Cedomir Todorovic

The Central Nucleus of the Amygdala and Corticotropin-Releasing Factor: Insights into Contextual Fear Memory

The central nucleus of the amygdala (CeA) has been traditionally viewed in fear conditioning to serve as an output neural center that transfers conditioned information formed in the basolateral amygdala to brain structures that generate emotional responses. Recent studies suggest that the CeA may also be involved in fear memory consolidation. In addition, corticotropin-releasing factor systems were shown to facilitate memory consolidation in the amygdala, which contains a high density of CRF immunoreactive cell bodies and fibers in the lateral part of the CeA (CeAl). However, the involvement of CeA CRF in contextual fear conditioning remains poorly understood. Therefore, we first conducted a series of studies using fiber-sparing lesion and reversible inactivation methods to assess the general role of the CeA in contextual fear. We then used identical training and testing procedures to compare and evaluate the specific function of CeA CRF using CRF antisense oligonucleotides (CRF ASO). Rats microinjected with ibotenic acid, muscimol, or a CRF ASO into the CeA before contextual fear conditioning showed typical levels of freezing during acquisition training but exhibited significant reductions in contextual freezing in a retention test 48 h later. Furthermore, CeA inactivation induced by either muscimol or CRF ASO administration immediately before retention testing did not impair freezing, suggesting that the previously observed retention deficits were caused by inhibition of consolidation rather than fear expression. Collectively, our results suggest CeA involvement in the consolidation of contextual fear memory and specifically implicate CeA CRF as an important mediator.

Suppression of the MEK/ERK signaling pathway reverses depression-like behaviors of CRF2-deficient mice

The neuropeptide corticotropin-releasing factor (CRF) plays a critical role in the proper functioning of the stress response system through its actions on its receptors, CRF receptor 1 (CRF1) and CRF receptor 2 (CRF2), located at multiple anatomical sites. Clinical data indicate that stress response dysfunctions, such as excessive CRF activity and hyperstimulation of CRF1, are present in a range of stress-related disorders, including depression and anxiety disorders. Our previous work along with that of other laboratories has demonstrated that mice deficient in CRF2 (CRF2−/−) display increased anxiety and depression-like behaviors. In this study, we found CRF2−/− mice display increased hippocampal levels of activated (phosphorylated) mitogen-activated protein kinase (MAP kinase)/ERK kinase (MEK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and ribosomal protein S6 kinases 1 (RSK1). These changes can be explained by overactive hippocampal CRF1, in view of the finding that the application of the nonselective CRF receptor antagonist [Glu11,16] astressin ([Glu11,16]Ast) into the dorsal hippocampus of mutant mice returned the levels of the phosphorylated proteins to baseline. Moreover, inhibition of the hippocampal MEK/ERK pathway with the specific MEK inhibitor U0126, decreased depression-like behaviors in the forced swim test and tail suspension test of CRF2−/− mice. Similarly, treatment with [Glu11,16]Ast reversed depression phenotype of CRF2−/− mice without affecting the phenotype of wild-type littermates. Our results support an involvement of CRF receptors in the development of depression, such that elevated hippocampal CRF1 activity, in the absence of CRF2, produces a depression-dominated phenotype through the activation of the MEK/ERK pathway.

Differential activation of CRF receptor subtypes removes stress-induced memory deficit and anxiety.

The objective of this study was to investigate the role of corticotropin‐releasing factor receptors 1 (CRF1) and 2 (CRF2) in anxiety‐like behavior and learning of C57BL/6J mice after exposure to a stressful stimulus. When C57BL/6J mice were exposed to immobilization (1 h) serving as stressful stimulus, context‐ and tone‐dependent fear conditioning were impaired if the training followed immediately after immobilization. The stress‐induced impairment of context‐dependent fear conditioning was prevented by specific blockade of CRF2 of the lateral septum (LS) with anti‐sauvagine‐30. Immobilization did not only affect conditioned fear, but also enhanced, through CRF2 of the LS, anxiety‐like behavior determined with the elevated plus maze. Recovery from stress‐induced anxiety and impairment of context‐dependent fear conditioning was observed after 1 h delay of training and required hippocampal CRF1, as indicated by the finding that this recovery was prevented by blockade of intrahippocampal CRF1. It was concluded that exposure to a stressor initially affected both anxiety‐like behavior and contextual conditioned fear through septal CRF2, while the later activation of hippocampal CRF1 resulted in the return to baseline levels of both processes. Intraventricular injection of mouse urocortin 2, a CRF2‐selective agonist, removed the stress‐induced anxiety and learning impairment, but did not reduce the activation of the hypothalamic pituitary adrenal axis indicative of the hormonal stress response. We propose that the enhanced anxiety is the component of the stress response responsible for the memory deficit.

The role of CRF receptors in anxiety and depression: Implications of the novel CRF1 agonist cortagine

Corticotropin-releasing factor (CRF), a 41 amino acid peptide exhibits its actions through two pharmacologically distinct CRF receptor subtypes CRF(1) and CRF(2). Regulation of the relative contribution of the two CRF receptors to central CRF activity may be essential in coordinating physiological responses to stress. To facilitate the analysis of their differential involvement, we recently developed a CRF(1)-selective agonist cortagine by synthesis of chimeric peptides derived from human/rat CRF, ovine CRF, and sauvagine. Cortagine was analyzed in behavioral experiments using male wild type and CRF(2)-deficient C57BL/6J mice for its action on anxiety- and depression-like behaviors. In contrast to the current hypothesis that increased CRF(1) activity facilitates the expression of anxiety- and depression-like behavior, cortagine combines anxiogenic properties with antidepressant effects. In this article, we show that antidepressant effects are partially mediated by CRF(1) of the dorsal hippocampus. Possible pathways responsible for the paradoxical antidepressant effects observed after CRF(1) activation are discussed.

Hippocampal c-Jun-N-Terminal Kinases Serve as Negative Regulators of Associative Learning

In the adult mouse, signaling through c-Jun N-terminal kinases (JNKs) links exposure to acute stress to various physiological responses. Inflammatory cytokines, brain injury and ischemic insult, or exposure to psychological acute stressors induce activation of hippocampal JNKs. Here we report that exposure to acute stress caused activation of JNKs in the hippocampal CA1 and CA3 subfields, and impaired contextual fear conditioning. Conversely, intrahippocampal injection of JNKs inhibitors sp600125 (30 μm) or d-JNKI1 (8 μm) reduced activity of hippocampal JNKs and rescued stress-induced deficits in contextual fear. In addition, intrahippocampal administration of anisomycin (100 μg/μl), a potent JNKs activator, mimicked memory-impairing effects of stress on contextual fear. This anisomycin-induced amnesia was abolished after cotreatment with JNKs selective inhibitor sp600125 without affecting anisomycins ability to effectively inhibit protein synthesis as measured by c-Fos immunoreactivity. We also demonstrated milder and transient activation of the JNKs pathway in the CA1 subfield of the hippocampus during contextual fear conditioning and an enhancement of contextual fear after pharmacological inhibition of JNKs under baseline conditions. Finally, using combined biochemical and transgenic approaches with mutant mice lacking different members of the JNK family (Jnk1, Jnk2, and Jnk3), we provided evidence that JNK2 and JNK3 are critically involved in stress-induced deficit of contextual fear, while JNK1 mainly regulates baseline learning in this behavioral task. Together, these results support the possibility that hippocampal JNKs serve as a critical molecular regulator in the formation of contextual fear.

Deletion of selenoprotein P results in impaired function of parvalbumin interneurons and alterations in fear learning and sensorimotor gating

One of the primary lines of defense against oxidative stress is the selenoprotein family, a class of proteins that contain selenium in the form of the 21st amino acid, selenocysteine. Within this class of proteins, selenoprotein P (Sepp1) is unique, as it contains multiple selenocysteine residues and is postulated to act in selenium transport. Recent findings have demonstrated that neuronal selenoprotein synthesis is required for the development of parvalbumin (PV)-interneurons, a class of GABAergic neurons involved in the synchronization of neural activity. To investigate the potential influence of Sepp1 on PV-interneurons, we first mapped the distribution of the Sepp1 receptor, ApoER2, and parvalbumin in the mouse brain. Our results indicate that ApoER2 is highly expressed on PV-interneurons in multiple brain regions. Next, to determine whether PV-interneuron populations are affected by Sepp1 deletion, we performed stereology on several brain regions in which we observed ApoER2 expression on PV-interneurons, comparing wild-type and Sepp1(-/-) mice. We observed reduced numbers of PV-interneurons in the inferior colliculus of Sepp1(-/-) mice, which corresponded with a regional increase in oxidative stress. Finally, as impaired PV-interneuron function has been implicated in several neuropsychiatric conditions, we performed multiple behavioral tests on Sepp1(-/-) mice. Our behavioral results indicate that Sepp1(-/-) mice have impairments in contextual fear extinction, latent inhibition, and sensorimotor gating. In sum, these findings demonstrate the important supporting role of Sepp1 on ApoER2-expressing PV-interneurons.

Behavior and severity of adjuvant arthritis in four rat strains

Previous research has suggested that behavioral traits of the histocompatible Lewis and Fischer strains of rats could be related to the difference in their susceptibility to adjuvant arthritis (AA). In the present study, the predictive value of behavioral markers in susceptibility to AA was investigated in nonhistocompatible inbred DA, Lewis, Albino Oxford (AO), and outbred Wistar strain. Behavioral profiles (open filed test and forced swim test) were determined prior to immunization with a single intradermal injection of complete Freunds adjuvant. Animals were daily scored for clinical signs of AA. The occurrence of certain behaviors and clinical indices of AA was significantly associated with strain membership. Discriminant analysis identified strain-related behavioral and illness profiles with very few overlaps among the phenotypes. Discriminant classification significantly exceeded the proportion of cases, which could have been correctly classified on the basis of chance. Open field behavior, in particular, exploration and grooming, differentiated among AA-susceptible and AA-resistant strains. Multiple regression analysis indicated that severity of AA (maximum clinical sign) can be predicted by the latency time and grooming behavior in the open field independently of strain membership. No clear distinction between AA-susceptible and AA-resistant strains was found with respect to forced swim test immobility. It was concluded that (a) strain-related genetic predisposition is important for the expression of certain behavioral traits and for susceptibility to AA and (b) open field behaviors, particularly grooming and latency, predict susceptibility to AA across different rat strains.

c-Jun N-terminal kinases in memory and synaptic plasticity.

Abstract The c-Jun N-terminal kinases (JNK) belong to the subfamily of mitogen-activated protein kinases (MAPK). JNK is an important signaling enzyme that is involved in many facets of cellular regulation including gene expression, cell proliferation and programmed cell death. Activation of JNK isoforms (JNK1, 2, and 3) is regarded as a molecular switch in stress signal transduction. The activation of JNK pathways is also critical for pathological death associated with neurodegenerative diseases. Considering that a variety of stressors activate JNK, it is surprising that the role of hippocampal JNK in memory and synaptic plasticity has not yet been systematically investigated. Here we summarize the emerging evidence for the functions of hippocampal JNK in memory and synaptic plasticity, including our recent demonstration that JNK isoforms play critical roles in regulation of contextual fear conditioning under stressful and baseline conditions. We postulate that sustained activation of the hippocampal JNK2 and JNK3 pathways is involved in the initial stress response that ultimately leads to deficits in memory and long-term potentiation, whereas transient JNK1 activation regulates baseline contextual fear conditioning. Results obtained within the framework of our recent findings will be used for future work, which will differentiate mechanisms underlying beneficial short-term JNK action from prolonged JNK activation that may lead to memory deficits and neurodegeneration.

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

Soluble β-amyloid has been shown to regulate presynaptic Ca2+ and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimers patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca2+ imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator.

Correlation between age-related changes in open field behavior and plaque forming cell response in da female rats

We investigated the relationship between immunological and behavioral changes during ageing in Dark Agouti female rats. Results showed that ageing was associated with decreased exploratory behavior and increased emotionality (open field test) and decreased pain perception (writhing assay), but not with altered depression-like behavior (forced swim test). The observed behavioral changes were paralleled with decreased innate immunity in middle-aged and old rats, as revealed by reduced peroxide production of peritoneal macrophages; and decreased specific immunity, measured by the plaque-forming cell response, in old rats in comparison with young rats. Correlation analyses between behavioral and immune parameters demonstrated a significant correlation between the lines crossed in the open field test and the plaque-forming cell response. Taken together, the demonstrated age-dependent association between exploratory behavior and specific immune response suggests a senescent decline of a common neuroimmune regulatory mechanism.