Julio M. Azcurra

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.

The circling training rat model as a behavioral teratology test

The properties of circling training (CT) for detecting behavioral teratologic drug-induced effects was evaluated by prenatal administration of two behavioral teratogenic drugs: vitamin A (80,000 IU/kg/day) and haloperidol (2.5 mg/kg/day). The circling training was started at 30 days of age and performed for 8 days in an automated apparatus. Statistically significant differences between drug-treated and control animals regarding the measured response (turns per minute) were found. Two components may affect the response measured by the CT: associative learning and motor performance. The incidence of these components was discriminated with behavioral and mathematical approaches. In the experimental conditions used the most affected parameter was motor performance. The results indicate that CT can be used as an instrumental conditioning test where the quantifiable endpoint is the on-going motor performance. Further applications of the CT for neurochemical evaluation of drug induced effects are also discussed.

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.

Different dopamine D1 and D2 receptors expression after motor activity in the striatal critical period

Circling training during rat striatum postnatal critical period (PN30 to 37 days) induces a life-lasting co-reduction of muscarinic acetylcholine receptors (mAChR) and dopamine D2 receptors (D2R) binding. Here, we evaluated the expression of D1R and D2R under similar experimental conditions. Trained rats showed a decrease of 40% in D2R binding sites (p<0.01) and of 45% in the D2R mRNA expression which involve short (p<0.05) and long (p<0.01) isoforms. In contrast, D1R binding sites nor its mRNA expression levels were affected by training, indicating a differential synaptic refinement during this ontogenetically fixed period.

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.

Developmental striatal critical period of activity-dependent plasticity is also a window of susceptibility for haloperidol induced adult motor alterations

Experience-dependent plasticity during critical periods of postnatal (PN) development shapes the adult brain anatomy and function. In rat motor system, there is a critical period of activity-dependent plasticity in the striatum (PN30-37). In this period, motor activity of running in a circular path induced in the Circling Training test (CT), elicits several plasticity changes on striatal synapses. It has been recently proposed that developmental critical periods might represent a unique pharmacological window of vulnerability to induce life-lasting behavioral modifications. In this paper we tested the hypothesis of existence of a pharmacological susceptibility to induce adult alterations on motor behavior during the striatal critical period. Due to its main action on the striatum and developmental motor behavioral effects, we applied the prototypical antipsychotic haloperidol to male rats (i.p. 0.7 or 2.5 mg/kg/day) before, during or after the period of plasticity (PN20-27, PN30-37 or PN40-47 respectively). Then, in the adulthood (PN80), we evaluated induced motor activity in the CT. The results showed that only rats exposed to the D2R blocker during the period PN30-37 increased the CT activity in comparison to control rats. Moreover, only these animals also showed an increase in the spontaneous locomotor activity at the open field test. These behavioral alterations were not accompanied by permanent striatal changes either on the number of D2R binding sites or on its mRNA expression levels. In conclusion, we have shown a pharmacological susceptibility of inducing adult motor behavior alterations by haloperidol during a natural critical period of activity-dependent plasticity (PN30-37) in rat striatum development. These results also emphasize the importance of behavioral screening for pharmacological agents to be used in developmental stages of maturation.

In vivo labeling of cytoplasmic RNA in neurons of the immature brain cortex

only the total and the nuclear RNAs were analyzed. The present report describes a procedure for the isolation of the neuronal cytoplasmic RNA and its application to the measurement of the approximate maturation time of its ribosomal RNA component in 8- and 20-day-old cerebral cortex perikarya. While this work was in progress a report describing the labeling pattern of neuronal and glial cytoplasmic RNA isolated from the whole brain of adult rats has appeared ~. Neuronal perikarya were isolated as described previously, except for the following modification: the solution employed for tissue suspension contained 7.5 ~ polyvinylpyrrolidone (w/v), 10 mM CaC12 and 4 ~ (w/v) instead of the 1 ~ bovine serum albumin previously used 1°. With this change in albumin concentration the suspension solution becomes isotonic. The yield and preservation of the perikaryal fraction and its purity were described 9,1°. In each experiment 10 rats were injected intrathecally with 10/~Ci of [5-aH]uridine (Amersham/Searle, 20 Ci/mmole). The neuronal perikarya were suspended in 3 ml of a solution consisting of 0.2 M sucrose- 0.06 M NaCI0.1 M Tris-HC1 buffer, pH 7.6, and the suspension was homogenized by means of 3 strokes in a glass-teflon loose-fitting homogenizer. The homogenate was centrifuged at 2,000 × g for 10 min and the supernatant was aspirated and stored in ice. This procedure was repeated and the supernatant pool was brought up to 0.25 ~ Macaloid (American Tansul Co., Houston, Texas), 0.01 M EDTA and 50/~g/ml of polyvinyl

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.

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.

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.