Acoustic overexposure increases the expression of VGLUT-2 mediated projections from the lateral vestibular nucleus to the dorsal cochlear nucleus

The dorsal cochlear nucleus (DCN) is a first relay of the central auditory system as well as a site for integration of multimodal information. Vesicular glutamate transporters VGLUT-1 and VGLUT-2 selectively package glutamate into synaptic vesicles and are found to have different patterns of organization in the DCN. Whereas auditory nerve fibers predominantly co-label with VGLUT-1, somatosensory inputs predominantly co-label with VGLUT-2. Here, we used retrograde and anterograde transport of fluorescent conjugated dextran amine (DA) to demonstrate that the lateral vestibular nucleus (LVN) exhibits ipsilateral projections to both fusiform and deep layers of the rat DCN. Stimulating the LVN induced glutamatergic synaptic currents in fusiform cells and granule cell interneurones. We combined the dextran amine neuronal tracing method with immunohistochemistry and showed that labeled projections from the LVN are co-labeled with VGLUT-2 by contrast to VGLUT-1. Wistar rats were exposed to a loud single tone (15 kHz, 110 dB SPL) for 6 hours. Five days after acoustic overexposure, the level of expression of VGLUT-1 in the DCN was decreased whereas the level of expression of VGLUT-2 in the DCN was increased including terminals originating from the LVN. VGLUT-2 mediated projections from the LVN to the DCN are likely to play a role in the head position in response to sound. Amplification of VGLUT-2 expression after acoustic overexposure could be a compensatory mechanism from vestibular inputs in response to hearing loss and to a decrease of VGLUT-1 expression from auditory nerve fibers.     

Signaling mechanisms of daidzein-induced axonal outgrowth in hippocampal neurons

We aim to study the mechanisms underlying the neurotrophic effect of daidzein (Dz) in hippocampal neurons. Dz-enhanced axonal outgrowths manifested growth cone formation and increased immunostaining intensity of growth-associated protein 43 (GAP-43) in growth cones. Consistent with this, Dz increased GAP-43 phosphorylation and its membrane translocation without affecting total GAP-43 levels. In the presence of Dz, significant increase in the immunoreactivity for estrogen receptor (ER) β, but not ERα, was observed on the membrane of cell bodies and growing axons. Dz also induced the activation of protein kinase C α (PKCα), which was inhibited by the ICI182,780 pretreatment. Similarly, Dz-promoted axonal elongation was blocked by ICI182,780 and Gö6976. Moreover, Dz-stimulated activation of GAP-43 was specifically abolished by Gö6976, suggesting PKCα being the upstream effector of GAP-43. Taken together, our data suggest that Dz triggers an ERβ/PKCα/GAP-43 signaling cascade to promote axonal outgrowths in cultured hippocampal neurons.     

5-HT(2C) receptors localize to dopamine and GABA neurons in the rat mesoaccumbens pathway

The serotonin 5-HT(2C) receptor (5-HT(2C)R) is localized to the limbic-corticostriatal circuit, which plays an integral role in mediating attention, motivation, cognition, and reward processes. The 5-HT(2C)R is linked to modulation of mesoaccumbens dopamine neurotransmission via an activation of γ-aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA). However, we recently demonstrated the expression of the 5-HT(2C)R within dopamine VTA neurons suggesting the possibility of a direct influence of the 5-HT(2C)R upon mesoaccumbens dopamine output. Here, we employed double-label fluorescence immunochemistry with the synthetic enzymes for dopamine (tyrosine hydroxylase; TH) and GABA (glutamic acid decarboxylase isoform 67; GAD-67) and retrograde tract tracing with FluoroGold (FG) to uncover whether dopamine and GABA VTA neurons that possess 5-HT(2C)R innervate the nucleus accumbens (NAc). The highest numbers of FG-labeled cells were detected in the middle versus rostral and caudal levels of the VTA, and included a subset of TH- and GAD-67 immunoreactive cells, of which >50% also contained 5-HT(2C)R immunoreactivity. Thus, we demonstrate for the first time that the 5-HT(2C)R colocalizes in DA and GABA VTA neurons which project to the NAc, describe in detail the distribution of NAc-projecting GABA VTA neurons, and identify the colocalization of TH and GAD-67 in the same NAc-projecting VTA neurons. These data suggest that the 5-HT(2C)R may exert direct influence upon both dopamine and GABA VTA output to the NAc. Further, the indication that a proportion of NAc-projecting VTA neurons synthesize and potentially release both dopamine and GABA adds intriguing complexity to the framework of the VTA and its postulated neuroanatomical roles.     

Aging in the rat hippocampus is associated with widespread reductions in the number of glutamate decarboxylase-67 positive interneurons but not interneuron degeneration

Increased excitability of principal excitatory neurons is one of the hallmarks of aging in the hippocampus, signifying a diminution in the number and/or function of inhibitory interneurons with aging. To elucidate this, we performed comprehensive GABA-ergic interneuron cell counts in all layers of the dentate gyrus and the CA1 and CA3 subfields, using serial sections from adult, middle-aged and aged Fischer 344 rats. Sections were immunostained for glutamate decarboxylase-67 (GAD-67, a synthesizing enzyme of GABA) and GAD-67 immunopositive interneurons were counted using an unbiased cell counting method, the optical fractionator. Substantial declines in the absolute number of GAD-67 immunopositive interneurons were found in all hippocampal layers/subfields of middle-aged and aged animals, in comparison with the adult animals. However, the counts were comparable between the middle-aged and aged groups for all regions. Interestingly, determination of the absolute number of interneurons using neuron-specific nuclear antigen (NeuN) expression in the strata oriens and radiatum of CA1 and CA3 subfields revealed an analogous number of interneurons across the three age groups. Furthermore, the ratio of GAD-67 immunopositive and NeuN positive interneurons decreased from adult age to middle age but remained relatively static between middle age and old age. Collectively, the results underscore that aging in the hippocampus is associated with wide-ranging decreases in the number of GAD-67 immunopositive interneurons and most of the age-related changes in GAD-67 immunopositive interneuron numbers transpire by middle age. Additionally, this study provides novel evidence that age-related reductions in hippocampal GAD-67 immunopositive interneuron numbers are due to loss of GAD-67 expression in interneurons rather than interneuron degeneration.     

Palmitoylation modification of Gαo depresses its susceptibility to GAP-43 activation

Interaction between GAP-43 (growth associated protein-43) and Gα_o (alpha subunit of Go protein) influences the signal transduction pathways leading to differentiation of neural cells. GAP-43 is known to increase guanine nucleotide exchange by Gα_o, which is a major component of neuronal growth cone membranes. However, it is not clear whether GAP-43 stimulation is related to the Gα_o palmitoylation or the conversion of Gα_o from oligmers to monomers, which was shown to be a necessary regulatory factor in GDP/GTP exchange of Gα_o. Here we expressed and purified GAP-43, GST-GAP-43 and Gα_o proteins, detected their stimulatory effect on [³⁵S]-GTPγS binding of Gα_o. It was found that the EC₅₀ of both GAP-43 and GST-GAP-43 activation were tenfold lower in case of depalmitoylated Gα_o than palmitoylated Gα_o. Non-denaturing gel electrophoresis and p-PDM cross-linking analysis revealed that addition of GST-GAP-43 induced disassociation of depalmitoylated Gα_o from oligomers to monomers, but did not influence the oligomeric state of palmitoylated Gα_o, which suggests that palmitoylation is a key regulatory factor in GAP-43 stimulation on Gα_o. These results indicated the interaction of GAP-43 and Gα_o could accelerate conversion of depalmitoylated Gα_o but not palmitoylated Gα_o from oligomers to monomers, so as to increase the GTPγS binding activity of Gα_o. Results here provide new evidence about how signaling protein palmitoylation is involved in the G-protein-coupled signal transduction cascade, and give a useful clue on the participation of GAP-43 in G-protein cycle by its preferential activation of depalmitoylated Gα_o.     

Activation of ERK1/2 and PI3K/Akt by IGF-1 on GAP-43 Expression in DRG Neurons with Excitotoxicity Induced by Glutamate In Vitro

Insulin-like growth factor-1 (IGF-1) is a neurotrophic factor and plays an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Whether IGF-1 influences growth-associated protein 43 (GAP-43) expression and activates the extracellular signal-regulated protein kinase (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in DRG neurons with excitotoxicity induced by glutamate (Glu) remains unknown. In this study, embryonic 15-day-old rat DRG explants were cultured for 48 h and then exposed to IGF-1, Glu, Glu + IGF-1, Glu + IGF-1 + PD98059, Glu + IGF-1 + LY294002, Glu + IGF-1 + PD98059 + LY294002 for additional 12 h. The DRG explants were continuously exposed to growth media as control. The levels of GAP-43 mRNA were detected by real time-PCR analysis. The protein levels of GAP-43, phosphorylated ERK1/2, phosphorylated Akt, total ERK1/2, and total Akt were detected by Western blot assay. GAP-43 expression in situ was determined by immunofluorescent labeling. Apoptotic cell death was monitored by Hoechst 33342 staining. IGF-1 alone increased GAP-43 and its mRNA levels in the absence of Glu. The decreased GAP-43 and its mRNA levels caused by Glu could be partially reversed by the presence of IGF-1. IGF-1 rescued neuronal cell death caused by Glu. Neither the ERK1/2 inhibitor PD98059 nor the PI3K inhibitor LY294002 blocked the effect of IGF-1, but both inhibitors together were effective. To validate the impact of GAP-43 expression by IGF-1, GAP-43 induction was blocked by administration of dexamethasone (DEX). IGF-1 partially rescued the decrease of GAP-43 and its mRNA levels induced by DEX. DEX induced an increase of cell apoptosis. IGF-1 may play an important role in neuroprotective effects on DRG neurons through regulating GAP-43 expression with excitotoxicity induced by Glu and the process was involved in both ERK1/2 and PI3K/Akt signaling pathways.     

Prenatal Ethanol Exposure Decreases GAP-43 Phosphorylation and Protein Kinase C Activity in the Hippocampus of Adult Rat Offspring

Abstract: Consumption of moderate quantities of ethanol during pregnancy produces deficits in long-term potentiation in the hippocampal formation of adult offspring. Protein kinase C (PKC)-mediated phosphorylation of the presynaptic protein GAP-43 is critical for the induction of long-term potentiation. We tested the hypothesis that this system is affected in fetal alcohol-exposed (FAE) rats by measuring GAP-43 phosphorylation and PKC activity in the hippocampus of adult offspring of rat dams that had consumed one of three diets throughout gestation: (a) a 5% ethanol liquid diet, which produced a maternal blood ethanol concentration of 83 mg/dl (FAE); (b) an isocalorically equivalent 0% ethanol diet (pair-fed); or (c) lab chow ad libitum. Western blot analysis using specific antibodies to PKC-phosphorylated GAP-43 revealed that FAE rats had an ∼50% reduction in the proportion of phosphorylated GAP-43. Similarly, we found that PKC-mediated incorporation of ³²P into GAP-43 was reduced by 85% in hippocampal slices from FAE rats compared with both control groups. FAE animals also showed a 50% reduction in total hippocampal PKC activity, whereas the levels of six major PKC isozymes did not change in any of the diet groups. These results suggest that GAP-43 phosphorylation deficits in rats prenatally exposed to moderate levels of ethanol are not due to alterations in the expression of either the enzyme or substrate protein, but rather to a defect in kinase activation.     

The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells

The RNA-binding protein HuD binds to a regulatory element in the 3' untranslated region (3' UTR) of the GAP-43 mRNA. To investigate the functional significance of this interaction, we generated PC12 cell lines in which HuD levels were controlled by transfection with either antisense (pDuH) or sense (pcHuD) constructs. pDuH-transfected cells contained reduced amounts of GAP-43 protein and mRNA, and these levels remained low even after nerve growth factor (NGF) stimulation, a treatment that is normally associated with protein kinase C (PKC)-dependent stabilization of the GAP-43 mRNA and neuronal differentiation. Analysis of GAP-43 mRNA stability demonstrated that the mRNA had a shorter half-life in these cells. In agreement with their deficient GAP-43 expression, pDuH cells failed to grow neurites in the presence of NGF or phorbol esters. These cells, however, exhibited normal neurite outgrowth when exposed to dibutyryl-cAMP, an agent that induces outgrowth independently from GAP-43. We observed opposite effects in pcHuD-transfected cells. The GAP-43 mRNA was stabilized in these cells, leading to an increase in the levels of the GAP-43 mRNA and protein. pcHuD cells were also found to grow short spontaneous neurites, a process that required the presence of GAP-43. In conclusion, our results suggest that HuD plays a critical role in PKC-mediated neurite outgrowth in PC12 cells and that this protein does so primarily by promoting the stabilization of the GAP-43 mRNA.     

Constitutive Expression of Calmodulin-Binding Phosphoprotein GAP-43 in Rat Serotonergic and Noradrenergic Cell Groups Which Project to the Spinal Cord

In situ hybridization was combined with serotonin (5-hydroxytryptamine, 5-HT) or tyrosine hydroxylase immunocytochemistry and with Fluoro-Gold retrograde labeling of bulbo-spinal pathways in order to investigate the expression of GAP-43 mRNA in monoamine cell groups of the adult rat brain stem. Consistent with previous reports, GAP-43 mRNA was observed in serotonin and dopamine cell groups in the pons. In addition, GAP-43 expressing cells were observed in all the major monoamine cell groups in the medulla. Thus the B1, B2 and B3 serotonin cell groups all showed high GAP-43 expression and all contained many GAP-43 expressing serotonin cells with spinal cord projections. The A1, A2, A5 and A6 noradrenalin cell groups also showed high GAP-43 expression, although cells with spinal cord projections were largely restricted to the A5 group and A6 subcoeruleus region. In all areas, GAP-43 expressing cells with spinal cord projections were also observed which were not serotonergic or noradrenergic.     

Anti-apoptotic role of retinoic acid in the inner ear of noise-exposed mice

Exposure to loud noise can induce temporary or permanent hearing loss, and acoustic trauma is the major cause of hearing impairment in industrial nations. However, the mechanisms underlying the death of hair cells after acoustic trauma remain unclear. In addition to its involvement in cellular stress and apoptosis, the c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is involved in cell survival, transformation, embryonic morphogenesis, and differentiation. JNK is primarily activated by various environmental stresses including noise, and the phenotypic result appears be to cell death. All-trans retinoic acid (ATRA) is an active metabolite of vitamin A that regulates a wide range of biological processes, including cell proliferation, differentiation, and morphogenesis. We evaluated the role of ATRA in preserving hearing in mice exposed to noise that can induce permanent hearing loss. Mice fed with ATRA before and during 3 consecutive days of noise exposure had a more preserved hearing threshold than mice fed sesame oil or saline. Histological and TUNEL staining of the cochlea showed significantly enhanced preservation of the organ of Corti, including outer hair cells and relatively low apoptotic nuclei, in mice-fed ATRA than in mice-fed sesame oil or saline. Phospho-JNK immunohistochemistry showed that ATRA inhibited the activation of JNK. These results suggest that ATRA has an anti-apoptotic effect on cochleae exposed to noise.     

Capsaicin-Induced Activation of ERK1/2 and Its Involvement in GAP-43 Expression and CGRP Depletion in Organotypically Cultured DRG Neurons

Low concentrations of capsaicin (CAP) stimulate and high concentrations of CAP can be toxic to the primary sensory neurons of the dorsal root ganglion (DRG). CAP induces the phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in DRG neurons. The effect of the activation of ERK1/2 by different concentrations of CAP on growth-associated protein 43 (GAP-43) expression and calcitonin gene-related peptide (CGRP) depletion in DRG neurons remains unknown. In the present study, organotypic embryonic 15-day-old rat DRG explants were used to determine the effect of different concentrations of CAP on GAP-43 expression and CGRP depletion. The results showed that, compared to unstimulated control cultures, GAP-43 and pERK1/2 protein levels increased at a low concentration (2 μmol/L) of CAP and decreased at a higher concentration (10 μmol/L). The number of CGRP-immunoreactive (IR) migrating neurons also decreased in CAP-treated cultures. The increase in GAP-43 levels and CGRP depletion could be blocked by the administration of ERK1/2 inhibitor PD98059. The results of the present study imply that CAP at different concentrations has different effects on GAP-43 expression and CGRP depletion. These effects were involved in the activation of ERK1/2 in organotypically cultured DRG neurons stimulated with CAP. These data may provide new insights for further development potential therapeutic applications of CAP with moderate dose on neurogenic inflammation.     

Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization,Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.     

Palmitoylcarnitine Affects Localization of Growth Associated Protein GAP-43 in Plasma Membrane Subdomains and its Interaction with Gαo in Neuroblastoma NB-2a Cells

Palmitoylcarnitine was observed previously to promote differentiation of neuroblastoma NB-2a cells, and to affect protein kinase C (PKC). Palmitoylcarnitine was also observed to increase palmitoylation of several proteins, including a PKC substrate, whose expression augments during differentiation of neural cells—a growth associated protein GAP-43, known to bind phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. Since palmitoylated proteins are preferentially localized in sphingolipid- and cholesterol-rich microdomains of plasma membrane, the present study has been focused on a possible effect of palmitoylcarnitine on GAP-43 localization in these microdomains. Palmitoylcarnitine treatment resulted in GAP-43 appearance in floating fractions (rafts) in sucrose gradient and increased co-localization with cholesterol and with PI(4,5)P₂, although co-localization of both lipids decreased. GAP-43 disappeared from raft fraction upon treatment with 2-bromopalmitate (an inhibitor of palmitoylating enzymes) and after treatment with etomoxir (carnitine palmitoyltransferase I inhibitor). Raft localization of GAP-43 was completely abolished by treatment with methyl-β-cyclodextrin, a cholesterol binding agent, while there was no change upon sequestration of PI(4,5)P₂ with neomycin. GAP-43 co-precipitated with a monomeric form of Gα_o, a phenomenon diminished after palmitoylcarnitine treatment and paralleled by a decrease of Gα_o in the raft fraction. These observations point to palmitoylation of GAP-43 as a mechanism leading to an increased localization of this protein in microdomains of plasma membrane rich in cholesterol, in majority different, however, from microdomains in which PI(4,5)P₂ is present. This localization correlates with decreased interaction with Gα_o and suppression of its activity—an important step regulating neural cell differentiation.     

Alterations in growth-associated protein (GAP-43) expression in lower urinary tract pathways following chronic spinal cord injury

Alterations in the expression of growth-associated protein 43 (GAP-43) were examined in lower urinary tract micturition reflex pathways 6 or 8 weeks following complete spinal cord transection (~ T9). In control animals, expression of GAP-43 was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the corticospinal tract; (3) the dorsal horn; and (4) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (5) in the lateral collateral pathway of Lissauer in L6-S1 spinal segments. Densitometry analysis has demonstrated significant increases (p 0.001; 1.3-6.4-fold increase) in GAP-43-immunoreactivity (IR) in these regions of the rostral lumbar (L1-L2) and caudal lumbosacral (L6-S1) spinal cord 6 weeks following spinal cord injury. Changes in GAP-43-IR were restricted to the L1-L2 and L6-S1 segments that are involved in lower urinary tract reflexes. Changes in GAP-43-IR were not observed at the L5 segmental level except for an increase in GAP-43-IR in the superficial, dorsal horn at 6 weeks post-injury. In all segments examined, GAP-43-IR was decreased (2-5-fold) in the corticospinal tract (dorsal division) 6 and 8 weeks following spinal cord injury. Eight weeks following spinal cord injury, changes in GAP-43-IR had returned to control levels except for the persistence of increased GAP-43-IR in the region of the sacral parasympathetic nucleus and the lateral collateral pathway in the S1 spinal segment. Alterations in GAP-43-IR following chronic spinal cord injury may suggest a reorganization of bladder afferent projections and spinal elements involved in urinary bladder reflexes consistent with alterations in urinary bladder function (hyperreflexia) observed in animals following spinal cord injury above the lumbosacral spinal cord.     

Alterations in growth-associated protein (GAP-43) expression in lower urinary tract pathways following chronic spinal cord injury

Alterations in the expression of growth-associated protein 43 (GAP-43) were examined in lower urinary tract micturition reflex pathways 6 or 8 weeks following complete spinal cord transection (approximately T9). In control animals, expression of GAP-43 was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the corticospinal tract; (3) the dorsal horn; and (4) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (5) in the lateral collateral pathway of Lissauer in L6-S1 spinal segments. Densitometry analysis has demonstrated significant increases (p < or =0.001; 1.3-6.4-fold increase) in GAP-43-immunoreactivity (IR) in these regions of the rostral lumbar (L1-L2) and caudal lumbosacral (L6-S1) spinal cord 6 weeks following spinal cord injury. Changes in GAP-43-IR were restricted to the L1-L2 and L6-S1 segments that are involved in lower urinary tract reflexes. Changes in GAP-43-IR were not observed at the L5 segmental level except for an increase in GAP-43-IR in the superficial, dorsal horn at 6 weeks post-injury. In all segments examined, GAP-43-IR was decreased (2-5-fold) in the corticospinal tract (dorsal division) 6 and 8 weeks following spinal cord injury. Eight weeks following spinal cord injury, changes in GAP-43-IR had returned to control levels except for the persistence of increased GAP-43-IR in the region of the sacral parasympathetic nucleus and the lateral collateral pathway in the S1 spinal segment. Alterations in GAP-43-IR following chronic spinal cord injury may suggest a reorganization of bladder afferent projections and spinal elements involved in urinary bladder reflexes consistent with alterations in urinary bladder function (hyperreflexia) observed in animals following spinal cord injury above the lumbosacral spinal cord.     

Evidence for a Glutamatergic Pathway from the Guinea Pig Auditory Cortex to the Inferior Colliculus

Abstract: We attempt to provide evidence that the projection from the guinea pig auditory cortex (AC) to the inferior colliculus (IC) may contain glutamatergic or GABAergic fibers. Seven days after unilateral AC aspiration, histological studies indicated almost complete AC destruction and preterminal degeneration of fibers and terminal fields in the dorsal cortex (DCIC), external cortex (ECIC), and central nucleus (CNIC) of the IC ipsilateral to the ablated AC. Contralaterally, degeneration appeared in the DCIC. AC ablation depressed the electrically evoked Ca²⁺-dependent release of d -[³H]aspartate ( d -[³H]Asp) in the ipsilateral DCIC, ECIC, and CNIC, and d -[³H]Asp uptake in the CNIC. Together with other evidence that the corticocollicular pathway is excitatory, these findings suggest that this projection may contain glufamatergic and/or aspartatergic (Glu/Asp-ergic) fibers. Glutamic acid decarboxylase immunoreactivity was not apparent in presumed pyramidal cells of layer V of the AC retrogradely labeled with biotinylated dextran injected into the ipsilateral IC. Thus, corticocollicular neurons probably do not synthesize GABA and may not be GABAergic. However, AC ablation depressed [¹⁴C]GABA release from the ipsilateral DCIC and ECIC, and [¹⁴C]GABA uptake in the DCIC. These findings are consistent with the atrophy or down-regulation of some subcortical neurons that mediate GABAergic transmission in the IC.     

Phosphorylation-site mutagenesis of the growth-associated protein GAP- 43 modulates its effects on cell spreading and morphology

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane- association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP- 43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin- containing cortical cytoskeleton in a regulatable manner.     

Identification of brain proteins BASP1 and GAP-43 in mouse oocytes and zygotes

The similarity between the calcium-activated signaling systems of oocytes and neuronal axon terminals has prompted us to test whether BASP1 and GAP-43 proteins, highly expressed in brain neurons, are present in oocytes. Using immunocytochemical techniques combined with confocal microscopy, we have for the first time demonstrated that both BASP1 and GAP-43 are present in mouse metaphase II (MII) oocytes and zygotes. BASP1 is localized to the plasma membrane and actin cortex of MII oocytes, which is similar to BASP1 distribution in neurons and other cell types. GAP-43 is generally regarded as a postmitotic membrane marker of nerve cells; however, GAP-43 in MII oocytes is associated with microtubules of the meiotic spindle. GAP-43 is also colocalized with γ-tubulin at the spindle poles (centrosomes) and at the discrete microtubule- organizing centers in the cytoplasm. The antibodies to Ser41-phosphorylated form of GAP-43 allowed for demonstration that GAP-43 in oocytes is subject to phosphorylation by protein kinase C. The presence of BASP1 and GAP-43 in oocytes is also confirmed by electrophoresis and western blotting. Microinjection of BASP1 (but not GAP-43) into the cytoplasm of mouse MII oocytes induces their exit from metaphase II arrest followed by parthenogenetic embryo development. This suggests putative BASP1 involvement in fertilization-induced oocyte activation, presumably, through regulation of local concentration of polyphosphoinositides in the plasma membrane. Recently it was found that GAP-43 is associated with centrosomes in asymmetrically dividing neuronal progenitors, which is similar to the localization of GAP-43 at the meiotic spindle and centrosomes in oocytes. Therefore we suggest that GAP-43 may be involved in regulation of spindle orientation and oocyte polarity.