Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts.

Although both c-Ret and GFRalpha1 are required for responsiveness to GDNF, GFRalpha1 is widely expressed in the absence of c-Ret, suggesting alternative roles for "ectopic" sites of GFRalpha1 expression. We show that GFRalpha1 is released by neuronal cells, Schwann cells, and injured sciatic nerve. c-Ret stimulation in trans by soluble or immobilized GFRalpha1 potentiates downstream signaling, neurite outgrowth, and neuronal survival, and elicits dramatic localized expansions of axons and growth cones. Soluble GFRalpha1 mediates robust recruitment of c-Ret to lipid rafts via a novel mechanism requiring the c-Ret tyrosine kinase. Activated c-Ret associates with different adaptor proteins inside and outside lipid rafts. These results provide an explanation for the tissue distribution of GFRalpha1, supporting the physiological importance of c-Ret activation in trans as a novel mechanism to potentiate and diversify the biological responses to GDNF.

Participation of hippocampal metabotropic glutamate receptors, protein kinase A and mitogen-activated protein kinases in memory retrieval.

The ability to recall past events is a major determinant of survival strategies in all species and is of paramount importance in determining our uniqueness as individuals. In contrast to memory formation, the information about the molecular mechanisms of memory retrieval is surprisingly scarce and fragmentary. Here we show that pretest inhibition of the specific upstream activator of mitogen-activated protein kinase kinase, or of protein kinase A in the hippocampus, blocked retrieval of long-term memory for an inhibitory avoidance task, a hippocampal-dependent learning task. An activator of protein kinase A enhanced retrieval. Mitogen-activated protein kinase activation increased in the hippocampus during retrieval, while protein kinase A activity remained unchanged. Pretest intrahippocampal blockade of metabotropic glutamate receptors or alpha-amino-3-hydroxy-5-methyl-4-isoxazolone propionic acid/kainate receptors, but not N-methyl-D-aspartate receptors or calcium/calmodulin dependent-protein kinase II, impaired retrieval. Thus, recall of inhibitory avoidance activates mitogen-activated protein kinase, which is necessary, along with metabotropic glutamate receptors, alpha-amino-3-hydroxy-5-methyl-4-isoxazolone propionic acid/kainate receptors, and protein kinase A, for long-term memory expression. Our results indicate that memory formation and retrieval may share some molecular mechanisms in the hippocampus.

Decreased GAP-43/B-50 phosphorylation in striatal synaptic plasma membranes after circling motor behavior during development.

We evaluated the in vitro phosphorylation of the presynaptic substrate of protein kinase C (PKC), GAP-43/B-50 and the PKC activity in the striatum of rats submitted to a circling training (CT) test during postnatal development. Motor activity at 30 days of age, but not at other ages, produced a unilateral reduction (-29.5%; p<0.001) in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum with respect to the ipsilateral side, while non-trained control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of trained animals also showed a significant reduction (-29.3%; p<0. 001) in the incorporation of 32P-phosphate into GAP-43. This decreased in vitro GAP-43 phosphorylation was seen at 30 min, but not immediately after circling motor behavior. This contralateral change in GAP-43 phosphorylation correlated with the running speed developed by the animals [(r=0.9443, p=0.0046, n=6, relative to control group) and (r=0.8813, p=0.0203, n=6, with respect to the ipsilateral side of the exercised animals)]. On the contrary, GAP-43/B-50 immunoblots did not show changes in the amount of this phosphoprotein among the different experimental groups. Back phosphorylation assays, performed in the presence of bovine purified PKC, increased the level of GAP-43/B-50 phosphorylation in the striatum contralateral to the sense of turning [(+22%; p<0.05, with respect to ipsilateral side of the same trained group) and (+21%; p<0.05, relative to control group)]. Taken together, these results demonstrate that the activity developed in the CT test induces a reduction in the phosphorylation state of GAP-43/B-50 in the specific site for PKC. We conclude that general markers of activity-dependent neuronal plasticity are also altered in the same period that long-lasting changes in striatal neuroreceptors are triggered by circling motor behavior.

Experience-dependent decrease in synaptically localized Fra-1.

The Fos family of transcription factors has been repeatedly shown to participate in the long-term neural responses associated with a variety of physiological stimuli, including activity-dependent plastic processes. Quite recently, several transcription factors have been found in synaptic regions, localized in dendrites and presynaptic terminals. Here we show that the transcription factor Fos-related antigen-1 (Fra-1) was detected in synaptosomes (Syn) and synaptic plasma membrane (SPM) fractions from the rat cerebral cortex and hippocampus as a single band migrating with M(r) 42-43 kDa. The 55-kDa c-Fos protein was also detected in syn and SPM fractions. Conversely, the inducible 62-65-kDa c-Fos is present in nuclear fractions from metrazole-treated animals (positive control), but not in Syn or SPM fractions. Furthermore, no Fra-2, Fos B or c-Jun immunoreactivities were detected in these same synaptic regions. DNA-mobility shift assays showed the presence of specific AP-1 binding activity in synaptic protein extracts. Immunoelectronmicroscopic analysis of cortical and hippocampal tissues revealed that Fra-1 and Fos-like immunoreactivities are localized in association with presynaptic plasma membranes. One trial inhibitory avoidance training, a hippocampal-dependent task, is associated with a time-dependent decrease (-31%) in Fra-1, but not in 55-kDa c-Fos, levels in hippocampal SPM fractions. In hippocampal homogenates, we do not detect significant changes in Fra-1 immunoreactivity, suggesting that this behavioural experience is probably accompanied by a subcellular redistribution of Fra-1 protein. These results suggest that Fra-1 may participate in the communication between synapse and the nucleus and in experience-dependent hippocampal plasticity.

Learning-associated activation of nuclear MAPK, CREB and Elk-1, along with Fos production, in the rat hippocampus after a one-trial avoidance learning: abolition by NMDA receptor blockade.

It is widely accepted that the formation of long-term memory (LTM) requires neuronal gene expression, protein synthesis and the remodeling of synaptic contacts. From mollusk to mammals, the cAMP/PKA/CREB signaling pathway has been shown to play a pivotal role in the establishment of LTM. More recently, the MAPK cascade has been also involved in memory processing. Here, we provide evidence for the participation of hippocampal PKA/CREB and MAPK/Elk-1 pathways, via activation of NMDA receptors, in memory formation of a one-trial avoidance learning in rats. Learning of this task is associated with an activation of p44 and p42 MAPKs, CREB and Elk-1, along with an increase in the levels of the catalytic subunit of PKA and Fos protein in nuclear-enriched hippocampal fractions. These changes were blocked by the immediate posttraining intra-hippocampal infusion of APV, a selective blocker of glutamate NMDA receptors, which renders the animals amnesic for this task. Moreover, no changes were found in control-shocked animals. Thus, inhibitory avoidance training in the rat is associated with an increase in the protein product of an IEG, c-fos, which occurs concomitantly with the activation of nuclear MAPK, CREB and Elk-1. NMDA receptors appear to be a necessary upstream step for the activation of these intracellular cascades during learning.

Co-alteration of dopamine D2 receptor and muscarinic acetylcholine receptor binding in rat striatum after circling training.

CIRCLING training (CT) decreases muscarinic acetyl-choline receptor (mAchR) binding in rat striatum. As cholinergic and dopaminergic systems interact strongly we evaluated the expression of D2-subtype dopamine receptor (DA D2) and mAchR together after CT. Animals trained from 30 to 37 days of age and sacrificed 2 months later showed an enduring drop in Bmax of 40% in DA D2 and 34% in mAchR. Plotting the percentage of binding drop of both receptors for each animal showed that the reduction of one system correlates with the other (r2 = 0.71, p < 0.01; n = 8). Neither mAchR nor DA D2 were affected when training started at 20 or 60 days. We conclude that the presence of a period where CT exerts long term alterations during development involves both cholinergic and dopaminergic systems.

Permanent alteration of muscarinic acetylcholine receptor binding in rat striatum after circling training during development.

We evaluated the effect of circling training (CT) in the expression of muscarinic acetylcholine receptor (mAchR) in developing rat striatum. For this, male and female rats were subjected to CT at 20, 30, 40 and 60 days of age during 7 days. Animals trained at 30 days but not at other ages showed an average decreased binding to mAchR of 33% in males and 24% in females, representing a significant difference with respect to control non-trained animals (males P < 0.001, females P < 0.005), and showing also a differential response between sex (P < 0.01). mAchR drop was found invariably either 2 months or 1 year after training indicating a long term plastic change due to circling training. Scatchard analysis showed that altered binding represents a variation of the total receptor number instead of its binding affinity, with no significant differences found among Kd (P > 0.1). mAchR variation was correlated with the motor performance accomplished in the test. Regarding total distance run, male rats trained for 3 days (300 meters. run), for 5 days (600 meters) and for 7 days (900 meters) showed a drop of 19, 28 and 33% respectively (r2 = 0.91, P < 0.001), while female changes were of 21, 23 and 24% (r2 = 0.78, P < 0.001). Nevertheless, no correlation with running speed was found (r2 = 0.13 male, r2 = 0.02 female; P > 0.1). In summary, these results demonstrate the presence of a limited sensitivity period during striatum development where mAchR expression may be affected by the activity performed during CT, representing a permanent alteration of the receptor levels.

Involvement of hippocampal PKCbetaI isoform in the early phase of memory formation of an inhibitory avoidance learning.

Several evidences demonstrate that protein kinase C (PKC) is involved in hippocampal long-term potentiation (LTP) and in different forms of learning, including inhibitory avoidance training in rats. Here, we evaluated the levels of conventional PKC isozymes (alpha, betaI, betaII, gamma) in synaptic plasma membrane (SPM) fractions isolated from hippocampus of rats subjected to a one-trial inhibitory avoidance paradigm. At 0, 30 and 120 min after training, there was a significant increase in the total amount of PKCbetaI. Densitometric analysis of the immunoblots showed an increase of 142+/-11% at 0 min, 193+/-16% at 30 min and 156+/-6% at 120 min after training relative to shocked control values. No changes were found in PKCbetaI levels in SPM fractions of the shocked animals relative to naive control values. No training-specific increments in the levels of PKCalpha, betaII and gamma were observed at any time point tested. However, an increase in PKCgamma levels was found in trained and shocked animals sacrificed 120 min after each experimental procedure. In addition, bilateral microinjections of a fairly selective inhibitor of PKCbetaI isozyme into the CA1 of the dorsal hippocampus produced amnesia when given 10 min before training, or 50, 110, but not 170 min, after training. Thus, the present findings demonstrate the participation of PKCbetaI in the early synaptic events responsible for the acquisition and consolidation of an inhibitory avoidance learning, and suggest a putative role of this presynaptic isozyme on the enhanced PKC-dependent B-50/GAP-43 phosphorylation previously detected by us during this associative learning.

Cold stress related alteration of RNA biosynthesis in brain cortex of mother-deprived newborn rats.

We studied the influence of maternal deprivation on the RNA biosynthesis in the brain cortex of 10 day-old rats. Mother-deprived pups, placed at 25 degrees C showed a reduction in body temperature of 6 +/- 1 degree C. After mother retrieval, RNA biosynthesis decreased 27% and 34% in total brain cortex and in isolated neurons, respectively. This fall is proportional to the body temperature reduction and can be avoided placing the pups at 37 degrees C immediately after the separation. Rethermostatization of offsprings, after one hour at 25 degrees C, showed an overshoot of RNA biosynthesis (145%) with further stabilization of synthesis rates to normal levels after 100 min. This classical physiological mechanism was further studied in vitro. Comparing in vivo and in vitro experiments, it is concluded that overshooting can not be observed in vitro if temperature reduction was not previously performed in vivo. Thus, this phenomenon seems to respond to humoral factors in order to be triggered. Afterwards, in vitro overshooting following cold stress in vivo, demonstrates that the depressed tissue by itself has the capability to turn back to normal RNA levels in the same way as observed in vivo.

The molecular basis of the self/nonself selectivity of a coelenterate toxin.

Coelenterates produce potent hemolysins inhibited by sphingomyelin (SM). Remarkably, instead of this lipid, their membranes contain a phosphono analogue of it. Using coelenterolysin (CL), a toxin produced by the sea anemone Phymactis clematis, we have examined a possible connection between these two peculiar traits. Our experiments showed that, while SM binds this lysin and inhibits its hemolytic activity, the endogenous PnSL do neither. In addition, liposomes made of bovine erythrocyte lipids are rapidly disrupted by CL, while those made of P. clematis lipids are completely resistant to it. However, if small amounts of SM are added to the P. clematis lipids, the resulting liposomes become sensitive to CL. Taken together, our results show for the first time that substitution of SM by its phosphono analogue is the molecular basis for the selectivity of an anthozoan toxin. We therefore propose that exotoxin production and membrane composition are coadapted traits that confer on the coelenterates a significant evolutionary advantage.

Decreased phosphorylation of GAP-43/B-50 in striatal synaptic plasma membranes after circling motor activity.

The effects of spontaneous circling motor activity on the in vitro phosphorylation of the protein kinase C substrate GAP-43/B-50 was studied on striatal membranes of developing rats (30 days of age). At this time of postnatal development, permanent plastic changes in cholinergic and dopaminergic systems are produced by physiological motor activity. Exercised animals showed a significant reduction of 31% in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum respect to the ipsilateral side (P < 0.01), while control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of exercised animals showed a 33% reduction in the incorporation of 32P-phosphate into GAP-43/B-50 30 minutes post-exercise (P < 0.01). This change in GAP-43/B-50 phosphorylation was correlated with the running speed developed by the animals (r:0.8986, P = 0.015). GAP-43/B-50 immunoblots revealed no changes in the amount of this protein in any group. Moreover, a significant variation of 25% (P < 0.05) in the PKC activity was seen between both exercised striata. Interhemispheric differences were not found in control animals. We conclude that endogenous phosphorylation of this protein is also altered by motor activity in the same period that permanent changes in striatal neuroreceptors are triggered after motor training.

Nerve growth factor preserves a critical motor period in rat striatum.

We previously found the occurrence of a critical motor period during rat postnatal development where circling training starting the 7-day schedule at 30 days-but not before or after-induces a lifetime drop in the binding to cholinergic muscarinic receptors (mAChRs) in striatum. Here, we studied whether nerve growth factor (NGF) participates in this restricted period of muscarinic sensitivity. For this purpose, we administered mouse salival gland 2.5S NGF (1.4 or 0.4 microg/day, infused by means of ALZA minipumps) by intrastriatal unilateral route between days 25 and 39, and then trained rats starting at 40 days. Under these conditions, NGF induced a long-term reduction in the striatal [3H] quinuclidilbenzylate (QNB) binding sites despite the fact that motor training was carried out beyond the natural critical period. Thus, at day 70, measurement of specific QNB binding in infused striata of trained rats showed decreases of 42% (p < .0004) and 33% (p < .02) after administration of the higher and lower NGF doses, respectively, with respect to trained rats treated with cytochrome C, for control. Noncannulated striata of the NGF-treated rats also showed a decrease in QNB binding sites (44%; p < .0001) only at the higher infusion rate. This effect was not found in the respective control groups. Our observations show that NGF modulates the critical period in which activity-dependent mAChR setting takes place during rat striatal maturation.

B-50/GAP-43 phosphorylation and PKC activity are increased in rat hippocampal synaptosomal membranes after an inhibitory avoidance training.

Several lines of evidence indicate that protein kinase C (PKC) is involved in long-term potentiation (LTP) and in certain forms of learning. Recently, we found a learning-specific, time-dependent increase in [3H]phorbol dibutyrate binding to membrane-associated PKC in the hippocampus of rats subjected to an inhibitory avoidance task. Here we confirm and extend this observation, describing that a one trial inhibitory avoidance learning was associated with rapid and specific increases in B-50/GAP-43 phosphorylation in vitro and in PKC activity in hippocampal synaptosomal membranes. The increased phosphorylation of B-50/GAP-43, was seen at 30 min (+35% relative to naive or shocked control groups), but not at 10 or 60 min after training. This learning-associated increase in the phosphorylation of B-50/GAP-43 is mainly due to an increase in the activity of PKC. This is based on three different sets of data: 1) PKC activity increased by 24% in hippocampal synaptosomal membranes of rats sacrificed 30 min after training; 2) B-50/GAP-43 immunoblots revealed no changes in the amount of this protein among the different experimental groups; 3) phosphorylation assays, performed in the presence of bovine purified PKC or in the presence of the selective PKC inhibitor CGP 41231, exhibited no differences in B-50/GAP-43 phosphorylation between naive and trained animals. In conclusion, these results support the contention that hippocampal PKC participates in the early neural events of memory formation of an aversively-motivated learning task.

Rapid and transient learning-associated increase in NMDA NR1 subunit in the rat hippocampus.

Several lines of evidence indicate that glutamate NMDA receptors are critically involved in long-term potentiation (LTP) and in certain forms of learning. It was previously demonstrated that memory formation of an inhibitory avoidance task in chick is specifically associated with an increase in the density of NMDA receptor in selected brain regions. Here we report on the effect of a one trial inhibitory avoidance training in rats, a hippocampal-dependent learning task, on the levels of different subunits of the glutamate NMDA receptor in synaptic plasma membranes (SPM) isolated from the hippocampus. Training rats on a one trial inhibitory avoidance task results in a rapid, transient and selective increase (+33%, p < 0.05) in NMDA NRI subunit expression in hippocampal SPM of rats sacrificed 30 min posttraining. No changes were observed at 0 or 120 min after training or in shocked animals in comparison to naive control rats. In addition, no training-associated increase in the levels of NMDA NR2A and NR2B or AMPA GluR 2/3 subunits was observed at any timepoint tested. In conclusion, the present findings support the hypothesis that alterations in expression of synaptic NMDA NR1 subunits in the hippocampus are specifically associated with memory formation of an inhibitory avoidance task and strongly suggest that hippocampal NMDA receptors are crucially involved in the neural mechanisms underlying certain forms of learning.

Experience-dependent increase in cAMP-responsive element binding protein in synaptic and nonsynaptic mitochondria of the rat hippocampus.

Cyclic AMP-responsive element binding protein (CREB) plays a pivotal role in the formation of long-term memory in Drosophila, Aplysia, mice and rats. Recently, we were able to demonstrate that CREB and its serine 133 phosphorylated form p-CREB are localized in synaptic and nonsynaptic mitochondria of the rat brain. Here we report on the effect of a one-trial inhibitory avoidance training procedure on mitochondrial CREB from the rat hippocampus. This aversively motivated training task is associated with a time-dependent increase (34-35%) in both p-CREB and CREB immunoreactivities detected in synaptic mitochondria of the hippocampus. In nonsynaptic mitochondria, p-CREB levels increased in both trained and shocked animals. In addition to CREB, two CRE-element binding repressors, CREB-2 and CREM-1, were also detected in purified brain mitochondria. No changes were observed in CREB-2 and CREM-1 immunoreactivities in hippocampal synaptic mitochondria after an inhibitory avoidance training. Taken together the present findings represent the first evidence showing that brain mitochondrial CREB may participate in plasticity-dependent changes associated with a behavioural training procedure.

Cyclic AMP-responsive element binding protein in brain mitochondria.

Cyclic AMP-responsive element binding protein (CREB) is critically involved in many important brain functions, including the formation of long-term memory. CREB is the best characterized member of a family of transcription factors (CREB/ATF family) recognized to be important nuclear targets for intracellular signal transduction systems. Here we show, by using different approaches, that CREB is unexpectedly localized to mitochondria of the rat brain. Controlled subcellular fractionation of hippocampus and cerebral cortex showed that both synaptic and nonsynaptic mitochondria exhibited immunoreactivity to the phosphorylated form of CREB (pCREB). Moreover, CREB extracted from synaptic mitochondria is able to be phosphorylated by the catalytic subunit of protein kinase A and dephosphorylated by protein phosphatase 1 or 2B. DNA mobility shift assays showed the presence of binding activity to the calcium-cyclic AMP-responsive element in mitochondrial extracts from hippocampus; this binding complex was specifically supershifted by an anti-CREB antibody. Immunoelectron microscopic analysis of hippocampal subcellular fractions revealed that pCREB immunoreactivity is localized in close association with the inner mitochondrial membrane. These results, together with recent findings describing the presence and phosphorylation of CREB in developing dendrites, suggest that CREB may participate in different mechanisms involved in the communication between extracellular signals and the expression of genes.