Generalized extinction of fear memory depends on co-allocation of synaptic plasticity in dendrites.

Memories can be modified by new experience in a specific or generalized manner. Changes in synaptic connections are crucial for memory storage, but it remains unknown how synaptic changes associated with different memories are distributed within neuronal circuits and how such distributions affect specific or generalized modification by novel experience. Here we show that fear conditioning with two different auditory stimuli (CS) and footshocks (US) induces dendritic spine elimination mainly on different dendritic branches of layer 5 pyramidal neurons in the mouse motor cortex. Subsequent fear extinction causes CS-specific spine formation and extinction of freezing behavior. In contrast, spine elimination induced by fear conditioning with >2 different CS-USs often co-exists on the same dendritic branches. Fear extinction induces CS-nonspecific spine formation and generalized fear extinction. Moreover, activation of somatostatin-expressing interneurons increases the occurrence of spine elimination induced by different CS-USs on the same dendritic branches and facilitates the generalization of fear extinction. These findings suggest that specific or generalized modification of existing memories by new experience depends on whether synaptic changes induced by previous experiences are segregated or co-exist at the level of individual dendritic branches.

Alpha-mangostin induces changes in glutathione levels associated with glutathione peroxidase activity in rat brain synaptosomes.

BACKGROUND: In a previous report, we have characterized the antiperoxidative properties of alpha-mangostin in different toxic models tested in nerve tissue preparations. OBJECTIVES: Here, the modulatory effects of this xanthone on the glutathione system (reduced glutathione (GSH) levels, glutathione peroxidase (GPx), and glutathione S-transferase (GST) activities) were tested in synaptosomal P2 fractions isolated from rat brains in order to provide further information on key mechanisms exerted by this antioxidant in the nervous system. METHODS: Synaptosomes were exposed to increasing concentrations of the xanthone, and also challenged to the toxic actions of a free radical generator, ferrous sulfate (FeSO(4)). For comparative purposes, the mitochondrial toxin 3-nitropropionic acid (3-NP) was also explored. RESULTS: Alpha-mangostin significantly decreased the levels of GSH, and increased GPx activity. DISCUSSION: This finding was interpreted as a modulatory action of the GSH system in preparation to exert antioxidant responses. Although FeSO(4) exhibited similar effects, these were interpreted as a compensatory response to the toxic actions of the pro-oxidant. We came to this conclusion based on our previous report where alpha-mangostin produced antiperoxidative effects and FeSO(4) produced oxidative damage to lipids. GST activity remained unaffected by both the antioxidant and the pro-oxidant. Our results suggest that alpha-mangostin is able to modulate GPx activity as a potential antioxidant strategy, thereby transiently consuming GSH levels.

Extinction memory in the crab Chasmagnathus: recovery protocols and effects of multi-trial extinction training.

A decline in the frequency or intensity of a conditioned behavior following the withdrawal of the reinforcement is called experimental extinction. However, the experimental manipulation necessary to trigger memory reconsolidation or extinction is to expose the animal to the conditioned stimulus in the absence of reinforcement. Recovery protocols were used to reveal which of these two processes was developed. By using the crab contextual memory model (a visual danger stimulus associated with the training context), we investigated the dynamics of extinction memory in Chasmagnathus. Here, we reveal the presence of three recovery protocols that restore the original memory: the old memory comes back 4 days after the extinction training, or when a weak training is administered later, or once the VDS is presented in a novel context 24 h after the extinction session. Another objective was to evaluate whether the administration of multi-trial extinction training could trigger an extinction memory in Chasmagnathus. The results evince that the extinction memory appears only when the total re-exposure time is around 90 min independently of the number of trials employed to accumulate it. Thus, it is feasible that the mechanisms described for the case of the extinction memory acquired through a single training trial are valid for multi-trial extinction protocols. Finally, these results are in agreement with those reports obtained with models phylogenetically far apart from the crab. Behind this attempt is the idea that in the domain of studies on memory, some principles of behavior organization and basic mechanisms have universal validity.

Memory reconsolidation and extinction in the crab: mutual exclusion or coexistence?

A conditioned stimulus (CS) exposure has the ability to induce two qualitatively different mnesic processes: memory reconsolidation and memory extinction. Previous work from our laboratory has shown that upon a single CS presentation the triggering of one or the other process depends on CS duration (short CS exposure triggers reconsolidation, whereas a long CS exposure triggers extinction), both being mutually exclusive processes. Here we show that either process is triggered only after CS offset, ruling out an interaction as the mechanism of this mutual exclusion. Also, we show here for the first time that reconsolidation and extinction can occur simultaneously without interfering with each other if they are serially triggered by respective short and long CS exposures. Thus, we conclude that (1) one single CS presentation triggers one single process, after CS offset, and (2) whether memory reconsolidation and extinction mutually exclude each other or whether they coexist depends only on whether they are triggered by single or multiple CS presentations.

Fear conditioning and extinction induce opposing changes in dendritic spine remodeling and somatic activity of layer 5 pyramidal neurons in the mouse motor cortex.

Multiple brain regions including the amygdala and prefrontal cortex are crucial for modulating fear conditioning and extinction. The primary motor cortex is known to participate in the planning, control, and execution of voluntary movements. Whether and how the primary motor cortex is involved in modulating freezing responses related to fear conditioning and extinction remains unclear. Here we show that inactivation of the mouse primary motor cortex impairs both the acquisition and extinction of freezing responses induced by auditory-cued fear conditioning. Fear conditioning significantly increases the elimination of dendritic spines on apical dendrites of layer 5 pyramidal neurons in the motor cortex. These eliminated spines are further apart from each other than expected from random distribution along dendrites. On the other hand, fear extinction causes the formation of new spines that are located near the site of spines eliminated previously after fear conditioning. We further show that fear conditioning decreases and fear extinction increases somatic activities of layer 5 pyramidal neurons in the motor cortex respectively. Taken together, these findings indicate fear conditioning and extinction induce opposing changes in synaptic connections and somatic activities of layer 5 pyramidal neurons in the primary motor cortex, a cortical region important for the acquisition and extinction of auditory-cued conditioned freezing responses.

Memory is not extinguished along with CS presentation but within a few seconds after CS-offset.

Prior work with the crab's contextual memory model showed that CS-US conditioned animals undergoing an unreinforced CS presentation would either reconsolidate or extinguish the CS-US memory, depending on the length of the reexposure to the CS. Either memory process is only triggered once the CS is terminated. Based on these results, the following questions are raised. First, when is extinction memory acquired, if not along extinction training, and how long does it take? Second, can acquisition and consolidation of extinction memory be pharmacologically dissected? Here we address these questions performing three series of experiments: a first one aimed to study systematically the relationship between extinction and increasing periods of unreinforced CS presentations, a second one to determine the time boundaries of the extinction memory acquisition, and the third one to assay the requirement for protein synthesis and NMDA-like receptors of acquisition and consolidation of extinction memory. Our results confirm that it is CS-offset and not the mere retrieval (CS-onset) that triggers acquisition of extinction memory and that it is completed in less than 45 sec after CS-offset. In addition, protein synthesis is required for consolidation but not for acquisition of this memory and, conversely, NMDA-like receptor activity is required for its acquisition but not for its consolidation. Finally, we offer an interpretative scheme of our results and we discuss to what extent it could apply to multitrial extinction.

Reactivation and reconsolidation of long-term memory in the crab Chasmagnathus: protein synthesis requirement and mediation by NMDA-type glutamatergic receptors.

Experiments with invertebrates support the view that intracellular events subserving the consolidation phase of memory are preserved across evolution. Here, we investigate whether such evolutionary persistence extends to reconsolidation mechanisms, which have recently received special attention in vertebrate studies. For this purpose, the memory model of the crab Chasmagnathus is used. A visual danger stimulus (VDS) elicits crab escaping, which declines after a few stimulus presentations. The long-lasting retention of this decrement, called context-signal memory (CSM), is mediated by an association between contextual cues of the training site and the VDS. The present results show amnesia for CSM in crabs re-exposed at 24 hr (day 2) for 5 min to the learning context, 24 hr after training, and injected with one of two amnesic agents, then tested 24 hr later. Agents and timing were either 15 microg of cycloheximide given between 1 hr before and 4 hr after re-exposure or 1 microg/gm (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine given between 1 hr before and 2 hr after re-exposure. The amnesic effects are specific to behavior that occurs a long time after reactivation but not a short time after. No CSM deficit is produced by such agents when crabs are exposed to a context different from that of training. Findings are consistent with those reported for vertebrates, with both showing that reactivation induces a recapitulation of the postacquisition cascade of intracellular events. The agreement between results from such phylogenetically disparate animals suggests that evolution may have adopted a given molecular cascade as the preferred means of encoding experiences in the nervous system.

Antipsychotics activate mTORC1-dependent translation to enhance neuronal morphological complexity.

Although antipsychotic drugs can reduce psychotic behavior within a few hours, full efficacy is not achieved for several weeks, implying that there may be rapid, short-term changes in neuronal function, which are consolidated into long-lasting changes. We showed that the antipsychotic drug haloperidol, a dopamine receptor type 2 (D2R) antagonist, stimulated the kinase Akt to activate the mRNA translation pathway mediated by the mammalian target of rapamycin complex 1 (mTORC1). In primary striatal D2R-positive neurons, haloperidol-mediated activation of mTORC1 resulted in increased phosphorylation of ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Proteomic mass spectrometry revealed marked changes in the pattern of protein synthesis after acute exposure of cultured striatal neurons to haloperidol, including increased abundance of cytoskeletal proteins and proteins associated with translation machinery. These proteomic changes coincided with increased morphological complexity of neurons that was diminished by inhibition of downstream effectors of mTORC1, suggesting that mTORC1-dependent translation enhances neuronal complexity in response to haloperidol. In vivo, we observed rapid morphological changes with a concomitant increase in the abundance of cytoskeletal proteins in cortical neurons of haloperidol-injected mice. These results suggest a mechanism for both the acute and long-term actions of antipsychotics.

Early changes in oxidative stress markers in a rat model of acute stress: effect of l-carnitine on the striatum.

This work focuses on the effect of acute stress on different markers of oxidative stress and mitochondrial dysfunction in the rat striatum. In addition, the effect of a single dose of l-carnitine (l-CAR, 300 mg/kg, i.p.) was evaluated in these animals. Immobilization (restraint) stress was induced to rats for 24 hr. The levels of lipid peroxidation (LP) and mitochondrial function (MF), as well as the superoxide dismutase (SOD) activity and content and reduced glutathione (GSH) levels, were all measured in striatal samples of animals subjected to stress. Our results indicate that acute stress is able to increase the striatal LP and reduced the levels of MF, while significantly lowered the manganese superoxide dismutase (Mn-SOD) activity. No changes were observed in the total striatal content of SOD, nor in GSH levels, but serum corticosterone content was increased by stress. l-CAR exhibited partial protective effects on the immobilized group, reducing the striatal LP and recovering the striatal MF and Mn-SOD activity. Our results suggest that acute restraint stress brings an accurate model for early pro-oxidant responses that can be targeted by broad-spectrum antioxidants like l-CAR.

Diazepam blocks striatal lipid peroxidation and improves stereotyped activity in a rat model of acute stress.

In this work, the effect of a single dose of diazepam was tested on different markers of oxidative damage in the striatum of rats in an acute model of immobilization (restraint) stress. In addition, the locomotor activity was measured at the end of the restraint period. Immobilization was induced to animals for 24 hr, and then, lipid peroxidation, superoxide dismutase activity and content, and mitochondrial function were all estimated in striatal tissue samples. Corticosterone levels were measured in serum. Diazepam was given to rats as a pre-treatment (1 mg/kg, i.p.) 20 min. before the initiation of stress. Our results indicate that acute stress produced enhanced striatal levels of lipid peroxidation (73% above the control), decreased superoxide dismutase activity (54% below the control), reduced levels of mitochondrial function (35% below the control) and increased corticosterone serum levels (86% above the control). Pre-treatment of stressed rats with diazepam decreased the striatal lipid peroxidation levels (68% below the stress group) and improved mitochondrial function (18% above the stress group), but only mild preservation of superoxide dismutase activity was detected (17% above the stress group). In regard to the motor assessment, only the stereotyped activity was increased in the stress group with respect to control (46% above the control), and this effect was prevented by diazepam administration (30% below the stress group). The preventive actions of diazepam in this acute model of stress suggest that drugs exhibiting anxiolytic and antioxidant properties might be useful for the design of therapies against early acute phases of physic stress.