Aryeh Routtenberg

GAP-43: an intrinsic determinant of neuronal development and plasticity

Several lines of investigation have helped clarify the role of GAP-43 (FI, B-50 or neuromodulin) in regulating the growth state of axon terminals. In transgenic mice, overexpression of GAP-43 leads to the spontaneous formation of new synapses and enhanced sprouting after injury. Null mutation of the GAP-43 gene disrupts axonal pathfinding and is generally lethal shortly after birth. Manipulations of GAP-43 expression likewise have profound effects on neurite outgrowth for cells in culture. GAP-43 appears to be involved in transducing intra- and extracellular signals to regulate cytoskeletal organization in the nerve ending. Phosphorylation by protein kinase C is particularly significant in this regard, and is linked with both nerve-terminal sprouting and long-term potentiation. In the brains of humans and other primates, high levels of GAP-43 persist in neocortical association areas and in the limbic system throughout life, where the protein might play an important role in mediating experience-dependent plasticity.

A membrane phosphoprotein associated with neural development, axonal regeneration, phospholipid metabolism, and synaptic plasticity

Abstract It has now been shown that a membrane phosphoprotein initially studied independently by several laboratories and given different designations, is in fact the same protein. Thus, the growth-associated proteins, GAP-43 and GAP-48, a phosphoprotein of the growth cone, pp46, a C-kinase substrate related to phosphatidylinositol turnover in synaptic membranes, B-50, and a C-kinase substrate associated with hippocampal long-term potentiation, F1, are identical. This protein may thus play a general role in the formation of synaptic relationships during development or regeneration, and a continuing role in the functional modulation of certain synapses throughout life.

Subfornical Organ: Site of Drinking Elicitation by Angiotensin II

Angiotensin II applied directly to the subfornical organ in a dose as small as 0.1 nanogram elicited short-latency drinking behavior in water-sated rats. Lesions in the body of this structure blocked drinking induced by angiotensin II applied to the basal telencephalon (including preoptic area). These results call attention to the subfornical organ as an important central nervous structure involved in the conrol of drinking behavior.

The role of protein kinase C in long-term potentiation: a testable model

With the use of appropriate reagents, LTP may be divided into at least two stages, induction and maintenance. Induction of LTP is dependent upon the activation of the NMDA receptor, and the consequent influx of calcium into the postsynaptic cell. Both correlational evidence (measures of PKC activity, protein F1 phosphorylation, and PI turnover) and interventive evidence (application of PKC inhibitors and activators) indicate that PKC activation is necessary for maintenance of the LTP response. An important regulatory pathway for PKC activation is the liberation of c-FAs from membrane phospholipids by PLA2. In LTP, activation of this pathway may stabilize PKC in an activated state, and thus contribute to maintenance of the potentiated response. LTP maintenance could result from presynaptic alteration (increased neurotransmitter release), postsynaptic alteration (increases in receptor number or sensitivity, or alterations of postsynaptic morphology), synapse addition, or any of these processes in combination. If LTP maintenance is mediated by presynaptic alteration, as has been indicated by measurement of glutamate release, then one must posit a signal that travels from the postsynaptic to the presynaptic membrane to activate presynaptic PKC. Alternatively, if LTP maintenance is mediated by postsynaptic alteration, a signal contained within the dendritic spine would suffice to activate postsynaptic PKC-mediated maintenance processes. We suggest that the contributions of presynaptic and postsynaptic processes to LTP maintenance may be determined by the differential distribution of PKC subtypes and substrates among hippocampal synaptic zones.

Direct activation of purified protein kinase C by unsaturated fatty acids (oleate and arachidonate) in the absence of phospholipids and Ca2

Unsaturated fatty acids (oleic acid and arachidonic acid) activate purified protein kinase C independently of phospholipid and Ca2+. Oleic acid activation of protein kinase C is as effective as phosphatidylserine and Ca2+. K a, values for oleic acid and arachidonic acid are 50 and 53 μM, respectively. In contrast to the cis fatty acids, a trans form (elaidic acid) or a saturated fatty acid (stearic acid) has little or no effect on protein kinase C activation. If cis fatty acid liberation is physiologically important, this suggests that another mechanism may exist for protein kinase C activation, in addition to phospholipase C/phosphatidylinositol turnover signaling, possibly via the liberation of cis fatty acids by the Ca2+‐dependent phospholipase A2 system.

Protein kinase C inhibitors eliminate hippocampal long-term potentiation

Recent findings suggest that protein kinase C (PKC) regulates the persistence of long-term potentiation (LTP). To test the hypothesis that PKC inhibition would decrease persistence of potentiation we applied PKC inhibitors (mellitin, polymyxin B, H-7) by micropressure ejection to the intact hippocampus either before or after LTP induction. When inhibitor was given 15 min before LTP, initial potentiation was unaffected, yet responses decayed to baseline levels by 60 min after the onset of potentiation. PKC inhibitor treatment 10 min after LTP onset induced decay of responses to pre-LTP baseline levels within 50 min of ejection. Inhibitor applied 60 min after LTP onset induced substantial decay but not to baseline levels. Potentiation was unaffected by inhibitor treatment 4 h after the induction of LTP. Measurement of PKC subcellular distribution revealed that inhibitor significantly reduced the proportion of PKC associated with the membrane. These findings represent the first demonstration that PKC inhibitors prevent persistence of potentiation. They also suggest that PKC regulates the persistence of synaptic enhancement beginning after its onset, and that PKCs role decreases with time after the induction of enhancement.

Gene expression of the transcription factor NF-κ 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.

Intracranial chemical injection and behavior: a critical review

The focus of this review of intracranial chemical injection (ICI) is on the methods used and how variations in the use of these methods affect the results obtained. A brief historical review indicates that investigators at the turn of the century were concerned with the local nature of ICI. Since this problem remains at this time, the logic of the protocols then used is considered applicable to present problems. After a review of the available methods, a checklist containing a standard set of recommended procedures is provided. These recommendations do not depart from current practices, but serve to identify sources of variability among experiments that could readily be reduced. A consideration of the sphere of influence of ICI focuses on the evaluation of the authors ventricular hypothesis. This hypothesis, as originally formulated, called attention to the potential of spread from sites of ICI to the ventricular system. This would blur the anatomical localization which is one of the major virtues of the ICI method. It is considered necessary to expand the view of spread from the application site to include an important contribution of the vasculature. The failure to find species generality is taken as one indicant that the critical sites of action of cholinergic agents which provoke drinking have not been discovered. Recent evidence indicates that the subfornical organ may be one important site of carbachol-induced drinking. Such evidence does not deny the possibility of a “thirst circuit” but it is clear that such a circuit has not been adequately defined. Discrepancies concerning the alpha- and beta-adrenergic systems involved in feeding and the influence of dopamine applied to the striatum on motor behavior is briefly reviewed in terms of the different methods used.

A selective increase in phosphorylation of protein F1, a protein kinase C substrate, directly related to three day growth of long term synaptic enhancement

Increased in vitro phosphorylation of the 47 kdalton, 4.5 pI protein F1 was observed in dorsal hippocampal tissue from animals exhibiting long term enhancement (LTE) three days after high frequency stimulation of the perforant pathway, as compared to tissue from low frequency stimulated controls or from unoperated animals. The increase in protein F1 phosphorylation was related to LTE rather than simple activation of perforant path-dentate gyrus synapses. This is the first report of a change in brain protein phosphorylation accompanying synaptic enhancement lasting days. The extent of growth of LTE over the three days following stimulation was directly related (r = +0.66, P less than 0.05) to protein F1 phosphorylation. Among the phosphoproteins studied this relationship between LTE and phosphorylation was selective for protein F1. This suggests that protein F1 may regulate growth of synaptic plasticity for at least a three day period. The mechanism for the LTE-related increase in protein F1 phosphorylation has not been established. However, recent evidence from this laboratory indicates: that protein F1 is phosphorylated by the calcium/phospholipid-dependent protein kinase C; and that kinase C is activated 1 h after LTE. Therefore, the increase in protein F1 phosphorylation following LTE may result from long term activation of protein C kinase.

Protein kinase C phosphorylates a 47 Mr protein (F1) directly related to synaptic plasticity

Ca2+-phospholipid-dependent protein kinase C, and activators of protein kinase C (phosphatidylserine, phorbol esters) stimulate the in vitro phosphorylation of a 47 kdalton phosphoprotein (protein F1) previously shown (Routtenberg, Lovinger and Steward, Behav. neural Biol., 43 (1985) 3-11) to be directly related to the plasticity of long-term potentiation. These data indicate that protein F1 serves as a protein kinase C substrate, and suggest the hypothesis that protein kinase C is involved in processes of long-term potentiation.