Sandra Peña de Ortiz

CREB required for the stability of new and reactivated fear memories

The cAMP-responsive element binding protein (CREB) family of transcription factors is thought to be critical in memory formation. To define the role of CREB in distinct memory processes, we derived transgenic mice with an inducible and reversible CREB repressor by fusing CREBS133A to a tamoxifen (TAM)–dependent mutant of an estrogen receptor ligand-binding domain (LBD). We found that CREB is crucial for the consolidation of long-term conditioned fear memories, but not for encoding, storage or retrieval of these memories. Our studies also showed that CREB is required for the stability of reactivated or retrieved conditioned fear memories. Although the transcriptional processes necessary for the stability of initial and reactivated memories differ, CREB is required for both. The findings presented here delineate the memory processes that require CREB and demonstrate the power of LBD-inducible transgenic systems in the study of complex cognitive processes.

Consolidation of Fear Extinction Requires Protein Synthesis in the Medial Prefrontal Cortex

Extinction of conditioned fear is thought to form a long-term memory of safety, but the neural mechanisms are poorly understood. Consolidation of extinction learning in other paradigms requires protein synthesis, but the involvement of protein synthesis in extinction of conditioned fear remains unclear. Here, we show that rats infused intraventricularly with the protein synthesis inhibitor anisomycin extinguished normally within a session but were unable to recall extinction the following day. Anisomycin-treated rats showed no savings in the rate of re-learning of extinction, consistent with amnesia for extinction training. The identical effect was observed when anisomycin was microinfused into the medial prefrontal cortex (mPFC) but not the insular cortex. Furthermore, we observed that extinction training increased c-Fos levels in the mPFC but not in the insular cortex, consistent with extinction-induced gene expression in the mPFC. These findings extend previous lesion and unit-recording data by demonstrating that the mPFC is a critical storage site for extinction memory, rather than simply a pathway for expression of extinction. Understanding consolidation of fear extinction could lead to new treatments for anxiety disorders in which fear extinction is thought to be compromised.

Hippocampal gene expression profiling in spatial discrimination learning.

Learning and long-term memory are thought to involve temporally defined changes in gene expression that lead to the strengthening of synaptic connections in selected brain regions. We used cDNA microarrays to study hippocampal gene expression in animals trained in a spatial discrimination-learning paradigm. Our analysis identified 19 genes that showed statistically significant changes in expression when comparing Nai;ve versus Trained animals. We confirmed the changes in expression for the genes encoding the nuclear protein prothymosin(alpha) and the delta-1 opioid receptor (DOR1) by Northern blotting or in situ hybridization. In additional studies, laser-capture microdissection (LCM) allowed us to obtain enriched neuronal populations from the dentate gyrus, CA1, and CA3 subregions of the hippocampus from Nai;ve, Pseudotrained, and spatially Trained animals. Real-time PCR examined the spatial learning specificity of hippocampal modulation of the genes encoding protein kinase B (PKB, also known as Akt), protein kinase C(delta) (PKC(delta)), cell adhesion kinase(beta) (CAK(beta), also known as Pyk2), and receptor protein tyrosine phosphatase(zeta/beta) (RPTP(zeta/beta)). These studies showed subregion specificity of spatial learning-induced changes in gene expression within the hippocampus, a feature that was particular to each gene studied. We suggest that statistically valid gene expression profiles generated with cDNA microarrays may provide important insights as to the cellular and molecular events subserving learning and memory processes in the brain.

Non‐homologous DNA end joining in the mature rat brain

Recent evidence suggests that DNA double strand breaks (DSBs) are introduced in neurons during the course of normal development, and that repair of such DSBs is essential for neuronal survival. Here we describe a non‐homologous DNA end joining (NHEJ) system in the adult rat brain that may be used to repair DNA DSBs. In the brain NHEJ system, blunt DNA ends are joined with lower efficiency than cohesive or non‐matching protruding ends. Moreover, brain NHEJ is blocked by DNA ligase inhibitors or by dATP and can occur in the presence or absence of exogenously added ATP. Comparison of NHEJ activities in several tissues showed that brain and testis share similar mechanisms for DNA end joining, whereas the activity in thymus seems to utilize different mechanisms than in the nervous system. The developmental profile of brain NHEJ showed increasing levels of activity after birth, peaking at postnatal day 12 and then gradually decreasing along with age. Brain distribution analysis in adult animals showed that NHEJ activity is differentially distributed among different regions. We suggest that the DNA NHEJ system may be utilized in the postnatal brain for the repair of DNA double strand breaks introduced within the genome in the postnatal brain.

DNA recombination as a possible mechanism in declarative memory: a hypothesis.

The durability of declarative memory suggests that it has either a chemical or a structural basis. Current models of long‐term memory are based on the general assumption that traces of memory are stored by structural modifications of synaptic connections, resulting in alterations in the patterns of neural activity. Changes in gene expression, regulated at both the transcriptional and the translational levels, are considered essential for structural synaptic modifications. Here we present an alternative hypothesis stating that permanent memory has a chemical rather than a structural basis. We suggest that the mechanism of memory coding in the brain is similar to that in the immune system so that the permanence of memories in the nervous system is ensured at the genomic level by a somatic recombination mechanism. Thus, we hypothesize that traces of permanent declarative memory might present within cerebral neurons in the form of novel proteins coded by the modified genes. This discussion is intended to provide evidence in support of a DNA recombination mechanism for memory storage in the brain and to stimulate further research working toward the evaluation of this hypothesis. J. Neurosci. Res. 63:72–81, 2001.

Different hippocampal activity profiles for PKA and PKC in spatial discrimination learning.

Protein kinases are considered essential for the processing and storage of information in the brain. However, the dynamics of protein kinase activation in the hippocampus during spatial learning are poorly understood. In this study, rats were trained to learn a holeboard spatial discrinmination task and the activity profiles for cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and Ca2+/ phospholipid-dependent protein kinase C (PKC) in the hippocampus were examined. Hippocampal PKA activity increased rapidly on Day 1 of spatial learning and remained moderately high at later stages of acquisition. In contrast, PKC activity increased in particulate fractions compared with cytosolic fractions after habituation training and was maximal at Day 3 of spatial acquisition. The results establish a temporal dissociation between PKA and PKC during acquisition of spatial discrimination learning.

An Inhibitor of DNA Recombination Blocks Memory Consolidation, But Not Reconsolidation, in Context Fear Conditioning

Genomic recombination requires cutting, processing, and rejoining of DNA by endonucleases, polymerases, and ligases, among other factors. We have proposed that DNA recombination mechanisms may contribute to long-term memory (LTM) formation in the brain. Our previous studies with the nucleoside analog 1-β-d-arabinofuranosylcytosine triphosphate (ara-CTP), a known inhibitor of DNA ligases and polymerases, showed that this agent blocked consolidation of conditioned taste aversion without interfering with short-term memory (STM). However, because polymerases and ligases are also essential for DNA replication, it remained unclear whether the effects of this drug on consolidation were attributable to interference with DNA recombination or neurogenesis. Here we show, using C57BL/6 mice, that ara-CTP specifically blocks consolidation but not STM of context fear conditioning, a task previously shown not to require neurogenesis. The effects of a single systemic dose of cytosine arabinoside (ara-C) on LTM were evident as early as 6 h after training. In addition, although ara-C impaired LTM, it did not impair general locomotor activity nor induce brain neurotoxicity. Importantly, hippocampal, but not insular cortex, infusions of ara-C also blocked consolidation of context fear conditioning. Separate studies revealed that context fear conditioning training significantly induced nonhomologous DNA end joining activity indicative of DNA ligase-dependent recombination in hippocampal, but not cortex, protein extracts. Finally, unlike inhibition of protein synthesis, systemic ara-C did not block reconsolidation of context fear conditioning. Our results support the idea that DNA recombination is a process specific to consolidation that is not involved in the postreactivation editing of memories.

Chronic lithium decreases Nurr1 expression in the rat brain and impairs spatial discrimination

Lithium (Li+) is a drug used for the treatment of neuropsychiatric disorders, whereas Nuclear receptor-related factor 1 (Nurr1) has been implicated in normal and aberrant cognitive processes. Li+s effects on cognition and Nurr1 expression were examined. Rats were exposed to Li+ in their diet for 4 weeks and only those reaching Li+ blood concentrations within the established clinically therapeutic range were used. Li+ decreased rearing activity in rats, but did not affect horizontal locomotion nor object recognition memory. In contrast, Li+ treated animals were significantly impaired in the initial, but not late, stages of acquisition of a hippocampal-dependent spatial discrimination task. In agreement with the behavioral results, chronic Li+ caused a significant downregulation of basal Nurr1 expression in several brain regions. In particular, a significant negative correlation between Li+ blood levels and Nurr1 expression was identified in the CA1 hippocampal subregion, but not in CA3, perirhinal cortex or the dorsal endopiriform nucleus. Upregulation of hippocampal Nurr1 levels to those of controls were observed in Li+ treated rats following training in the spatial task. Overall, the results suggest that the effects of Li+ on the brain may be particularly relevant to hippocampal-dependent cognitive processes involving Nurr1 expression.

Spatial learning in rats is impaired by microinfusions of protein kinase C-γ antisense oligodeoxynucleotide within the nucleus accumbens

The nucleus accumbens (NAcc) has been shown to play a role in motor and spatial learning. Protein kinase C (PKC) has been implicated in the mechanisms of initiation and maintenance of long-term potentiation that is thought to be involved in the storage of long-term memory. In the present study, the importance of de novo synthesis of PKC-gamma within the NAcc in the acquisition and retention of spatial discrimination learning was assessed using an antisense knockdown approach. Separate groups of Long-Evans rats were exposed to acute microinfusions (6microg/microl) of PKC-gamma antisense oligodeoxynucleotide (AS-ODN), control oligodeoxynucleotide (C-ODN) or vehicle into the NAcc at 24 and 3h before each training session. Behavioral findings showed that the blockade of NAcc-PKC-gamma translation caused impairments in the early phase of learning and retention of spatial information. Biochemical experiments showed that PKC-gamma expression was reduced and Ca(2+)/phospholipid-dependent protein kinase C (PKC) activity was blocked significantly in the AS-ODN-treated rats in comparison with control rats. The present findings suggest that NAcc-PKC-gamma plays a role during the early acquisition of spatial learning. Also, retention test results suggest that NAcc-PKC-gamma may be working as an intermediate factor involved in the onset of molecular mechanisms necessary for spatial memory consolidation within the NAcc.

Intracranial self-stimulation facilitates active-avoidance retention and induces expression of c-Fos and Nurr1 in rat brain memory systems

Intracranial self-stimulation (ICSS), a special form of deep brain stimulation in which subjects self-administered electrical stimulation in brain reward areas as the lateral hypothalamus, facilitates learning and memory in a wide variety of tasks. Assuming that ICSS improves learning and memory increasing the activation of memory-related brain areas, the present work examined whether rats receiving an ICSS treatment immediately after the acquisition session of a two-way active avoidance conditioning (TWAA) show both an improved retention and a pattern of increased c-Fos and Nurr1 protein expression in the amygdala, hippocampus, dorsal striatum and/or lateral hypothalamus. The response of both activity-induced IEGs to ICSS was examined not only as markers of neural activation, but because of their reported role in the neural plasticity occurring during learning and memory formation. Results showed that the TWAA conditioning alone increased the expression of the two analysed IEGs in several hippocampal areas, and TWAA retention increased Nurr1 expression in amygdala. ICSS treatment increased the number of c-Fos and Nurr1 positive cells in almost all the brain regions studied when it was measured 70min, but not 48h, after the stimulation. Post-training ICSS treatment, as expected, facilitated the 48h retention of the conditioning. It is noteworthy that in CA3 conditioning and ICSS separately increased c-Fos expression, but this increasing was greater when both, conditioning and ICSS, were combined. Present results suggest that rapid and transient increased expression of these two synaptic plasticity and memory related IEGs in some hippocampal areas, such as CA3, could mediate the facilitative effects of ICSS on learning and memory consolidation.