The extracellular protease matrix metalloproteinase-9 is activated by inhibitory avoidance learning and required for long-term memory

Matrix metalloproteinases (MMPs) are a family of extracellularly acting proteolytic enzymes with well-recognized roles in plasticity and remodeling of synaptic circuits during brain development and following brain injury. However, it is now becoming increasingly apparent that MMPs also function in normal, nonpathological synaptic plasticity of the kind that may underlie learning and memory. Here, we extend this idea by investigating the role and regulation of MMP-9 in an inhibitory avoidance (IA) learning and memory task. We demonstrate that following IA training, protein levels and proteolytic activity of MMP-9 become elevated in hippocampus by 6 h, peak at 12-24 h, then decline to baseline values by approximately 72 h. When MMP function is abrogated by intrahippocampal infusion of a potent gelatinase (MMP-2 and MMP-9) inhibitor 3.5 h following IA training, a time prior to the onset of training-induced elevation in levels, IA memory retention is significantly diminished when tested 1-3 d later. Animals impaired at 3 d exhibit robust IA memory when retrained, suggesting that such impairment is not likely attributed to toxic or other deleterious effects that permanently disrupt hippocampal function. In anesthetized adult rats, the effective distance over which synaptic plasticity is impaired by a single intrahippocampal infusion of the MMP inhibitor of the kind that blocks IA memory is approximately 1200 microm. Taken together, these data suggest that IA training induces a slowly emerging, but subsequently protracted period of MMP-mediated proteolysis critical for enabling long-lasting synaptic modification that underlies long-term memory consolidation.     

Memory-enhancing corticosterone treatment increases amygdala norepinephrine and Arc protein expression in hippocampal synaptic fractions

Considerable evidence indicates that glucocorticoid hormones enhance the consolidation of memory for emotionally arousing events through interactions with the noradrenergic system of the basolateral complex of the amygdala (BLA). We previously reported that intra-BLA administration of a β-adrenoceptor agonist immediately after inhibitory avoidance training enhanced memory consolidation and increased hippocampal expression of the protein product of the immediate early gene activity-regulated cytoskeletal-associated protein (Arc). In the present experiments corticosterone (3 mg/kg, i.p.) was administered to male Sprague-Dawley rats immediately after inhibitory avoidance training to examine effects on long-term memory, amygdala norepinephrine levels, and hippocampal Arc expression. Corticosterone increased amygdala norepinephrine levels 15 min after inhibitory avoidance training, as assessed by in vivo microdialysis, and enhanced memory tested at 48 h. Corticosterone treatment also increased expression of Arc protein in hippocampal synaptic tissue. The elevation in BLA norepinephrine appears to participate in corticosterone-influenced modulation of hippocampal Arc expression as intra-BLA blockade of β-adrenoceptors with propranolol (0.5 μg/0.2 μL) attenuated the corticosterone-induced synaptic Arc expression in the hippocampus. These findings indicate that noradrenergic activity at BLA β-adrenoceptors is involved in corticosterone-induced enhancement of memory consolidation and expression of the synaptic-plasticity-related protein Arc in the hippocampus.     

Networks of neurons, networks of genes: an integrated view of memory consolidation

Investigations into the mechanisms of memory formation have abided by the central tenet of the consolidation theory-that memory formation occurs in stages which differ in their requirement for protein synthesis. The current most widely accepted hypothesis posits that new memories are encoded as neural activity-induced changes in synaptic efficacy, and stabilization of these changes requires de novo protein synthesis. However, the basic assumptions of this view have been challenged by concerns regarding the specificity of the effects of the protein synthesis inhibitors used to support the claim. Studies on immediate-early genes (IEGs), in particular Arc, provide a distinct and independent perspective on the issue of the requirement of new protein synthesis in synaptic plasticity and memory consolidation. The IEG Arc and its protein are dynamically induced in response to neuronal activity, and are directly involved in synaptic plasticity and memory consolidation. Although we provide extensive data on Arc's properties to address the requirement of genomic and proteomic responses in memory formation, Arc is merely one element in a network of genes that interact in a coordinated fashion to serve memory consolidation. From gene expression and other studies, we propose the view that the stabilization of a memory trace is a continuous and ongoing process, which does not have a discrete endpoint and cannot be reduced to a single deterministic "molecular cascade". Rather, memory traces are maintained within metastable networks, which must integrate and update past traces with new ones. Such an updating process may well recruit and use many of the plasticity mechanisms necessary for the initial encoding of memory.     

Involvement of the rostral anterior cingulate cortex in consolidation of inhibitory avoidance memory: interaction with the basolateral amygdala

Previous findings suggest that the rostral anterior cingulate cortex (rACC) is involved in memory for emotionally arousing training. There is also extensive evidence that the basolateral amygdala (BLA) modulates the consolidation of emotional arousing training experiences via interactions with other brain regions. The present experiments examined the effects of posttraining intra-rACC infusions of the cholinergic agonist oxotremorine (OXO) on inhibitory avoidance (IA) retention and investigated whether the BLA and rACC interact in enabling OXO effects on memory. In the first experiment, male Sprague-Dawley rats were implanted with bilateral cannulae above the rACC and given immediate posttraining OXO infusions. OXO (0.5 or 3 ng) induced significant enhancement of retention performance on a 48-h test. In the second experiment, unilateral posttraining OXO infusions (0.5, 3.0 or 10 ng) enhanced retention when infused into rACC, but not caudal ACC, consistent with previous evidence that ACC is composed of functionally distinct regions. A third experiment investigated the effects of posttraining intra-rACC OXO infusions (0.5 or 10 ng) in rats with bilateral sham or NMDA-induced lesions of the BLA. The BLA lesions did not impair IA retention, but blocked the enhancement induced by posttraining intra-rACC OXO infusions. Lastly, unilateral NMDA lesions of rACC blocked the enhancement of IA retention induced by posttraining ipsilateral OXO infusions into the BLA. These findings support the hypothesis that the rACC is involved in modulating the storage of emotional events and provide additional evidence that the BLA modulates memory consolidation through interactions with efferent brain regions, including the cortex.     

Inhibition of mTOR by rapamycin in the amygdala or hippocampus impairs formation and reconsolidation of inhibitory avoidance memory

Mammalian target of rapamycin (mTOR), a central regulator of protein synthesis in neurons, has been implicated in synaptic plasticity and memory. Here we show that mTOR inhibition by rapamycin in the basolateral amygdala (BLA) or dorsal hippocampus (DH) impairs both formation and reconsolidation of memory for inhibitory avoidance (IA) in rats. Male Wistar rats received bilateral infusions of vehicle or rapamycin into the BLA or DH before or after IA training or retrieval. Memory retention was tested at different time points after drug infusion. Rapamycin impaired long-term IA retention when given before or immediately after training or retrieval into the BLA. When infused into the DH, rapamycin produced memory impairment when given before training or immediately after retrieval. The impairing effects of post-retrieval rapamycin required memory retrieval and were not reversed by a reminder shock. The results provide the first evidence that mTOR in the BLA and DH might play a role in IA memory reconsolidation.     

Consolidation and translation regulation

mRNA translation, or protein synthesis, is a major component of the transformation of the genetic code into any cellular activity. This complicated, multistep process is divided into three phases: initiation, elongation, and termination. Initiation is the step at which the ribosome is recruited to the mRNA, and is regarded as the major rate-limiting step in translation, while elongation consists of the elongation of the polypeptide chain; both steps are frequent targets for regulation, which is defined as a change in the rate of translation of an mRNA per unit time. In the normal brain, control of translation is a key mechanism for regulation of memory and synaptic plasticity consolidation, i.e., the off-line processing of acquired information. These regulation processes may differ between different brain structures or neuronal populations. Moreover, dysregulation of translation leads to pathological brain function such as memory impairment. Both normal and abnormal function of the translation machinery is believed to lead to translational up-regulation or down-regulation of a subset of mRNAs. However, the identification of these newly synthesized proteins and determination of the rates of protein synthesis or degradation taking place in different neuronal types and compartments at different time points in the brain demand new proteomic methods and system biology approaches. Here, we discuss in detail the relationship between translation regulation and memory or synaptic plasticity consolidation while focusing on a model of cortical-dependent taste learning task and hippocampal-dependent plasticity. In addition, we describe a novel systems biology perspective to better describe consolidation.     

Task-dependent role for dorsal striatum metabotropic glutamate receptors in memory

The effect of post-training intradorsal striatal infusion of metabotropic glutamate receptor (mGluR) drugs on memory consolidation processes in an inhibitory avoidance (IA) task and visible/hidden platform water maze tasks was examined. In the IA task, adult male Long-Evans rats received post-training intracaudate infusions of the broad spectrum mGluR antagonist α-methyl-4-carboxyphenylglycine (MCPG; 1.0, 2.0 mM/0.5 μL), the group I/II mGluR agonist 1-aminocyclopentane-1,3-carboxylic acid (ACPD; 0.5 or 1.0 μM/0.5 μL), or saline immediately following footshock training, and retention was tested 24 h later. In the visible- and hidden-platform water maze tasks, rats received post-training intracaudate infusions of ACPD (1.0 μM), MCPG (2.0 mM), or saline immediately following an eight-trial training session, followed by a retention test 24 h later. In the IA task, post-training infusion of ACPD (0.5 and 1.0 μM) or MCPG (1.0 and 2.0 mM) impaired retention. In the IA and visible-platform water maze tasks, post-training infusion of ACPD (1.0 μM), or MCPG (2.0 mM) impaired retention. In contrast, neither drug affected retention when administered post-training in the hidden-platform task, consistent with the hypothesized role of the dorsal striatum in stimulus-response habit formation. When intradorsal striatal injections were delayed 2 h post-training in the visible-platform water maze task, neither drug affected retention, indicating a time-dependent effect of the immediate post-training injections on memory consolidation. It is hypothesized that MCPG impaired memory via a blockade of postsynaptic dorsal striatal mGluR's, while the impairing effect of ACPD may have been caused by an influence of this agonist on presynaptic “autoreceptor” striatal mGluR populations.     

Post-training intra-basolateral amygdala infusions of norepinephrine block sevoflurane-induced impairment of memory consolidation and activity-regulated cytoskeletal protein expression inhibition in rat hippocampus

Considerable evidence indicates that the noradrenergic system of the basolateral amygdala (BLA) participates in the consolidation of various types of emotionally arousing memories. We previously reported that administration of an anesthetic-dose of sevoflurane immediately after continuous multiple-trail inhibition avoidance (CMIA) training impaired memory consolidation. This experiment investigated whether posttraining noradrenergic activation of the BLA is sufficient to reverse the memory impairing effect of sevoflurane. Adult male Sprague-Dawley rats received bilateral injections of norepinephrine (NE 0.3, 1.0, or 3.0 μg/0.5 μl) or normal saline (NS 0.5 μl) immediately after training in a CMIA paradigm. Subsequently, the rats were exposed to sevoflurane (2% inspired) or air for 2h. Norepinephrine produced a dose-dependent enhancement of memory consolidation on a 24-h retention test. The highest dose of NE tested (3.0 μg/0.5 μl) blocked sevoflurane-induced impairment of memory consolidation and reversed the inhibitory effect of sevoflurane on activity-regulated cytoskeletal protein (Arc) expression in the hippocampus 2h after training. These findings provide evidence that the mechanism mediating the memory-impairing effect of sevoflurane involves a network interaction between the BLA noradrenergic system and modulation of Arc protein expression in the hippocampus.     

Modulation of memory consolidation by the basolateral amygdala or nucleus accumbens shell requires concurrent dopamine receptor activation in both brain regions

Previous findings indicate that the basolateral amygdala (BLA) and the nucleus accumbens (NAc) interact in influencing memory consolidation. The current study investigated whether this interaction requires concurrent dopamine (DA) receptor activation in both brain regions. Unilateral, right-side cannulae were implanted into the BLA and the ipsilateral NAc shell or core in male Sprague-Dawley rats ( approximately 300 g). One week later, the rats were trained on an inhibitory avoidance (IA) task and, 48 h later, they were tested for retention. Drugs were infused into the BLA and NAc shell or core immediately after training. Post-training intra-BLA infusions of DA enhanced retention, as assessed by latencies to enter the shock compartment on the retention test. Infusions of the general DA receptor antagonist cis-Flupenthixol (Flu) into the NAc shell (but not the core) blocked the memory enhancement induced by the BLA infusions of DA. In the reverse experiment, post-training intra-NAc shell infusions of DA enhanced retention and Flu infusions into the BLA blocked the enhancement. These findings indicate that BLA modulation of memory consolidation requires concurrent DA receptor activation in the NAc shell but not the core. Similarly, NAc shell modulation of memory consolidation requires concurrent DA receptor activation in the BLA. Together with previous findings, these results suggest that the dopaminergic innervation of the BLA and NAc shell is critically involved in the modulation of memory consolidation.     

mTOR signaling in the hippocampus is necessary for memory formation

It is widely accepted that the formation of long-term memory (LTM) requires mRNA translation, but little is known about the cellular mechanisms in the brain that regulate this process. Mammalian target of rapamycin (mTOR) is a key regulator of translational efficacy and capacity. Here, we show that LTM formation of one-trial inhibitory avoidance (IA) in rats, a hippocampus-dependent fear-motivated learning task, requires mTOR activation. IA training is specifically associated with a rapid increase in the phosphorylation state of mTOR and its substrate ribosomal S6 kinase (p70S6K). Bilateral intra-CA1 infusion of rapamycin, a selective mTOR inhibitor, 15 min before, but not immediately after training completely hinders IA LTM without affecting short-term memory (STM) retention. Therefore, our findings indicate that the regulation of hippocampal mRNA translation is a major control step in memory consolidation.     

Endogenous BDNF is required for long-term memory formation in the rat parietal cortex

Information storage in the brain is a temporally graded process involving different memory phases as well as different structures in the mammalian brain. Cortical plasticity seems to be essential to store stable long-term memories, although little information is available at the moment regarding molecular and cellular events supporting memory consolidation in the neocortex. Brain-derived neurotrophic factor (BDNF) modulates both short-term synaptic function and activity-dependent synaptic plasticity in hippocampal and cortical neurons. We have recently demonstrated that endogenous BDNF in the hippocampus is involved in memory formation. Here we examined the role of BDNF in the parietal cortex (PCx) in short-term (STM) and long-term memory (LTM) formation of a one-trial fear-motivated learning task in rats. Bilateral infusions of function-blocking anti-BDNF antibody into the PCx impaired both STM and LTM retention scores and decreased the phosphorylation state of cAMP response element-binding protein (CREB). In contrast, intracortical administration of recombinant human BDNF facilitated LTM and increased CREB activation. Moreover, inhibitory avoidance training is associated with a rapid and transient increase in phospho-CREB/total CREB ratio in the PCx. Thus, our results indicate that endogenous BDNF is required for both STM and LTM formation of inhibitory avoidance learning, possibly involving CREB activation-dependent mechanisms. The present data support the idea that early sensory areas constitute important components of the networks subserving memory formation and that information processing in neocortex plays an important role in memory formation.     

Involvement of the basolateral amygdala in muscarinic cholinergic modulation of extinction memory consolidation

Previous studies have reported that drugs affecting neuromodulatory systems within the basolateral amygdala (BLA), including drugs affecting muscarinic cholinergic receptors, modulate the consolidation of many kinds of training, including contextual fear conditioning (CFC). The present experiments investigated the involvement of muscarinic cholinergic influences within the BLA in modulating the consolidation of CFC extinction memory. Male Sprague Dawley rats implanted with unilateral cannula aimed at the BLA were trained on a CFC task, using footshock stimulation, and 24 and 48 h later were given extinction training by replacing them in the apparatus without footshock. Following each extinction session they received intra-BLA infusions of the cholinergic agonist oxotremorine (10 ng). Immediate post-extinction BLA infusions significantly enhanced extinction but infusions administered 180 min after extinction training did not influence extinction. Thus the oxotremorine effects were time-dependent and not attributable to non-specific effects on retention performance. These findings provide evidence that, as previously found with original CFC learning, cholinergic activation within the BLA modulates the consolidation of CFC extinction.     

Participation of hippocampal cholinergic system in memory persistence for inhibitory avoidance in rats

Memory persistence needs a new event of consolidation 12h after the acquisition. We investigated the role of the cholinergic activity on the persistence of memory. For this purpose, we performed the treatments 9 or 12h after acquisition and the memory tested 2 or 7 days after inhibitory avoidance (IA) training. Here we report that activity of medial septum, by transitorily inactivating this structure with lidocaine 12h after IA training, is essential for memory persistence at the 7th day, but not for the formation at the 2nd day. We also report that muscarinic and nicotinic cholinergic receptors of CA1 area are engaged on memory persistence. Since scopolamine (mAChRs antagonist) and mecamylamine (nAChRs blocker) infusions, 12h post-training, demonstrated impairment on long term memory (LTM), persistence on the 7th day but no effect on LTM formation was found on the 2nd day in the IA test. The same effects were found with pirenzepine, an M1 antagonist. No effects on the formation and persistence of memory on the 2nd and 7th days were demonstrated after DHβE infusions (nAChRs subtype antagonist α4β2, α3β2). These findings suggest that mAChR and nAChR at the CA1 area, and also MS activation, are required for the persistence of memory.     

Enhancement of inhibitory avoidance and conditioned taste aversion memory with insular cortex infusions of 8-Br-cAMP: involvement of the basolateral amygdala

There is considerable evidence that in rats, the insular cortex (IC) and amygdala are involved in the learning and memory of aversively motivated tasks. The present experiments examined the effects of 8-Br-cAMP, an analog of cAMP, and oxotremorine, a muscarinic agonist, infused into the IC after inhibitory avoidance (IA) training and during the acquisition/consolidation of conditioned taste aversion (CTA). Posttraining infusion into the IC of 0.3 microg oxotremorine and 1.25 microg 8-Br-cAMP enhanced IA retention. Infusions of 8-Br-cAMP, but not oxotremorine, into the IC enhanced taste aversion. The experiments also examined whether noradrenergic activity in the basolateral amygdala (BLA) is critical in enabling the enhancement of CTA and IA memory induced by drug infusions administered into the IC. For both CTA and IA, ipsilateral infusions of beta-adrenergic antagonist propranolol administered into the BLA blocked the retention-enhancing effect of 8-Br-cAMP or oxotremorine infused into the IC. These results indicate that the IC is involved in the consolidation of memory for both IA and CTA, and this effect requires intact noradrenergic activity into the BLA. These findings provide additional evidence that the BLA interacts with other brain regions, including sensory cortex, in modulating memory consolidation.     

Hippocampal c-fos is necessary for long-term memory of a socially transmitted food preference

The present article examined the requirement of hippocampal c-Fos for learning a socially transmitted food preference (STFP). We reported previously that expression of the c-Fos protein is increased in the dorsal and ventral hippocampus of rats trained on the STFP (Countryman, Orlowski, Brightwell, Oskowitz, & Colombo, 2005). Pretraining intrahippocampal antisense to the immediate early gene c-fos was administered to adult male Long-Evans rats to determine if c-fos expression is necessary for either short- or long-term memory for STFP. Guide cannulae were implanted bilaterally into the dorsal hippocampus. Antisense oligodeoxynucleotides (ODNs) were administered unilaterally either 6.5, 8.5, 10.5, or 12.5 h prior to STFP training while either sense ODNs or saline were infused into the opposite hemisphere. Immunocytochemistry was performed, and cells showing c-Fos immunoreactivity (ir) were counted from the antisense-treated hemisphere and compared to cell counts from the control hemisphere. The results indicated significant suppression of learning-induced c-Fos protein at the 8.5 and 10.5 infusion-train intervals. Additional rats were implanted with cannulae into the dorsal and ventral hippocampus, and antisense ODNs, sense ODNs, or saline were administered bilaterally 8.5h prior to training. Rats were tested immediately and 14 days after training. Rats in all groups showed a significant preference for the demonstrated food at the short-term memory test. At the long-term memory test, however, rats infused with c-fos antisense showed no preference for the demonstrated food whereas rats infused with either sense or saline maintained their preference. The present findings suggest that c-fos is necessary for consolidation of non-spatial hippocampal-dependent memory.     

Conditioned taste aversion modifies persistently the subsequent induction of neocortical long-term potentiation in vivo

The ability of neurons to modify their synaptic strength in an activity-dependent manner has a crucial role in learning and memory processes. It has been proposed that homeostatic forms of plasticity might provide the global regulation necessary to maintain synaptic strength and plasticity within a functional dynamic range. Similarly, it is considered that the capacity of synapses to express plastic changes is itself subject to variation dependent on previous experience. In particular, training in several behavioral tasks modifies the possibility to induce long-term potentiation (LTP). Our previous studies in the insular cortex (IC) have shown that induction of LTP in the basolateral amygdaloid nucleus (Bla)-IC projection previous to conditioned taste aversion (CTA) training enhances the retention of this task. The aim of the present study was to analyze whether CTA training modifies the ability to induce subsequent LTP in the Bla-IC projection in vivo. Thus, CTA trained rats received high frequency stimulation in the Bla-IC projection in order to induce LTP 48, 72, 96 and 120 h after the aversion test. Our results show that CTA training prevents the subsequent induction of LTP in the Bla-IC projection, for at least 120 h after CTA training. We also showed that pharmacological inhibition of CTA consolidation with anisomycin (1 μl/side; 100 μg/μl) prevents the CTA effect on IC-LTP. These findings reveal that CTA training produces a persistent change in the ability to induce subsequent LTP in the Bla-IC projection in a protein-synthesis dependent manner, suggesting that changes in the ability to induce subsequent synaptic plasticity contribute to the formation and persistence of aversive memories.     

The NO-cGMP-PKG signaling pathway regulates synaptic plasticity and fear memory consolidation in the lateral amygdala via activation of ERK/MAP kinase

Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and long-term potentiation (LTP) at thalamic and cortical input pathways to the LA. In behavioral experiments, rats given intra-LA infusions of either the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibited dose-dependent impairments or enhancements of fear memory consolidation, respectively. In slice electrophysiology experiments, bath application of Rp-8-Br-PET-cGMPS or the guanylyl cyclase inhibitor LY83583 impaired LTP at thalamic, but not cortical inputs to the LA, while bath application of 8-Br-cGMP or the guanylyl cyclase activator YC-1 resulted in enhanced LTP at thalamic inputs to the LA. Interestingly, YC-1-induced enhancement of LTP in the LA was reversed by concurrent application of the MEK inhibitor U0126, suggesting that the NO-cGMP-PKG signaling pathway may promote synaptic plasticity and fear memory formation in the LA, in part by activating the ERK/MAPK signaling cascade. As a test of this hypothesis, we next showed that rats given intra-LA infusion of the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibit impaired or enhanced activation, respectively, of ERK/MAPK in the LA after fear conditioning. Collectively, our findings suggest that an NO-cGMP-PKG-dependent form of synaptic plasticity at thalamic input synapses to the LA may underlie memory consolidation of Pavlovian fear conditioning, in part, via activation of the ERK/MAPK signaling cascade.