barrel patterning. Our findings indicate that cortex acts to cluster afferents from a given whisker, even in the absence of normal topography. By contrast, single-whisker stimulation revealed normal adult topographic organization in WT and HZ mice. However, we found that functional representations of adult HZ barrels are larger than those found in WT mice. Since histological HZ barrels recover normal dimensions by postnatal day 26, the altered circuit function in GAP-43 HZ cortex could be a secondary consequence of the rescue of barrel dimensions.

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.

Transmission of Food Preference does Not Require Socially Relevant Cues in a Mouse Strain with Low Sociability

The social transmission of food preference task (STFP) is based on the principle that dietary information can be communicated between rodents during social interaction (Galef and Kennett, 1987). Briefly, a demonstrator mouse consumes a novel flavor, and then freely interacts with an observer mouse. The observer mouse is now “socially cued” toward that flavor, and will prefer it in a choice paradigm over another novel “un-cued” flavor. Socially relevant cues are required for this transmission of food preference in adult rats (Galef and Kennet, 1987) and C57BL/6J mice (Ryan et al., 2008). This evidence indicates that the STFP task is an appropriate measure of social communication in rodents. Since impaired communication is a diagnostic criterion for autism (DSMIV), several studies have utilized this protocol to investigate autistic-like behavior in mice (Boylan et al., 2007; McFarlane et al., 2008; Ryan et al., 2010). We performed the STFP task, as previously described (McFarlane et al., 2008), to evaluate social communication in mice with a mixed C57BL/6J 129S3/SvImJ background (B6129S3) (Zaccaria et al., 2010). This mouse strain exhibits low social approach and lack of preference for social novelty (Zaccaria et al., 2010). Therefore, it was surprising that B6129S3 mice consumed significantly more cued than non-cued food (t38 = 2.41, p < 0.05 – Figure ​Figure1A).1A). According to McFarlane et al. (2008), this indicates intact social communication, which is difficult to reconcile with strain-specific low sociability. Figure 1 Food consumption during the STFP and NSTFP tasks. (A) In both tasks, B6129S3 mice consumed significantly more cued food than non-cued food (*p < 0.05). (B) To control for differences in total food intake, the ratio of cued food ... Our findings could be explained if transmission of preference was in response to olfactory cues alone, and did not require the social component of the task. We tested this hypothesis by performing a non-social transmission of food preference (NSTFP) task, which removed socially relevant cues (Zaccaria et al., 2010). The STFP protocol was modified by substituting a novel-scented object (interlocking blocks) for the novel-scented mouse. In this NSTFP task, B6129S3 mice ate significantly more cued than non-cued food (t28 = 2.12, p  60% in both tasks (Figure ​(Figure1B,1B, Zaccaria et al., 2010), demonstrating that mice developed preference for the cued food both with and without socially relevant cues. These results indicate that social interaction is not necessary for transmission of food preference in this background strain, contradicting findings from the more social C57BL/6J mouse strain (Ryan et al., 2008). This discrepancy is likely due to differences in strain-specific levels of sociability. These findings highlight the importance of isolating the effects of olfactory vs. social cues when evaluating transmission of food preference. To confirm that transmission requires social communication, the NSTFP task should be performed as a control when evaluating positive results from the STFP task. This appears to be critical in strains with low sociability. These findings also reveal a previously undescribed abnormality in social communication among mice. The STFP task can only evaluate whether transmission of food preference occurs in a social setting – not whether social cues are actually required. By performing the NSTFP task, we also evaluated whether the presence of a novel food in a non-social setting can lead to similar transmission. We found that our low-sociability mice do not distinguish between social and non-social cues for determining food preference. In a natural habitat, this NSTFP could be severely maladaptive. To learn what foods are safe to eat, rodents rely heavily on communication of dietary information through social contact (Valsecchi et al., 1996). In this context, choosing food based on non-social cues (i.e., presence of a food on an inanimate object) could place a rodents survival at risk. By this logic, we propose that lack of discrimination between social and non-social cues for transmission of food preference is a novel example of impaired communication in mice.