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Biomedical subjects

A Routtenberg

Publications and source records attributed to A Routtenberg.

At least 37 records · Page 2Linked to original sources
PubMed1996

Rapid induction by kainic acid of both axonal growth and F1/GAP-43 protein in the adult rat hippocampal granule cells.

Hippocampal granule cells do not normally express the axonal growth- and plasticity-associated protein F1/GAP-43 in the adult rat. Using three different methods that lead to hypersynchronous activity in limbic circuits, expression of F1/GAP-43 mRNA can be induced in granule cells which is followed by sprouting in mossy fibers, the axons of granule cells. F1/GAP-43 mRNA expression in granule cells was induced in the temporal, but not septal, hippocampus beginning at 12 hours after kainic acid (KA) subcutaneous injection (10 mg/kg). Beginning 2 days after KA treatment, mossy fiber sprouts restricted to the temporal hippocampus were observed in the supragranular layer. In the same animal we also observed that levels of protein F1/GAP-43 immunoreactivity in this layer apparently increased at this same 2 day time point and same ventral hippocampal location. F1/GAP-43 protein levels and mossy fiber sprouting showed an increase up to 10 days after KA treatment. Sprouting was at a maximum at 40 days, the longest time point studied. These events parallel axonal regeneration with one critical difference: granule cell axons are not damaged by kainate. The rapid onset of axonal growth in the adult is striking and occurs earlier than reported previously (2 days vs. 12 days). Such growth closely associated with elevated levels of protein F1/GAP-43 may occur as a result of a) reactive synaptogenesis caused by the availability of post-synaptic surface on granule cell dendrites at the supragranular layer, b) Hebbian co-activation of the post-synaptic granule cells and their presynaptic afferents, and c) loss of target-derived inhibitory growth factor.

Animals↗
PubMed1996

Is the lack of protein F1/GAP-43 mRNA in granule cells target-dependent?

Protein F1/GAP-43 is differentially expressed in brain with high levels present in regions associated with memory functions. However, in hippocampus the granule cells lack F1/GAP-43 expression. To determine if this lack of expression is due to inhibitory signals from the target cells, we selectively destroyed CA3 pyramidal cells unilaterally using microinjections of excitotoxins. Kainate lesions induced F1/GAP-43 mRNA expression bilaterally in granule cells at 24 h post-injection. Since the induction contralateral to the lesion was not due to loss of target cells, that induction may be ascribed to consequences of seizure activity. However, F1/GAP-43 mRNA hybridization decreased by 3 d post-lesion and was at background levels by 6 d, indicating that the lack of F1/GAP-43 expression in granule cells is restored despite a lack of target neurons. Unilateral lesions of CA3 cells using ibotenate, which are not as complete as kainate but do not cause seizures, did not induce F1/GAP-43 mRNA in granule cells on either the contralateral or, in 4 of 5 cases, the ipsilateral side. Taken together, these data suggest that the CA3 target is not essential for the absence of F1/GAP-43 expression in granule cells. To compare the extent of damage caused by the lesions, we investigated the location of astrocytes undergoing reactive gliosis, employing as a reporter glial fibrillary acidic protein (GFAP) gene expression. After both kainate and ibotenate injections GFAP hybridization increased in the lesioned area as well as in the contralateral hippocampus. These results indicate that injections of kainate, and possibly ibotenate to a lesser extent, may affect behavior not only by damaging cells at the injection site, but also by altering gene expression in cells at distant sites.

Animals↗
PubMed1996

MARCKS and protein F1/GAP-43 mRNA in chick brain: effects of imprinting.

The phosphorylation of MARCKS, but not protein F1/GAP-43, is increased in the intermediate and medial portion of the hyperstriatum ventrale (IMHV) after chick imprinting. Here we investigated if MARCKS, but not F1/GAP-43, gene expression would also be altered after imprinting. We first investigated the constitutive mRNA distribution of MARCKS and F1/GAP-43 in chick brain. MARCKS mRNA was expressed in most cells and exhibited a relatively homogeneous distribution. In contrast, F1/GAP-43 mRNA levels were elevated in discrete brain regions, as we had observed in mammals. The highest F1/GAP-43 mRNA levels in the chick brain were in sensory and associational structures such as the hyperstriatal complex and neostriatum, and lower levels were in structures involved in motor control, such as paleostriatum. These results in chick are consistent with the previously drawn generalization that F1/GAP-43 mRNA is expressed in those brain regions which exhibit synaptic plasticity. After imprinting, MARCKS mRNA levels in IMHV were higher in good learners than poor learners. In contrast, analysis of F1/GAP-43 mRNA levels revealed no differences related to training in any brain region sampled. These selective results for MARCKS but not F1/GAP-43 parallel the prior findings on their phosphorylation, and are consistent with our hypothesis that the very same proteins that are post-translationally modified in association with learning and memory also undergo alterations in their gene expression.

Analysis of Variance↗
PubMed1996

Gene expression of the transcription factor NF-kappa B in hippocampus: regulation by synaptic activity.

NF-kappa B is a potent transcriptional activator that resides in latent form in the cytoplasm complexed to its inhibitor I kappa B. Phosphorylation of I kappa B by protein kinase C (PKC) releases NF-kappa B, enabling its translocation to the nucleus. Since PKC can activate NF-kappa B and PKC is activated by long-term potentiation (LTP), we investigated NF-kappa B expression after hippocampal LTP induced in vivo. We first described the expression of the NF-kappa B subunits, p50 and p65, and I kappa B alpha mRNAs, in each cell field of the hippocampus. In other brain locations I kappa B alpha mRNA exhibited a more selective expression than p50 and p65. We then demonstrated specific NF-kappa B-like DNA-binding activity in hippocampal whole-cell extracts and in synaptosomes using electrophoretic mobility shift assays by the following criteria: (1) latent binding was revealed after deoxycholate treatment; (2) binding was competed off by unlabeled kappa B oligonucleotides; and (3) antibodies to either p50 or p65 blocked binding. Since p50 gene expression is auto-regulated by NF-kappa B, we used its expression as a reporter for NF-kappa B activity using quantitative in situ hybridization. Both p50 and p65 increased their expression in response to either LTP-inducing or low-frequency control stimulation, although the increase in p65 mRNA levels was greater after LTP than control stimulation. In contrast to p50 and p65, I kappa B alpha hybridization levels were not increased, but were inversely correlated with the magnitude of LTP. Since NF-kappa B subunit gene expression in the hippocampus is increased by augmented synaptic activity, NF-kappa B activation may contribute to alterations in target gene expression that accompany activity-dependent synaptic plasticity, but only in a combinatorial fashion with other transcription factors.

Animals↗
PubMed1996

Prolonged alteration in E-box binding after a single systemic kainate injection: potential relation to F1/GAP-43 gene expression.

The presence in hippocampus of a basic helix-loop-helix (bHLH) family of transcription factors (TFs) specifically binding in an electrophoretic mobility shift assay (EMSA) to the E-box recognition element was established by selective blockade of binding both by cold competition and by an antibody to MyoD1, an E-box TF. Protein source was from a micro-dissected preparation enriched in hippocampal granule cells. Specific E-box binding of hippocampal transcription factors was significantly reduced in kainate acid (KA) treated animals. This was observed at 24 and 72 h, but not before (3, 6 h) or after (96 h). This is the first report to our knowledge to study functional regulation of E-box binding protein in adult hippocampus. To determine the generality of this E-box regulatory event, we studied four other situations, in addition to kainate treatment, where axonal growth is known or has been suggested to increase: NGF treatment of PC12 cells, unilateral hilar lesions, long-term potentiation after 1 h, and postnatal rat hippocampal development. In all four cases, decreased E-box binding was observed. The recent link of F1/GAP-43 mRNA induction in hippocampal granule cells by KA to growth of their axons, the mossy fibers in the adult rat, suggests a potential role for the F1/GAP-43 5' flanking promoter region in regulating neurite outgrowth. Since in all cases decreased E-box binding preceded increased F1/GAP-43 mRNA expression, it is suggested that E-box binding to the F1/GAP-43 promoter in hippocampal granule cells could negatively regulate F1/GAP-43 gene expression. Indeed, analysis of recognition elements on the F1/GAP-43 gene revealed an arrangement, previously described in other genes, of multiple adjacent E-box elements. E-box binding of bHLH transcription factors is likely to occur on several different genes in addition to F1/GAP-43. It is, therefore, attractive to think that E-box binding is regulated by in vivo activation of the adult brain and that this gene regulatory event participates in the orchestration of molecular and cellular responses underlying axonal growth.

Amino Acid Sequence↗
PubMed1995

Protein F1/GAP-43 and PKC gene expression patterns in hippocampus are altered 1-2 h after LTP.

Three days after long-term potentiation (LTP) there is a decrease in the gene expression of protein F1 (GAP-43) and gamma-PKC in CA3 pyramidal cells that is correlated with the magnitude of LTP. We predicted these decreases would be preceded by an increment in gene expression. At 1 h, but not at 2 h after LTP, F1/GAP-43 and gamma-PKC mRNA hybridization were increased, but increases were also observed after control stimulation. At both 1 and 2 h after LTP, changes in F1/GAP-43 hybridization were positively correlated with gamma-PKC hybridization and negatively correlated with LTP magnitude. These data indicate that correlated alterations in F1/GAP-43 gene expression and synaptic efficacy can occur as early as 1 h after LTP and persist for days.

Animals↗
PubMed1995

Long-term potentiation in vivo in the intact mouse hippocampus.

We describe the characteristics of long-term potentiation (LTP) in the intact mouse. Perforant path stimulation evokes both a population excitatory postsynaptic potential (pop-EPSP) and a population spike potential (pop-spike) from the hippocampal dentate gyrus in urethane anesthetized animals. LTP, as measured by increased pop-spike amplitude and pop-EPSP slope, was successfully induced and reliably maintained at a stable level for at least 12 h, the longest time tested. The LTP-inducing stimulus (3 trains of 400 Hz, 8 0.4 ms pulses/train) used in two strains of mice was less by half than that used in rat. These parameters for inducing LTP were also successfully applied to obtain LTP in two different transgenic mouse strains: one bearing a F1/Gap-43 promoter-lacZ fusion gene and another which overexpresses the S100 beta gene. We also examined the effects of protein synthesis inhibitors, cycloheximide (CXM) and anisomycin (ANI). When CXM or ANI was given 30 min before LTP induction, there was no persistent loss of LTP at the 4 h time point. However, if CXM was given 4 h before LTP induction, significant decay of the potentiated responses occurred 90 min after induction. Half of the animals receiving CXM but not ANI showed a complete and sudden elimination of the entire response after the LTP-inducing stimulus. It was speculated that loss of a constitutively-expressed housekeeping protein, for example a calcium buffering protein, with an estimated half-life of 2 h would lead to an inability to buffer LTP-induced alterations, such as intracellular calcium elevation, increasing intracellular calcium to toxic levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

NMDA receptor blockade prevents kainate induction of protein F1/GAP-43 mRNA in hippocampal granule cells and subsequent mossy fiber sprouting in the rat.

Granule cells in the adult rat hippocampus do not constitutively express the growth-related axonal protein F1 (a.k.a. B-50, GAP-43, neuromodulin, pp46), yet kainic acid (KA) can induce extensive growth of granule cell axons, the mossy fibers, into the supragranular layer. Does this KA-induced growth occur in the absence of protein F1/GAP-43? Using quantitative in situ hybridization, we found that 16-24 h after KA (10 mg/kg, s.c.) F1/GAP-43 mRNA was in fact induced in granule cells and remained elevated above control levels for at least 20 days. The induction of F1/GAP-43 mRNA in granule cells was blocked either by MK-801 or pentobarbital pretreatment. If pentobarbitol was given 55 min, but not 90 min, after KA, F1/GAP-43 mRNA was also blocked. Since induction of F1/GAP-43 occurred when pentobarbitol was given 90 min after KA, a 35 min window of activation is required, beyond the initial 55 min, for F1/GAP-43 mRNA induction. As both MK-801 and pentobarbital blocked behavioral seizures their anti-convulsant action may be important for blocking F1/GAP-43 mRNA induction. Mossy fiber sprouting observed 30 days after KA was also blocked when either MK-801 or pentobarbital was given prior to KA. These results are consistent with the proposal that protein F1/GAP-43 promotes axonal growth in the adult brain in an input-dependent manner, and may also be of clinical relevance to the molecular mechanisms underlying structural remodeling in epilepsy.

Animals↗
PubMed1994

Glial-derived S100b protein selectively inhibits recombinant beta protein kinase C (PKC) phosphorylation of neuron-specific protein F1/GAP43.

Protein F1/GAP43 is neuron-specific, associated with neurite outgrowth during development and a substrate for PKC. This protein is present in high levels in serotonergic neurons which in culture sprout in response to the glial-derived S100b, the beta-beta homodimer. As an initial step in determining whether S100b acts on F1/GAP43 we studied the regulation by S100b of PKC phosphorylation of F1/GAP43. Either the S100b or a mixture of S100a and S100b, both from a brain glial cell source, inhibited in vitro phosphorylation of purified F1/GAP43 by purified PKC in a dose-dependent manner. Using recombinant PKC subtypes, purified S100b preferentially inhibited the F1/GAP43 phosphorylation by the beta subtype. The IC50 of S100b for beta I and beta II PKC was 8 microM while for alpha and gamma PKC it was 64 microM. S100b inhibition was thus subtype-selective. Histone III-S phosphorylation by the four PKC subtypes was not inhibited by S100b. S100b inhibition was thus substrate-selective. Moreover, the effect of S100b on phosphorylation could not be explained by a direct inhibition of kinase activity. Together with earlier studies implicating a role for S100 in synaptic plasticity and neurite outgrowth, the present results suggest that S100b may regulate such functions through its inhibition of neuron-specific PKC substrate (F1/GAP43) phosphorylation. The regulation of this neuron-specific substrate phosphorylation by glial S100 suggests the potential for a novel neuro-glial interaction. Finally, the location of S100 gene on chromosome 21, trisomic in Down's syndrome, and over-expressed in this disorder, as well as in Alzheimer's disease, suggests a link to cognitive impairments in human.

Animals↗
PubMed1993

Protein kinase C and F1/GAP-43 gene expression in hippocampus inversely related to synaptic enhancement lasting 3 days.

The mRNA levels of protein F1 (also known as GAP-43), and protein kinase C (PKC) subtypes were measured 3 days after the induction of long-term enhancement (also known as long-term potentiation) in the hippocampus of chronically prepared conscious rats by quantitative in situ hybridization. Altered mRNA levels correlated significantly with alternations in synaptic efficacy; such correlations have not been reported previously. Rats with greater synaptic enhancement had lower gene expression in the CA3 subfield of F1/GAP-43 and both beta-PKC and gamma-PKC, but not alpha-PKC. For microtubule-associated protein 2 (MAP-2), neurogranin, and the glutamate receptor subtype B-flip, no correlation was observed in any cell field between synaptic enhancement and hybridization to the mRNA. To our surprise, alterations in mRNA levels of F1/GAP-43 and gamma-PKC were highly correlated (r = +0.928, P < 0.001), suggesting coordinate regulation. Since F1/GAP-43 is associated with neurite growth, its lowered expression at 3 days would reduce potential growth, leading to synaptic stabilization. We propose that long-term synaptic change is mediated by gene expression of the very same proteins initially modified posttranslationally.

Animals↗
PubMed1993

Learning selectively increases protein kinase C substrate phosphorylation in specific regions of the chick brain.

The effect of imprinting, an early form of exposure learning, on the phosphorylation state of the protein kinase C substrates myristoylated alanine-rich C-kinase substrate (MARCKS) and protein F1/43-kDa growth-associated protein (F1/GAP-43) was studied in two regions of the chick forebrain. One region, the intermediate and medial part of the hyperstriatum ventrale (IMHV), is probably a site of long-term memory; the other, the wulst, contains somatic sensory and visual projection areas. After imprinting, a significant increase in MARCKS protein phosphorylation was observed in the left IMHV but not the right IMHV. No significant alteration in F1/GAP-43 was observed in IMHV. MARCKS was resolved into two acidic components of pI approximately 5.0 and approximately 4.0. Phosphorylation of the pI approximately 5.0 MARCKS but not the pI approximately 4.0 MARCKS was significantly altered by imprinting. The partial correlation between preference score (an index of learning) and phosphorylation, holding constant the effect of approach activity during training, was significant only for the pI approximately 5.0 MARCKS in the left IMHV. A significant negative partial correlation between preference score and F1/GAP-43 phosphorylation in the right wulst was observed. Because the imprinting-induced alteration in MARCKS is selective with respect to phosphoprotein moiety, hemispheric location, and brain region, we propose that these alterations may be central to the learning process.

Animals↗
PubMed1993

Induction of F1/GAP-43 gene expression in hippocampal granule cells after seizures [corrected].

In the adult rat hippocampus mRNA of F1/GAP-43, an axonal growth-associated protein, is highly expressed in pyramidal cells, but is absent in granule cells. To determine whether granule cells can be induced to express mRNA of F1/GAP-43, transcript levels were studied after limbic seizures, which can induce sprouting of granule cell mossy fibers. Seizure-inducing electrolytic lesions were made in the dentate gyrus hilus with stainless-steel electrodes and mRNA levels were measured in contralateral hippocampus by quantitative in situ hybridization. Induction of F1/GAP-43 mRNA expression was observed in granule cells at 24 h, but not at 6 or 12 h, after the hilar lesion. When equivalent sized hilar lesions were made with platinum electrodes, which do not induce seizures, no hybridization was apparent over the granule cells. Hybridization over granule cells had declined by 48 h post-lesion, but even at 10 days it was still slightly higher than in control rats. F1/GAP-43 mRNA expression was also increased 2-fold in CA1 pyramidal cells with peak expression at 48 h post-lesion. These are the first data to our knowledge that demonstrate that F1/GAP-43 gene expression can be altered in neurons located within the adult brain. Induction of F1/GAP-43 mRNA expression in the granule cells may be important for the sprouting of mossy fibers and could be triggered by the elevated levels of brain-derived neurotrophic factor in CA3 cells which precede the increased F1/GAP-43 gene expression in granule cells.

Animals↗
PubMed1993

Brief exposure to a novel environment enhances binding of hippocampal transcription factors to their DNA recognition elements.

The behavioral regulation of transcription factor function in hippocampus, a brain region activated by novelty and important for information storage, has not been previously studied. Sixty min after a 4 min exploration of a novel space by adult albino rats, electrophoretic mobility shift assays revealed a selective increase in specific binding to particular consensus recognition elements. For both AP-1 and CRE, an upper and lower band were observed in the electrophoretic mobility shift assay. The upper band showed increased binding restricted to the 60 min time point after exploration; no increase was observed 30 min or less, or 120 min or more after the novel experience. Increased binding to the lower of either AP-1 or CREB was observed at 30 and 120 min. When the time of exploration was increased from 4 to 15 min, no alterations in either the upper or lower band were observed in the transcription factor binding to either the AP-1 or the CRE element. Since the animals ceased to explore the environment after 10 min, the lack of binding may reflect behavioral habituation, leading to transcription factor deactivation. We conclude that brief, naturalistic stimulation can activate brain transcription factors in a time-delimited fashion, suggesting post-translational control of protein-DNA binding. We propose that promoter elements of target genes critically involved in the storage of information are turned on by environmentally-activated transcription factors.

Animals↗
PubMed1992

Postsynaptic then presynaptic protein kinase C activity may be necessary for long-term potentiation.

Protein kinase C inhibitor was injected intracellularly by iontophoresis into CA1 somata either before or after long-term potentiation in the hippocampal slice preparation. Two different protein kinase C inhibitors, polymyxin B (PMXB) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), injected 10 min before long-term potentiation induction caused potentiated responses to return to baseline 15-35 min after induction without significantly affecting the initial magnitude of potentiation. There was no effect on long-term potentiation persistence when H-7 or PMXB was injected intracellularly 5 min after long-term potentiation induction. In contrast, focal extracellular micro-pressure ejection of protein kinase C inhibitor in the stratum radiatum, 15 or 30 min, but not 60 min after long-term potentiation induction caused decay of long-term potentiation to baseline. This is probably a presynaptic action since intracellular inhibitors injected postsynaptically were ineffective 5 min after long-term potentiation induction. Focal application to stratum pyramidale produced a weaker decay than to stratum radiatum suggesting a Schaffer collateral presynaptic terminal site of action. We propose that activation of postsynaptic protein kinase C activity is necessary for long-term potentiation persistence but this activity persists for less than 5 min after induction. Presynaptic protein kinase C activity is also necessary for persistence and is time-limited to less than 60 min. It is attractive to think that these two events are sequentially activated and employ different protein kinase C subtypes differentially localized to presynaptic or postsynaptic elements.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Phosphorylation of pyruvate dehydrogenase in the hippocampal slice: time course of response to cellular depolarization.

In the rat hippocampal slice, we evaluated the effects of potassium depolarization on phosphorylation of the alpha subunit of the pyruvate dehydrogenase complex (E1 alpha, mol. wt. approximately 41 kDa) using an in vitro back-titration assay. The in vitro back-titration phosphorylation of El alpha was maximal following 30 s of depolarization and remained elevated for up to 3 min following termination of the depolarizing pulse. Possible significance of and mechanisms for the depolarization-induced changes in E1 alpha phosphorylation are discussed.

Animals↗
PubMed1991

Contrasting patterns of protein phosphorylation in human normal and Alzheimer brain: focus on protein kinase C and protein F1/GAP-43.

We introduce a new procedure to study kinase substrates in postmortem human brain. By adding purified exogenous protein kinase C (PKC) and the phospholipid phosphatidylserine to brain homogenates in vitro we are able to analyze PKC substrates. A human 53-kDa phosphoprotein is described that appears to be homologous to rat and monkey protein F1 (GAP-43). This identity is based on molecular weight, isoelectric point, phosphorylation by exogenous protein kinase C, enhancement of its phosphorylation by three activators (phospholipids, calcium and phorbol esters), phosphopeptide maps, and cross-reactivity with an antibody raised against rat protein F1. Protein F1 is a PKC substrate associated with synaptic plasticity and nerve growth. Its phosphorylation in rat brain has been correlated with long-term potentiation, an electrophysiological model of memory. In the present study of normal brain, human protein F1 shows an occipitotemporal in vitro phosphorylation gradient. This is consistent with previous observations in nonhuman primates. This gradient is less pronounced in Alzheimer's disease (AD). Changes in the in vitro phosphorylation pattern of three other non-PKC substrates in Alzheimer's disease, including one with characteristics similar to microtubule-associated protein tau, are also reported. These results suggest that protein phosphorylation can be studied in postmortem human brain and that PKC-mediated phosphorylation of protein F1, already linked to synaptic plasticity and memory, may be altered in AD.

Alzheimer Disease↗