Novelty enhances retrieval: molecular mechanisms involved in rat hippocampus.

Rats exposed to a novel environment just prior to or 1-2 h, but not 4 or 6 h, before retention testing exhibited an enhanced retrieval of a one-trial inhibitory avoidance training. The bilateral intrahippocampal infusion of PD098059, an inhibitor of mitogen-activated protein kinase (MAPK), the specific upstream activator of p42 and p44 MAPKs, given 10 min before the exposure to the novel environment, blocked the enhancing effect of novelty on memory retrieval. In addition, prenovelty infusion of DL-2-amino-5-phosphonovalerate (APV), an antagonist of glutamate NMDA receptors, produced similar effects. The exposure to the novel environment is associated with an activation of p42 and p44 MAPKs and an increase in the phosphorylation state of the transcription factor cAMP response element binding protein (CREB). No changes were observed in cAMP-dependent protein kinase (PKA) activity or in alpha-CAMKII activation. Taken together, our results indicate that novelty activates hippocampal MAPKs, which are necessary, along with glutamate NMDA receptors, for the enhancing effect of novelty on retrieval.

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.

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.

Learning-specific, time-dependent increases in hippocampal Ca2+/calmodulin-dependent protein kinase II activity and AMPA GluR1 subunit immunoreactivity.

Ca2+/calmodulin-dependent protein kinase II (CAMK II) and one of its target, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), glutamate receptors have been shown to participate in both long-term potentiation (LTP) in the hippocampus, and in spatial, as well as in a variety, of learning paradigms. Recently, we were able to demonstrate that the intrahippocampal infusion of a specific inhibitor of CAMK II (KN62) provoked full retrograde amnesia of an inhibitory avoidance learning in rats when given immediately, but not 120 or 240 min, after training. Furthermore, this task is accompanied by a rapid, selective and reversible increase in hippocampal [3H] AMPA receptor binding. Here we report the effect of this aversively motivated learning task on CAMK II activity, and AMPA GluR1 subunit phosphorylation and immunoreactivity in the hippocampus. One trial inhibitory avoidance training is associated with a learning-specific, time-dependent increase (25-78%) in both total and Ca2+-independent activities of CAMK II in the hippocampus of rats killed immediately (0 min), but not 120 min, after training. In addition, immunoblotting experiments showed an increment in the amount of the alpha-subunit of CAMK II at 0, 30 and 120 min after training. An increase in the in vitro phosphorylation of alpha- and beta-subunits of CAMK II was also observed in hippocampal synaptosomal membranes (SPM) of trained rats killed immediately and 30 min post-training. In addition, inhibitory avoidance is accompanied by a 20% increase in GluR1 phosphorylation and a 33% increase in GluR1 immunoreactivity 120 min after training. No significant changes were observed in shocked animals. Phosphorylation of hippocampal SPM from naive control animals in conditions suitable for CAMK II activation resulted in a large increase in the density of [3H] AMPA binding (+ 100%). Taken together, these findings confirm and extend previous data suggesting that CAMK II and AMPA glutamate receptors in the hippocampus participate in the early phase of memory formation of an inhibitory avoidance learning.

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.

Effects of prenatal diazepam on two-way avoidance behavior, swimming navigation and brain levels of benzodiazepine-like molecules in male Roman high- and low-avoidance rats.

Utilizing psychogenetically selected Roman high- and low-avoidance rats (RHA/Verh and RLA/Verh), the present experiments investigated the effects of prenatally administered vehicle and diazepam (1 and 3 mg/kg per day, SC) on the behavior and neurochemistry of adult, male offspring. Active, two-way avoidance behavior was analyzed in 96 rats, at 6 months of age, and swimming navigation in 68 others, at 11 months. Three weeks after testing, selected brain areas from the latter animals were immunoassayed for benzodiazepine (BZD)-like molecules. The 3 mg/kg dose of diazepam both decreased freezing behavior in the shuttle box and reduced the hippocampal content of BZD-like molecules in the RLA/Verh male rats. Swimming navigation (spatial learning), at which the RLA/Verh rats were more adept, was not specifically affected by prenatal diazepam in either rat line. The possibility exists that an increased hippocampal release of BZD-like substances may be necessary to alter shuttle box behavior in RLA/Verh rats.

Inhibitory avoidance training induces rapid and selective changes in 3[H]AMPA receptor binding in the rat hippocampal formation.

The AMPA receptor has been shown to participate in the synaptic mechanisms involved in certain forms of learning and memory. We have previously demonstrated that the posttraining infusion of 6-cyano-7-nitroquinoxaline-2,3-dione, an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor blocker, into the dorsal hippocampus of rats, causes retrograde amnesia of an inhibitory avoidance training. Here, we report on the effect of this learning task on 3[H]AMPA binding to frozen rat brain sections. By using a quantitative autoradiographic analysis, we were able to demonstrate that the binding of 3[H]-AMPA was increased by 40-80% in the CA1, CA2, CA3, and dentate gyrus subregions of the hippocampal formation of rats trained in a step-down inhibitory avoidance paradigm, compared to naive, shocked, and free exploration controls. This effect was evident between 30 and 180 min after training, and it was mainly due to an increase in the density, but not in the affinity of binding sites. No alterations in 3[H]AMPA binding were observed either in those animals that received only the footshock (shocked group) or in animals that were submitted to 1 min of free exploration of the training box (free exploration group). In the rest of the brain regions, including the frontal cortex, entorhinal cortex, striatum, amygdala, cerebellum, and thalamus, the 3[H]AMPA binding remained unchanged. In addition, the binding of 3[H]muscimol and 3[H]-flunitrazepam to the GABAA/benzodiazepine receptor complex was unaltered in all the experimental groups. In conclusion, rats submitted to a one-trial inhibitory avoidance training showed a rapid, selective, and specific increase in 3[H]AMPA binding in the hippocampal formation. The present findings support the hypothesis that hippocampal AMPA receptors are involved in the neural mechanisms underlying certain forms of learning and memory.

Apigenin, a component of Matricaria recutita flowers, is a central benzodiazepine receptors-ligand with anxiolytic effects.

The dried flower heads of Matricaria recutita L. (Asteraceae) are used in folk medicine to prepare a spasmolytic and sedative tea. Our fractionation of the aqueous extract of this plant led to the detection of several fractions with significant affinity for the central benzodiazepine receptor and to the isolation and identification of 5,7,4'-trihydroxyflavone (apigenin) in one of them. Apigenin competitively inhibited the binding of flunitrazepam with a Ki of 4 microM and had no effect on muscarinic receptors, alpha 1-adrenoceptors, and on the binding of muscimol to GABAA receptors. Apigenin had a clear anxiolytic activity in mice in the elevated plusmaze without evidencing sedation or muscle relaxant effects at doses similar to those used for classical benzodiazepines and no anticonvulsant action was detected. However, a 10-fold increase in dosage produced a mild sedative effect since a 26% reduction in ambulatory locomotor activity and a 35% decrement in hole-board parameters were evident. The results reported in this paper demonstrate that apigenin is a ligand for the central benzodiazepine receptors exerting anxiolytic and slight sedative effects but not being anticonvulsant or myorelaxant.

Isolation of pharmacologically active benzodiazepine receptor ligands from Tilia tomentosa (Tiliaceae).

Tilia species are traditional medicinal plants widely used in Latin America as sedatives and tranquilizers. For this purpose, the infusion of their inflorescences is used to prepare a tea. In this study extracts of inflorescences from Tilia tomentosa Moench, one of the species found in the market, were purified using a benzodiazepine (BZD) binding assay to detect BZD receptor ligands in the different fractions. One of the ligands was identified as kaempferol, but it had low affinity (Ki = 93 microM) for this receptor, and did not produce sedative or anxiolytic effects in mice. On the other hand, a complex fraction, containing as yet unidentified constituents, but probably of a flavonoid nature, when administered intraperitoneally in mice, had a clear anxiolytic effect in both the elevated plus-maze and holeboard tests, two well validated pharmacological tests to measure anxiolytic and sedative compounds. This active fraction had no effect on total and ambulatory locomotor activity. In conclusion, our results demonstrate the occurrence of active principle(s) in, at least, one species of Tilia that may explain its ethnopharmacological use as an anxiolytic.

In vivo formation of benzodiazepine-like molecules in mammalian brain.

The possible cerebral formation of benzodiazepine (BDZ)-like molecules was evaluated in rats bilaterally microinjected into either the lateral ventricles (ICV) or the hippocampus (IH), with 10-70 microCi of different radiolabeled amino acids. After 3, or 24 h, the rats were sacrificed and BDZ-like molecules from total brain or hippocampus were purified by reversed phase HPLC. At 24 h. but not at 3 h of the ICV or IH microinjections with [3H] tryptophan, a peak of radioactivity containing material that inhibited the binding of [3H] flunitrazepam to both the BDZ receptor and the anti -BDZ monoclonal antibody MAb 21-7F9 was obtained. This active labeled fraction eluting just before diazepam also bound directly and specifically to the BDZ receptor and to MAb 21-7F9. No peak of radioactivity containing BDZ-like material was obtained when [3H] phenylalanine, [14C] glycine, [14C] methionine or [14C] tyrosine alone or in different combinations were microinjected into the hippocampus. The present results, together with our previous findings on the in vitro production of BDZ-like molecules in rat brain homogenates or slices (11), strongly suggest that the mammalian brain is capable of synthesizing low molecular weight substances possessing BDZ-like activity.

Anxiogenic effects of the intraamygdala injection of flumazenil, a benzodiazepine receptor antagonist.

The effects of bilateral intraamygdala microinjection of flumazenil, a benzodiazepine receptor (BZD-R) antagonist, on the exploratory activity in an elevated plus maze were examined in chronically implanted rats. This compound induced a significant decrease in the time spent in the open arms, which is consistent with an anxiogenic action. No effect was observed after intrastriatal injections of flumazenil. Naive rats exposed to the elevated plus maze showed a rapid and selective decline in the content of BZD-like molecules in amygdala (-68%) but not in striatum and hippocampus. These data suggest that the anxiogenic effects of the intraamygdala injection of flumazenil is probably due to the blockade of BZD-like molecules released during the performance.

Production of benzodiazepine-like compounds in bovine rumen.

The presence of benzodiazepine-like molecules was detected radioimmunologically in bovine rumen contents and in incubates of ruminal contents with homogenates of several common grasses. A similar production was found "in vivo" in samples obtained from a grazing cow with a rumen cannula.

Habituation and inhibitory avoidance training alter brain regional levels of benzodiazepine-like molecules and are affected by intracerebral flumazenil microinjection.

The effects of habituation and inhibitory avoidance training on the rat brain regional levels of benzodiazepine (BZD)-like molecules and on central type BZD binding sites were examined. BZD-like immunoreactivity was decreased by 26-50% in the amygdala, cerebral cortex and septum of rats sacrificed immediately after stepping-down from the platform of an inhibitory avoidance apparatus (non-trained group) as compared to naive controls. Rats submitted to a second step-down session 20 h later (habituated group) have significantly lower BZD-like immunoreactivity in the septum (-60%) as compared to non-trained animals. Rats exposed to an inhibitory avoidance training, i.e. stepping-down and receiving a footshock (trained group), showed a significant reduction in the content of BZD-like molecules in cerebral cortex (-44%), amygdala (-68%), septum (-80%) and hippocampus (-82%) as compared to non-trained rats. In addition, the density of central type BZD binding sites was slightly increased in the hippocampus and septum of trained rats. No changes were observed in the apparent dissociation constant. No changes were observed in parallel measurements of [3H]-L-quinuclidinyl benzylate binding constants at cholinergic muscarinic binding sites. The immediate posttraining intrahippocampal bilateral injection of the central type BZD receptor antagonist flumazenil (10 nmol/hippocampus), enhanced the retention of habituation but not when injected in the amygdala or septum. In contrast, retention of the inhibitory avoidance task was significantly increased by flumazenil administered bilaterally into any of the 3 brain structures.(ABSTRACT TRUNCATED AT 250 WORDS)

S-adenosyl methionine increases ?(1) adrenoceptors in rat cerebral cortex membranes.

A crude synaptosomal membrane fraction of rat cerebral cortex was submitted to binding with [(3)H]prazosin for ?(1) adrenoceptors. Methylation with 100 ?M S-adenosyl methionine (SAM) resulted in a 43 +/- 7% increase in the binding of 3 nM [(3)H]prazosin. The effect of SAM was stimulated by 8mM Ca(2+), and was not inhibited by S-adenosyl-homocysteine. Time incubation, as well as concentration curves, showed a biphasic effect of SAM. In saturation experiments SAM caused a large increase in the B(max) with little effect on the K(D). Displacement curves with noradrenaline showed that the affinity and proportion of agonist biding sites was not altered by SAM. These findings are discussed on the basis of what is known about the action of SAM on ? adrenoceptors and benzodiazepine receptors. It is concluded that the effect of SAM on ?(1) adrenoceptors is probably mediated by a direct action on the lipoprotein structure of the membrane and not on an effect of methyltransferases.

Chrysin (5,7-di-OH-flavone), a naturally-occurring ligand for benzodiazepine receptors, with anticonvulsant properties.

Chrysin (5,7-di-OH-flavone) was identified in Passiflora coerulea L., a plant used as a sedative in folkloric medicine. Chrysin was found to be a ligand for the benzodiazepine receptors, both central (Ki = 3 microM, competitive mechanism) and peripheral (Ki = 13 microM, mixed-type mechanism). Administered to mice by the intracerebroventricular route, chrysin was able to prevent the expression of tonic-clonic seizures induced by pentylenetertrazol. Ro 15-1788, a central benzodiazepine receptor antagonist, abolished this effect. In addition, all of the treated mice lose the normal righting reflex which suggests a myorelaxant action of the flavonoid. The presence in P. coerulea of benzodiazepine-like compounds was also confirmed.

Benzodiazepine-like molecules, as well as other ligands for the brain benzodiazepine receptors, are relatively common constituents of plants.

The presence of benzodiazepine (BZD)-like molecules as well as of other substances with affinity for the brain BZD-receptors was explored in eight non-flowering plants known to contain biflavonoids, three flowering plants used as sedatives in folkloric medicine and one plant extensively used in Argentina, Uruguay, Brazil and Paraguay as a tea substitute. All the plants examined contained substances which bound to the central BZD-receptors and the majority of them also had BZD-like compounds detected by their specific interaction with a monoclonal antibody against BZDs. In various cases this last type of compound was present in amounts which exceeded trace levels (0.5-1.0 ng/g). The biological or clinical significance for humans of all these substances should be explored.

In vivo and in vitro modulation of central type benzodiazepine receptors by phosphatidylserine.

The in vivo and in vitro modulation of central benzodiazepine binding sites (BDZ-R) by phosphatidylserine purified from bovine cerebral cortex (BC-PS) was studied. Five days i.p. administration of 15 mg/kg/day of BC-PS liposomes increased the maximal number of binding sites (Bmax) for [3H]flunitrazepam in cerebral cortical membranes. In contrast, the density of hippocampal benzodiazepine recognition binding sites decreased. In cerebellar membranes, BC-PS treatment did not alter the characteristics of [3H]flunitrazepam binding. Similar experiments using phosphatidylcholine extracted from bovine brain (BC-PC) resulted in no changes in the [3H]flunitrazepam binding in the 3 neural structures studied. Confirming previous results, rats submitted to an acute swimming stress showed a decrease in the density of cerebral cortex BDZ-R. Animals treated with BC-PS liposomes before stress showed cortical [3H]flunitrazepam binding significantly below treated, unstressed animals but not below controls. The effects of BC-PS liposomes appeared to be selective for the central type of BDZ-R since no changes were observed in [3H]RO 5-4864 binding, a radioligand specific for the peripheral type BDZ-R. Preincubation of cerebral cortical and cerebellar synaptosomal membranes with BC-PS liposomes (1-300 micrograms per assay) significantly increased in a concentration-dependent manner (up to 100 micrograms) the [3H]flunitrazepam binding. Scatchard analysis revealed changes in the apparent affinity without alterations in the Bmax. Very similar results were obtained using a purified PS from spinal cord. BC-PC, phosphatidylinositol, phosphatidic acid and the lyso derivatives of PS and PC (lysoPS and lysoPC) were found to be ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective increase of alpha 1-adrenoceptors and muscarinic cholinergic receptors in rat cerebral cortex after chronic haloperidol.

The effect of chronic administration of haloperidol on alpha 1-, alpha 2-, and beta-adrenoceptors, cholinergic muscarinic, GABAA and benzodiazepine receptors in the cerebral cortex of the rat was investigated. Doses of 0.3 and 2 mg/kg of haloperidol during 7 days increased markedly the density of alpha 1-adrenoceptors without changes in affinity. The alpha 2- and beta-adrenoceptors were not modified after neuroleptic administration. The number of muscarinic receptors were also increased after haloperidol treatment (2 mg/kg/day). However, the GABAA and benzodiazepine binding sites remained unchanged. In the brainstem an increment in the alpha 1-, but not the beta-adrenoceptors was observed. The well known increase in the dopamine receptors in the striatum was confirmed. These observations demonstrate a multireceptor effect of haloperidol in the cerebral cortex.

Effect of chronic administration of haloperidol on secretory response mediated by cholinergic receptors in rat submandibular glands.

Administration of haloperidol influences peripheral non-dopaminergic receptors. The sialagogue response of the submandibular glands of the rats to methacholine was enhanced by chronic administration of haloperidol. The binding of [3H]QNB to muscarinic receptors in the submandibular glands was not changed by chronic haloperidol. The supersensitivity of postsynaptic cholinergic receptors to drugs in haloperidol treated rats is not related to changes in the number or affinity of such receptors. This paper confirmed the sialagogue supersensitivity to adrenergic drugs related to an increase in alpha 1-adrenoceptors in the submandibular glands of haloperidol injected rats.

Thyroid hormone regulation of adrenergic receptors and beta-adrenergic responsiveness in the rat submandibular gland.

The effects of altered thyroid function on the sensitivity of isoproterenol induced secretion of saliva and in the characteristics of adrenergic receptors from the rat submandibular gland were examined. Hyperthyroidism produced an increased sensitivity to beta-adrenergic stimulation of the gland, and this phenomenon was associated with an increase in the number of beta and alpha 1-adrenoceptors. On the other hand, surgical thyroidectomy produced a decrease sensitivity to isoproterenol stimulation of the submandibular gland and a diminished density of beta-adrenoceptors. In this case, no changes in alpha-adrenoceptors were observed. These results are discussed emphasizing the correlation between the functional control of saliva secretion and the adrenergic receptors in different thyroid states.