Exogenous NGF alters a critical motor period in rat striatum.

In previous studies we found that there is a critical period during rat postnatal development when motor training starting at age 30 days (P30) but not before or after this age, induces a bilateral lifetime drop in Bmax of the muscarinic radioligand [3H]QNB in striatum. We examined the possibility that striatal NGF level would be a determining factor for the normal occurrence of this synaptic plasticity. With this aim, rats underwent training at P30-37 with or without simultaneous NGF perfusion into the left striatum. At P70, we found the expected bilateral enduring fall of striatal [3H]QNB sites in trained controls. While the non-cannulated side of NGF-treated trained rats showed a similar drop in [3H]QNB binding, the perfused striata from these animals were not affected by training. Thus, the findings add new evidence in favour of a major role of NGF in this critical period of activity-dependent permanent adjustment in the striatal muscarinic system.

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.

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.

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.

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.

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.