Role of hippocampal signaling pathways in long-term memory formation of a nonassociative learning task in the rat.

Long-term habituation to a novel environment is one of the most elementary forms of nonassociative learning. Here we studied the effect of pre- or posttraining intrahippocampal administration of drugs acting on specific molecular targets on the retention of habituation to a 5-min exposure to an open field measured 24 h later. We also determined whether the exposure to a novel environment resulted in the activation of the same intracellular signaling cascades previously shown to be activated during hippocampal-dependent associative learning. The immediate posttraining bilateral infusion of CNQX (1 microg/side), an AMPA/kainate glutamate receptor antagonist, or of muscimol (0.03 microg/side), a GABA(A) receptor agonist, into the CA1 region of the dorsal hippocampus impaired long-term memory of habituation. The NMDA receptor antagonist AP5 (5 microg/side) impaired habituation when infused 15 min before, but not when infused immediately after, the 5-min training session. In addition, KN-62 (3.6 ng/side), an inhibitor of calcium calmodulin-dependent protein kinase II (CaMKII), was amnesic when infused 15 min before or immediately and 3 h after training. In contrast, the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, and the protein synthesis inhibitor anisomycin, at doses that fully block memory formation of inhibitory avoidance learning, did not affect habituation to a novel environment. The detection of spatial novelty is associated with a sequential activation of PKA, ERKs (p44 and p42 MAPKs) and CaMKII and the phosphorylation of c-AMP responsive element-binding protein (CREB) in the hippocampus. These findings suggest that memory formation of spatial habituation depends on the functional integrity of NMDA and AMPA/kainate receptors and CaMKII activity in the CA1 region of the hippocampus and that the detection of spatial novelty is accompanied by the activation of at least three different hippocampal protein kinase signaling cascades.

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 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.

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.

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.

Role of hippocampal NO in the acquisition and consolidation of inhibitory avoidance learning.

Nitric oxide (NO), an unconventional neurotransmitter in the brain, has been postulated as a retrograde intercellular messenger necessary for the induction, but not the maintenance phase, of activity-dependent forms of synaptic plasticity in the hippocampus. Here we report on the effects of an inhibitory avoidance learning task on hippocampal NO synthase (NOS) activity and on the effects of intrahippocampal infusion of a NOS inhibitor in the acquisition and consolidation of this task in rats. NOS activity increases by 45% in the hippocampus immediately after training (0 min) but not at 60 min after training. No changes were observed in cerebellar NOS activity. The bilateral intrahippocampal microinjection of nitro-arginine (NO-arg), an NOS inhibitor, provoked retrograde amnesia for the inhibitory avoidance when given 10 min before or immediately after training, but not 60 min after training. These results suggest that NO-regulated processes in the hippocampus play an important role at the time of training or very shortly thereafter of an inhibitory avoidance learning.

Evidence for the involvement of hippocampal CO production in the acquisition and consolidation of inhibitory avoidance learning.

Carbon monoxide (CO), produced through the action of haem oxygenase (HO) isoenzymes, has been recently postulated as a retrograde messenger in the early stages of long-term potentiation (LTP). In the present study, rats submitted to an inhibitory avoidance task there is a significant increase (+76%) in hippocampal HO activity immediately after training (0 min), but not at 60 min post-training. No changes were observed in cerebral cortical and cerebellar HO activity. Bilateral intrahippocampal infusion of the HO inhibitor zinc-protoporphyrin-IX (ZnPP) (2 micrograms side-1) caused full amnesia for inhibitory avoidance when given 10 min before training or immediately after training, but not 60 min after training. These findings provide evidence that CO production in the hippocampus is important for the early stages of memory processing of an inhibitory avoidance training.

Effect of various training procedures on performance in an elevated plus-maze: possible relation with brain regional levels of benzodiazepine-like molecules.

Rats submitted to one, two, or seven sessions of exploration to a new environment (habituation) or exposed to an inhibitory avoidance training showed different degrees of anxiety, evaluated by the elevated plus-maze test. Also, the brain regional levels of benzodiazepine (BDZ)-like molecules in rats submitted to one, two, or seven sessions of habituation were differentially decreased with respect to nontrained rats. The percentage of time spent in the open arms of the elevated plus-maze for each group correlates with the data of decrease in the BDZ-like immunoreactivity in amygdala (r = 0.77, p < 0.0005), hippocampus (r = 0.68, p < 0.0005), and septum (r = 0.57, p < 0.005). These results suggest that the limbic system responds to anxiogenic experiences by changing the BDZ-like molecule levels in relation to the degree of anxiety and/or stress that accompany these experiences.

Benzodiazepines in the brain. Their origin and possible biological roles.

Great progress has been made in the last 5 yr in demonstrating the presence of benzodiazepines (BDZs) in mammalian tissues, in beginning studies on the origin of these natural compounds, and in elucidating their possible biological roles. Many unanswered questions remain regarding the sources and biosynthetic pathways responsible for the presence of BDZs in brain and their different physiological and/or biochemical actions. This essay will focus on recent findings supporting that: (1) BDZs are of natural origin; (2) mammalian brain contains BDZs in concentrations ranging between 5 x 10(-10)-10(-8) M; (3) dietary source of BDZs might be a plausible explanation for their occurrence in animal tissues, including man; (4) the formation of BDZ-like molecules in brain is a possibility, experimentally supported; (5) BDZ-like molecules including diazepam and N-desmethyldiazepam are elevated in hepatic encephalopathy; and (6) natural BDZs in the brain are involved in the modulation of memory processes. Future studies using the full range of biochemical, physiological, behavioral, and molecular biological techniques available to the neuroscientist will hopefully continue to yield exciting and new information concerning the biological roles that BDZs might play in the normal and pathological functioning of the brain.

n-[3H]butyl-beta-carboline-3-carboxylate, a putative endogenous ligand, binds preferentially to subtype 1 of central benzodiazepine receptors.

Synthetic n-butyl beta-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]beta-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD = 2.7 +/- 0.1 nM) with a Bmax of 1.16 +/- 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax = 2.36 +/- 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10(-11)-10(-5) M butyl beta-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KD1 = 0.6-1.5 nM and KD2 = 12-18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl beta-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic haloperidol causes increase in salivary response and alpha 1-adrenoceptors in submandibular gland of the rat.

The effect of chronic haloperidol on the receptor-secretion coupling of the submandibular glands of the rat was studied. After injection of 2 mg/kg haloperidol daily for 7 days, the dose-response curve to L-noradrenaline was displaced to the left, with lowering of the threshold and enhancement of the maximal response. This was accompanied by a 73% increase in alpha 1-adrenoceptors in the glands. The effect was selective, since no changes were observed in alpha 2- and beta-adrenoceptors.